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Studies on interferon production

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Title:
Studies on interferon production
Creator:
Goorha, Rakesh Mohan, 1940-
Publication Date:
Language:
English
Physical Description:
ix, 114 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Actinomycin ( jstor )
Bottles ( jstor )
Cell culture techniques ( jstor )
Embryos ( jstor )
Incubation ( jstor )
Infections ( jstor )
Interferons ( jstor )
Ribosomes ( jstor )
RNA ( jstor )
Semliki forest virus ( jstor )
Interferon Inducers ( mesh )
Interferons ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1969.
Bibliography:
Includes bibliographical references (leaves 107-113).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Rakesh Mohan Goorha.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
25700798 ( OCLC )
ocm25700798
00898272 ( ALEPH )

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Full Text












STUDIES ON INTERFERON PRODUCTION














By -A
RAKESH MOHAN GOORHA


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY














UNIVERSITY OF FLORIDA
1969




STUDIES ON INTERFERON PRODUCTION
By
RAKESH MOHAN GOORHA
)
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA


ACKNOWLEDGMENTS
I acknowledge my deepest appreciation to Dr. G. E. Gifford
for his encouragement, supervision and guidance throughout the
period of this investigation and during the preparation of the manu
script. I also thank the members of my advisory committee,
Dr. H. E. Kaufman, Dr. L. W. Clem and Dr. P. Byvoet for
their suggestions and encouragement. I am grateful to Dr. P. A.
Small for critically reviewing the manuscript. I would like to
thank Dr. I. Rosen who helped me in many ways during my studies.
I would also like to thank Mrs. P. Jones for the generous
supply of cell cultures and other materials. I am also grateful
to Mr. M. Fruitstone, Mrs. B. Asch, Mrs. J. Curry and fellow
graduate students for their suggestions, aid and criticisms
during this investigation.
\
11


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS
LIST OF TABLES iv
LIST OF FIGURES v
KEY TO ABBREVIATIONS viii
INTRODUCTION 1
REVIEW OF LITERATURE 4
Production of Interferon . . 12
MATERIALS AND METHODS ; 23
Materials 23
Methods . . J 27
RESULTS 58
DISCUSSION 90
SUMMARY v 105
REFERENCES 107
VITA 114


LIST OF TABLES
Table Page
1. Comparison of Viral and Non-Viral
Induced Interferons. 5
2. The Effect of Two Multiplicities of
Virus on Interferon Production ..... 2
3. Interferon Production in Presence
or Absence of Actinomycin D ..... 76
4. Association of Chick Interferon with
Mouse and Chick Embryo Ribosomes .... 87
5. Association of Chick Interferon with
Ribosomal Subunits 89
iv


LIST OF FIGURES
Figure
1. Heat inactivation of Semliki Forest virus at 37C.
Aliquots were removed at various times during
incubation and assayed for residual virus.
3
2. H-uridine incorporation in uninfected chick embryo
cell cultures exposed to various concentrations of
^H-uridine and incubated at 37C for 30 minutes ..
3
3. H-uridine incorporation in uninfected chick embryo
cell cultures exposed to 15 l1 C of ^H-uridine for
various periods of time ....
4. Effect of calf serum on JH-uridine incorporation of
uninfected chick embryo cell cultures with or with
out actinomycin D. The cell cultures were exposed
to 20 pC of ^H-uridine for 30 minutes.
5. H-uridine incorporation, with or without calf (CS)
serum in uninfected chick embryo cell cultures. The
cell cultures were incubated at 37C for various per-
iods and then exposed to 20 pC of H-uridine for
30 minutes ....... . .
3
6. Inhibition of H-uridine incorporation in uninfected
chick embryo cell cultures with various concentra
tions of actinomycin D. The cell cultures were
exposed to 20 p C of H-uridine for 30 minutes at
the indicated times ... : . .
3
7. Inhibition of H-uridine incorporation in uninfected
chick embryo cell cultures with various concentra
tions of actinomycin D. The values plotted were
obtained from Figure 6 at 10 hours after the expo
sure to actinomycin D.
Page
35
38
39
40
42
43
44
8. Linearity of sucrose gradient as measured by refrac
tive index of each fraction with an Abbe refractometer 47
v


Figure
Page
9.Sucrose gradient analysis of chick embryo ribosomes.
One ml of crude ribosomal preparation was layered
on 8 to 25% linear sucrose gradient and then centri
fuged at 24, 000 rpm for 8 hours. The other details
have been described under Materials and Methods . 49
10. Separation of chick embryo ribosomes into their
sub-units. The purified ribosomal preparation was
mixed with l/lOth volume of 4 M sodium chloride and
incubated at 4C for 5, 15 or 30 minutes and then
centrifuged at 24, 000 rpmina8 to 25% linear sucrose
gradient for 12 hours . . . ... 51
11. Separation of chick embryo ribosomes into their sub
units. The NaCl concentration of ribosomal prepara
tion was raised to 0. 2 M, 0. 3 M, 0. 4 M or 0. 6 M and
incubated at 4C for 15 minutes. The samples were
then centrifuged ina 8 to 25% sucrose gradient containing
the same concentration of NaCl as that of sample and
centrifuged at 24, 000 rpm for 12 hours . ... 52
12. Interferon yields at 24 hours in chick embryo cell
cultures infected with various input multiplicities
of Semliki Forest virus ... ..... 59
13. Kinetics of interferon production in chick embryo
cell cultures infected with Semliki Forest virus at
multiplicities of 10 or 0.1 pfu/cell. . ... 60
14. RNA synthesis in chick embryo cell cultures in
fected with Semliki Forest virus at a multiplicity
of 10 pfu/cell with or without actinomycin D.
Actinomycin D sensitive RNA was plotted as the
difference between total RNA synthesis and
actinomycin D resistant RNA synthesis ... 63
15. Correlation of viral RNA synthesis with growth of
Semliki Forest virus (with or without actinomycin
D) in chick embryo cell cultures at a multiplicity
of 10 pfu/cell. . . . ... 66
16. Kinetics of interferon production in chick embryo
cell cultures exposed to heat inactivated Semliki
Forest virus. The cell cultures received the
equivalent of either 10 or 0. 1 pfu/cell of the virus . 68
vi


Figure Page
17. Kinetics of interferon production with heat
inactivated and live Semliki Forest virus
with cell cultures ......... 70
18. Kinetics of interferon production in presence
of immune serum prepared against Semliki
Forest virus. Details have been described in
the text . ......... 73
19. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
infected for 2 hours with Semliki Forest virus.
^H-uridine (20 P C) was added for 45 minutes, and
RNA was extracted. L cell RNA was employed
as carrier RNA ......... 78
20. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
infected for 4 hours with Semliki Forest virus.
^H-uridine (20 p C) was added for 45 minutes, and
RNA was then extracted. L cell RNA was employed
as carrier RNA ........ 79
21. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
infected for 6 hours with Semliki Forest virus.
^H-uridine (20 pC) was added for 45 minutes, and
RNA was then extracted. L cell RNA was employed
as carrier RNA .... ..... 80
22. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
exposed for 2 hours with heat inactivated Semliki
Forest virus. ^H-uridine (20 P C) was added for
45 minutes and RNA was then extracted. L cell
RNA was employed as carrier RNA . ... 82
23. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
exposed to heat inactivated Semliki Forest virus
for 4 or 6 hours. ^H-uridine (20 pC) was added
for 45 minutes, and RNA was then extracted. L
cell RNA was employed as carrier RNA ... .83
24. RNA synthesis in chick embryo cell cultures exposed
to heat inactivated or live Semliki Forest virus in
presence or absence of actinomycin D .85
vu


KEY TO ABBREVIATIONS
The following abbreviations were used in the text.
BSS
Balanced salt solution
CAM
Chorioallantoic membrane
CE
Chick embryo
CS
Calf serum
DNA
Deoxyribonucleic acid
FPA
Fluor ophenyalanine
MEM
Minimum essential medium
MM
Maintenance medium
MMM
Modified maintenance medium
m-RNA
Messenger ribonucleic acid
NDV
Newcastle disease virus
Pi
Post infection
PDD50
50 per cent plaque depressing dose
pfu
Plaque forming unit
rA/rU
Riboadenylic acid/ribouridylic acid
rl/rC
Riboinosinic acid/ribocytidylic acid
RNA
Ribonucleic acid
RNAse
Ribonuclease
RSB
Rabbit reticulocyte buffer
vm


SDS
S
TIP
vsv
w/v
Sodium dodecyl sulphate
Sedimentation coefficient
Translational inhibitory protein
Vesicular stomatitis virus
Weight per volume
ix


INTRODUCTION
One of the intriguing aspects of viral induced interferon production
is the role of multiplicity of infection. It has been known that high
multiplicities of certain viruses inhibit interferon production while
higher yields of interferon are obtained with lower multiplicities of
infection. (De Maeyer and De Somer, 1962; Gifford, 1963; Aurelian
and Roizman, 1965). Similar results were reported by Toy and Gifford
(1967a) who observed that maximal amount of interferon was obtained
in Semliki Forest virus (SFV) infected chick embryo (CE) cells at a
multiplicity of 0. 2 pfu/cell while multiplicity of 10 pfu/cell resulted in
marked reduction of interferon yield. An attempt was made to explain
this phenomenon by studying the effect of these two multiplicities of
infection on host cell macromolecular synthesis in the infected cells.
The study of RNA synthesis was suitable since cellular RNA could be
differentiated from viral RNA by the use of actinomycin D, an anti
biotic which is known to inhibit cellular RNA synthesis but has no
effect on Semliki Forest virus replication or its RNA synthesis. The
other factors considered in this study were: (1) effect of progeny virus
on interferon production, and (2) production of interferon by heat
inactivated virus.
The physico-chemical nature of the interferon inducers has, so
1


2
far, proved to be elusive. Recently, several investigators (Field et al. ,
1967; Vilcek et al. 1968; Dianzani et al. 1968) have reported that inter
feron production can be induced by complexes of polyriboinosinic acid
and polyribocytidylic acid (rl/rC) and other synthetic, double stranded
RNA preparations, in vivo as well as in vitro. However, the role of
single stranded RNA in interferon induction has been a subject of con
troversy. Field et al. (1967) reported that single stranded RNA did not
induce interferon production. These results have been confirmed by
Vilcek et al. (1968), Tytell et al. (1967) and Hilleman (1969). However,
Baron et al. (1968) and De Clercq and Merigan (1969a) found that certain
single stranded polyribonucleotide preparations such as polyriboinosinic
acid and polyribogunylic acid were able to induce small amounts of
interferon. Since heat inactivated SFV, though unable to replicate,
induces interferon production, the question that naturally presents itself
is: does the heat inactivated noninfectious SFV synthesize a double
stranded, replicative form when it induces interferon production in
CE cells or is the single stranded viral RNA in itself the inducing
agent? Part of the dissertation deals with an attempt to answer this
question.
The antiviral action of interferon in cells is due to the de novo
synthesis of another protein which apparently confers on ribosomes
the capability to distinguish between the cellular and viral messenger
RNA. These ribosomes are capable of forming polysomes with cellu
lar messenger RNA but bind poorly to viral messenger RNA and thus


the production of viral RNA directed proteins is inhibited (Marcus and
Salb, 1966; Carter and Levy, 1967). However, it is not known whether
this interferon induced protein acts alone or in conjunction with inter
feron. If interferon does play a direct role in the maintenance of a
virus resistant state in the cells, it is reasonable to assume that it
would also combine with ribosomes for the expression of antiviral
activity. To investigate this possibility, chick embryo and mouse
liver ribosomes and their subunits were exposed to various interferon
preparations and were examined for their capability to combine with
interferon.


REVIEW OF LITERATURE
Interferon is a term given to a group of antiviral proteins that can
be induced in vivo and in vitro by a wide variety of.agents. It was
first described by Isaacs and Lindenmann (1957) as a protective factor
against viral replication in chorio-allantoic membranes induced by
heat inactivated influenza virus. Since then, interferon has been in
duced by every major group of viruses and by a variety of nonviral
inducers of interferon-like substances. Thus, it has become evident
that production of interferon by cells is a general nonspecific response
to various stimuli. Interferons, in general, can be divided into two
broad classes. (1) Interferons whose synthesis is dependent upon RNA
and protein synthesis in the induced cells. Interferons produced in
response to viruses form a major group in this class. (2) Interferons
whose appearance after induction is not dependent upon RNA and pro
tein synthesis and seem to be released from certain cells (preformed
interferon). The other differences in the two classes of interferons
are summarized in Table 1. However, some inducers may invoke both
types of response and characterization of such preparations of inter
feron becomes difficult.
The inhibition of viral induced interferon production by actinomycin
D was first reported by Heller (1963) and Wagner (1963). This finding
4


TABLE 1
COMPARISON OF VIRAL AND NON-VIRAL INDUCED INTERFERONS
Characteristics
Viral
Non-viral
Reference
Kinetics of production (serum)
Late peak
Early peak
Youngner, J. Hallum, J. and
Stinebring, W. (1966)
Molecular weight (serum)
often > 50, 000
often < 50, 000
Ibid.
Sensitivity of Production:
Protein synthesis inhibitors
sensitive
not sensitive
Ho, M. and Breinig, M. K.
(1965)
Actinomycin D
sensitive
not sensitive
Finkelstein, M. S. Bausek,
G. H. and Merigan, T. C. (1968)
Presence of spleen during
induction
not essential
es sential
De Somer, P. and Billiau, A.
(1966)
Effect of BCG sensitization
on production
no influence
increased
production
Youngner, J. Hallum, J. and
Stinebring, W. (1966)
Elevated temperature
increased
production
no influence
Isaacs, A. (1961)--viral, De Some
P. and Billiau, A. (1966)--non-
viral


was interpreted as evidence for the coding of interferon by the cellular
genome rather than by the inducer virus. A similar effect of other
DNA dependent RNA synthesis inhibitors like 4, 5, 6-trichloro-l- D-
ribofuranosyl benzimidazole and 8-azaguanine on interferon synthesis
has been reported by Walters, Burke and Skehel (1967). The other lines
of evidence which support this hypothesis are provided by the obser
vations that (1) interferons are species specific, and (2) DNA as well
as RNA viruses induced apparently identical interferons in chick cells
(Lampson et al. 1965). Thus, it seems that in interferon induction by
viruses, a stimulus leads to cell directed RNA synthesis which then
controls the synthesis of the new protein, presumably different from the
other proteins of the cell.
The concept of viral induced interferon formation as a newly syn
thesized protein is supported by the work of Buchan and Burke (1965,
1966) who reported that 25 p g/ml of p-flurophenylalanine, in phenyl-
alanine-free medium, completely inhibited subsequent interferon for
mation in chick embryos exposed to ultraviolet-inactivated influenza
virus. This substance acts as an antagonist of phenylalanine and is
incorporated into proteins. Thus, the proteins might have a reduced
or no biological activity depending upon the importance of the phenyl
alanine residue for biological activity (Richmond, I960, 1963). Simi
larly, puromycin at low concentrations has also been found to com
pletely inhibit interferon production in various cell-virus systems.
It was reported by Wagner and Huang (1965) that once interferon


7
production has started in the Krebs-2 carcinoma cells-Newcastle
disease virus (NDV) system, the addition of puromycin resulted in the
immediate cessation of interferon production. Thus, the rate at
which interferon appears in cell culture fluids reflects the actual rate
of synthesis and there seems to be no build-up of the product in the
cells. Similar results have been obtained by Buchan and Burke (1965,
1966) employing chick embryos infected with ultraviolet-inactivated
virus.
This overall similarity of interferon production with Jacob and
Monod's operon model led Burke (1966) to propose a hypothetical scheme
in which virus invasion is presumably followed by uncoating and re
lease of an interferon inducer which induces the production of inter
feron specific messenger-RNA. This m-RNA then directs the syn
thesis of interferon. However, there is no well established case for
the operon model's counterpart in the mammalian system. In the inter
feron system, the exact nature of the inducer, the mechanism of inter
action between inducer and host genome, and the nature of the repres
sor molecule, if present, are largely unknown. Attempts to obtain
direct evidence for interferon specific messenger RNA have not been
successful (Burke and Low, 1965; Wagner and Huang, 1966). However,
Wagner and Huang (1965, 1966) reported that in the Krebs-2 carcinoma
cell-NDV system, practically all the interferon specific messenger
is synthesized within 6 hours after infection and is relatively stable
for at least 10 hours.


The discovery of substances other than viruses that can elicit
interferon production was first reported by Isaacs, Cox and Rotem
(1963). They found that chick, mouse and rabbit ribosomal RNA did
not induce interferon in homologous cells unless the RNA was first
made "foreign" by treatment with nitrous acid. Ribonucleic acid from
E. coli, turnip yellow mosaic virus and rat liver DNA though "foreign"
were not effective inducers of interferon in chick cells. Rotem, Cox
and Isaacs (1963) also showed that chick liver RNA inhibited the growth
of vaccinia virus in mouse cell cultures and mouse liver RNA inhibited
vaccinia virus in chick cell cultures.
Youngner and Stinebring (1964) reported the production of low
amount of interferon in chickens infected with Brucella abortus.
Stinebring and Youngner (1964) also found that intravenous injections
of endotoxin, Salmonella typhimurium, or Serratia marcescens caused
a rapid appearance of interferon in the blood stream of mice. Ho (1964)
also reported the production of interferon in rabbits injected with endo
toxin. Youngner and Stinebring (1965) found that endotoxin or Brucella
abortus induced interferon formation was unaffected by concentrations
of actinomycin and cyclohexamide which inhibited more than 97% of
RNA and protein synthesis, respectively, in mouse liver. They also
observed that pretreatment of animals with puromycin or cyclohexa
mide enhanced and prolonged the interferon response. Gifford (1965)
found that yeast RNA, hydrolysate of RNA, and mononucleotides in
hibited vaccinia and chikungunya virus in cell cultures but interferon


was not present in high enough titers to account for all the viral in
hibitory effects. Field et al. (1967) reported the formation of inter
feron in rabbits and their spleen cell cultures after induction by syn
thetic polyribonucleotides. Only double stranded polyribonucleotides,
such as polyinosinic acid/polycytidylic acid (rl/rC), were found to be
effective inducers while double stranded DNA polymers or single
stranded RNA polymers were unable to elicit an interferon response.
Similar results have also been obtained by Vilcek et al. (1968), Falcoff
and Bercoff (1968), Tytell et al. (1968), and Field et al. (1968).
However, it has also been claimed that single stranded polyribocy-
tidylic acid as well as polyriboinosinic acid can induce interferon for-
mation (Baron et al. 1968; Levy et al. 1968; Finkelstein, Bausek and
Merigan, 1968). It was then suggested that the inducing ability of
single stranded polyribonucleotides may be due to the contamination of
these preparations with double stranded polymers (Field et al. 1968;
Tytell et al. 1968). But De Clercq and Merigan (1969a) reportd that
the single stranded homopolymers, polyriboguanylic acid, polyribo
inosinic acid and polyriboxanthylic acid, at neutral pH and polyribo-
adenylic acid and polyribocytidylic acid at acid pH can induce inter
feron production. The preparations were apparently free of double
stranded polymers and the inducibility characteristic was related to
the stability of the secondary structure of homopolymers as indicated
by their high temperature of melting (Tm) values. However, triple
stranded complexes of polyribonucleotides, having higher Tm values,


10
were much less active than the double stranded polymers. It was post
ulated that differences in the inductive capability of various polyribo
nucleotides may be due to the differences in their affinity for the site
of initial interferon formation inside the cells, even though cells seem
to have similar permeabilities to various effective and ineffective RNA
polymers (Field, Tytell and Hilleman, 1969) and such polymers are
equally suceptible to nuclease degradation inside the cells (Colby and
Chamberlin, 1969). De Clercq, Eckstein and Merigan (1969) recently
reported that substitution of the phosphate group by a thiophosphate
group in an alternating copolymer, rA/rU, resulted in a 2-20 fold in
crease in its ability to induce interferon production. This increase in
inducibility was accompanied by a 10-100 fold increase in resistance to
pancreatic ribonuclease. It has been suggested (De Clercq and Merigan,
1969b) that the common structural requirement for polyribonucleotides
and other synthetic polyanion interferon inducers include (a) high
molecular weight, (b) a stable, primary and long, carbon to carbon
backbone, and (c) a regular and dense sequence of negative charges on
the backbone. It has been found by several investigators that polybasic
substances such as protamine sulphate, neomycin sulphate and DEAE
dextran significantly increase the yields of interferon in vitro as well
as in in vitro (Vilcek et al. 1968; Falcoff and Bercoff, 1968) by poly
ribonucleotides. The mechanism of enhancement is not well under
stood although it has been postulated that enhancement is due to the
increased penetrability of the polyribonucleotides into the cells.


11
The effect of actinomycin D on polyribonucleotide induced inter
feron formation has been a subject of controversy. Vilcek et al. (1968)
observed that 2P g/ml of actinomycin D inhibited the production of
interferon in rabbit kidney cells induced by poly rl/rC. Similar re
sults were obtained by Falcoff and Bercoff (1968) in human leukocytes
and in amniotic membranes. However, Finkelstein, Bausek and Merigan
(1968) observed that the concentration of actinomycin D which suppressed
NDV-induced interferon production had no effect on the interferon for
mation induced by poly rl/rC, pyran, and endotoxin in human skin fibro
blast and mouse peritoneal macrophage cultures. It was also found that
actinomycin suppressed polyribonucleotide-induced interferon formation
only at concentrations which were cytotoxic. Vilcek, Rossman and
Varacalli (1969) observed that actinomycin D, if applied two hours or
later after the induction with poly rl/rC, does not affect the yields of
interferon in rabbit kidney cell cultures. The formation of polyribo
nucleotides-induced interferon is not inhibited by protein synthesis in
hibitors like p-flurophenylalanine and puromycin in vitro (Finkelstein,
Bausek and Merigan, 1968) or in vivo (Youngner and Hallum, 1968)
while viral-induced interferon synthesis was suppressed under similar
conditions.
Differences between the two classes of interferons (summarized in
Table 1 ) raises the question of whether or not the mechanism of induc
tion in both classes (preformed versus synthesized) is similar. The
question becomes more pertinent since the two responses also occur


in morphologically uniform population of clls like human skin fibro
blast indicating that the same cell is capable of responding in two
different ways. It has been postulated that there may be two differ
ent processes of interferon induction, i. e., de novo synthesis as well
as release of already formed interferon (Ho and Kono, 1965; Ho, Postic
and Ke, 1968; Youngner, 1968; Flnkelstein, Bausek and Merigan, 1968).
It has also been suggested by Finkelstein, Bausek and Merigan (1968)
that both responses may involve de novo synthesis but that one requires
an additional step, e. g.t virus uncoating which is snsitive to metabolic
inhibitors; or, that both responses may require de novo interferon syn
thesis and both have similar biological activity but possess different
sensitivities to metabolic inhibitors and are released or formed at dif
ferent rates. Youngner (1968) suggested that the two responses may
result in release of preformed interferon but the viral-induced type
may require an interferon activating enzyme whose synthesis is sup
pressed by metabolic inhibitors. However, this possibility is ex
cluded by the work of Fhucker (1969) who reported, on the basis of
radiolabelled pulse experiments, that interferon produced in L cells
by UV-unactivated NDV is essentially a product of de novo synthesis.
A definite explanation for the mechanism of the two types of responses
may be possible once radiolabelled amino acid incorporation studies
on polyribonucleotide induced-interferon become available.
Production of Interferon
Interferon is a product of the interaction of cell and inducer. Both


13
elements of interaction seem to be equally important determinants of
whether interferon is produced and how much. Production would also
be dependent upon the type of cellular environment in which this inter
action takes place.
Cellular Aspects of Interferon Production
Genetic control
The direct evidence for the genetic control of interferon production
by cells comes from the observation of Desmyter, Melnick and Rawls
(1968) that Vero cells, a line of African green monkey kidney cells, was
unable to produce interferon when infected with NDV, Sendai, Sindbis
or Rubella virus. This apparently qualitative defectiveness in inter
feron production has also been noted when Vero cells were exposed to
poly rl/rC with or without protamine sulphate or neomycin sulphate
(Schaffer and Lockart, 1969). The cell line, though incapable of pro
ducing interferon, is sensitive to the action of exogenous interferon.
There is no information in regard to the size of the locus or the num
ber of genes involved in this defectiveness. Cogniaux-Le Clerc, Levy
and Wagner (1966) reported that the loss of ability to produce inter
feron as a function of increasing UV dose showed first order kinetics
and it was suggested that a single site on cellular DNA controls the
formation of the interferon specific messenger RNA. Similar results
were obtained by Burke and Morrison (1966).
Types of cellular response
Endotoxin, when administered into intact animals, elicits significant


14
amounts of interferon but is unable to induce interferon in vitro except
in leukocytic cell cultures (Smith and Wagner, 1967). Lackovic et al.
(19o7) reported failure of L cells to produce interferon when treated
with mannan, a yeast polysaccharide, although a good response was
obtained with mouse peritoneal macrophage cultures. Similarly,
Finkelstein, Dausek and Merigan (1968) observed that pyran, a poly-
carboxylate polymer, and poly rl/rC induced interferon in mouse
peritoneal macrophages but not in L cells. Thus, it appears that
there are several types of cellular responses to inducers, with
aneuploid cell lines being most restrictive. The type of cellular re
sponse, at least in part, seems to depend upon the penetrability of
the inducer into the cell since L cells have been reported to produce
significant amount of interferon with poly rl/rC after prior treatment
with diethylaminoethyl (DEAE) dextran (Dianzani et al. 1968). Other
factors which may contribute to variation in types of cellular responses
have not been elucidated.
Host macromolecular synthesis
The evidence available indicates that synthesis of interferon is a
latent cellular function, induced by viral infection or certain nonviral
agents. Thus, the role of cellular macromolecular synthesis in the
production of interferon becomes obvious. Several investigators have
reported that virulent viruses induce very low yields of interferon.
Ruiz-Gomez and Isaacs (1963) reported that NDV, a cytopathic virus
in CE cells, grew in high titers but produced very little interferon.


15
However, when mouse embryo fibroblast or human thyroid cell cultures
were infected with the same virus, no cytopathology was observed and
high yields of interferon were obtained. Similar results were obtained
with vesicular stomatitis virus (VSV) (Wagner et al. 1963), foot and
mouth disease virus (Sellers, 1963, 1964), polyoma virus (Friedman
and Rabson, 1964) and various arboviruses (Ruiz-Gomez and Isaacs,
1963). Virulent myxovirus has been reported to inhibit interferon syn
thesis induced by UV-inactivated (Lindenmann, I960) or avirulent
myxoviruses (Hermodsson, 1963). However, there is not always a
correlation between the virulence of virus and interferon production:
e. g. some strains of influenza virus (Inglot, Kochman and Mastalerz,
1965; Link et al. 1965a), Sindbis virus (Vilcek, 1964),vaccinia virus
(Link et al. 1965b) and NDV (Baron, 1964) do not follow this pattern.
The mechanism by which certain viruses can inhibit interferon
production was investigated by Wagner and Huang (1966). They induced
interferon production in suspension cultures of Krebs-2 carcinoma
cells by employing an avirulent strain of NDV. Interferon was first
detected 3 to 4 hours after the infection and production increased al
most linearly, reaching a peak at 20 hours. However, interferon syn
thesis was terminated when cells were superinfected with VSV (50
pfu/cell) within 4 hours after infection with NDV. Vesicular stoma
titis virus does not induce interferon in this cell system. Krebs-2
carcinoma cells infected with VSV exhibited a rapid and almost immedi
ate decline in the rate of cellular RNA synthesis but NDV,. at the


16
multiplicity employed, had no appreciable effect for at least 3 hours
after infection. Therefore, it was postulated that VSV prevented inter
feron synthesis by inhibiting cellular RNA synthesis. Similar results
were reported by Aurelian and Roizman (1965) employing a strain of
herpes virus which caused an abortive infection in dog kidney cells.
Infection at a multiplicity of 100 pfu/cell led to the formation of viral
DNA and antigen without any production of interferon, whereas infec
tion at lower multiplicity (12 pfu/cell) resulted in the production of
interferon, but not virus. In the former case, virus infection caused
a rapid decline in cellular RNA synthesis and, hence, the cessation of
interferon production. At the lower multiplicity, virus induced inhi
bition of RNA synthesis did not occur until 5 to 6 hours after infection
and interferon production was thus permitted.
Bolognesi and Wilson (1966) have reported a rapid and profound
decline in the rate of cellular protein synthesis in NDV infected CE
cells. Most probably, marked inhibition of protein synthesis is the
reason for the lack of interferon production in this system. The in
hibition of protein synthesis can be ascribed to either reduction in
cellular RNA synthesis or inhibition of messenger RNA translation in
NDV infected CE cells. However, the relationship between interferon
production and the rate of cellular protein synthesis is complex;
Friedman (1966a) observed that interferon yields were reduced in SFV
infected CE cells under conditions when overall cell protein synthesis
was augmented and postulated that increased protein synthesis resulted


17
in the accumulation of an interferon repressor in the cells thereby
decreasing interferon yields.
Factors Influencing Interferon Production
All the conditions which favor the optimal production of interferon
are not yet known. However, there are some factors which have been
reported to influence interferon synthesis and are considered in this
section.
t
Priming
The priming effect is obtained when cells treated either with in
activated virus or with interferon respond by an enhanced production of
interferon on subsequent induction. Burke and Isaacs (1958) observed
that influenza virus could induce interferon formation in chick embryo
chorio-allantoic membrane only if the tissue had first been primed with
heat-inactivated virus. Similar results have been obtained with Eastern
equine encephalitis virus (Mahdy and Ho, 1964) and Sindbis virus (Ho
and Breinig, 1962). Isaacs and Burke (1958) also described the poten
tiating effect of interferon on subsequent interferon formation. However,
Vilcek and Rada (1962) and Paucker and Cantell (1963) reported that
pretreatment of cells with interferon inhibited the subsequent yields
of interferon when challenged with a viral inducer. Later it was found
(Lockart, 1963; Taylor, 1964; Friedman, 1966b) that pretreatment of
cells with interferon may indeed have both effects depending upon the
i
amount used and multiplicities of virus subsequently employed to
elicit interferon production. Levy, Buckler and Baron (1966) and


Friedman (1966b) showed that "priming" not only increased the yields
but interferon was synthesized more rapidly in the primed cells.
Friedman (1966b) has also shown that enhancement of interferon pro
duction is dependent upon active protein synthesis in the cells during
the priming period. However, synthesis of interferon specific mes
senger RNA was not detected during the period of priming. It was
also reported that interferon-cell interaction for priming could occur
at 4C but would be expressed only if cells were further incubated at
37C for several hours before challenging with virus.
Temperature
Interferon production can only occur within a certain range of
temperature as would be expected for any cellular metabolic process.
Its synthesis was inhibited when cells were incubated at 4C (Isaacs,
1963). Ruiz-Gomez and Isaacs (1963a, b) observed that the optimum
temperature for interferon production was generally higher than that
for virus replication. For example, more interferon was induced by
chikungunya virus at 42C, an unfavorable temperature for virus
replication, than at 39C or 35C. Burke, Skehel and Low (1967)
found that SFV induced optimal yields of interferon at 42C when no
virus replication was detected. However, the cells have to be pre
incubated with virus at 37C for some time to presumably allow its
adsorption and uncoating before shifting the temperature to 42C.
Ruiz-Gomez and Isaacs (1963) also suggested thatthis increased inter
feron production at higher temperature may constitute a defense


19
mechanism which prevents the pathogenic effect of viruses during
febrile conditions. The results obtained by Ruiz-Gomez and Sosa-
Martinez (1965) tend to support this hypothesis. Mice inoculated with
Coxsackie B virus were kept at 4C, 11C and 25C. Those kept at
4C developed viremia with high titers in heart and liver and died
3 to 4 days after infection. Their livers had very little interferon
except on the first day after infection. However, the mice kept at
25C survived, virus did not replicate in any tissue, and livers con
tained high titers of interferon.
The other aspect of this relationship was reported by Siegert, Shu
and Kholhage (1967) who found that production of interferon in rabbits
infected with myxovirus was accompanied by fever. Similarly,
Merigan (1968) reported that interferon inducing doses of pyran also
caused fever in man. However, this correlation was not observed
with measles vaccine which induced interferon production without
concomittant induction of fever in human beings. Conversely, bac
terial endotoxin or etiocholanalone induced fever without the production
of interferon. However, endotoxin which is capable of inducing inter
feron production in mice (Stinebring and Youngner, 1964) as well as
in rabbits (Ho, 1964) is also known to produce fever in rabbits.
Multiplicity of infection
In several instances of cell-virus interaction, the multiplicity of
infection seems to play a significant role in determining the amount
of interferon synthesized by the induced cells. De Maeyer and De Somer


20
(1962) reported that rat tumor cells infected with Sindbis virus pro
duced maximal amounts of interferon when a multiplicity of 0. 1 pfu/
cell was employed, and much lower yields were obtained at a multi
plicity of 10 pfu/cell. Gifford (1963) observed that chikungunya virus
best induced interferon production when the input multiplicity of in
fection was 0.1 pfu/cell while higher multiplicity reduced the yields of
interferon. The observations of Aurelian and Roizman (1965) in the
dog kidney cell-herpes virus system and Toy and Gifford (1967a) in
the SFV-CE system tend to support the concept that higher multiplicity
of infection often inhibits interferon production whereas more interferon
is produced with lower multiplicities.
Nature of Inducing Agent
The physico-chemical nature of the agents which induce interferon
has not been well elucidated. Isaacs (1961) suggested that the induc
tion of interferon by viruses might be due to their "foreign" nucleic
acid. This concept was in agreement with the observations that
viruses, composed of nucleic acid and protein only, could induce inter
feron production, but incomplete viruses containing less nucleic cid
failed to induce.
Low yields of interferon have been obtained in various cell systems
by Rotem, Cox and Isaacs (1963) with heterologous RNA, and by Isaacs,
Cox and Rotem (1963) using nitrous acid treated homologous ribosomal
RNA. The few known properties of nucleic acid-induced viral inhibi
tors suggested identity with viral-induced interferon, but the physico
chemical characterization of these preparations has not been carried


21
out in detail. Moreover, several investigators were unable to confirm
these findings. However, the work of Skehel and Burke (1968a) supports
the hypothesis that complete viral nucleic acid is essential for the
induction of interferon production. These authors studied the effect
of hydroxylamine on SFV. Hydroxylamine has been extensively em
ployed to inactivate the infectivity of viruses by reacting with viral
nucleic acid; it has no effect on viral antigenicity (Schafer and Rott,
1962). The inactivation of SFV by 0. 2 M hydroxylamine at 25C fol
lowed a pattern of first order kinetics, and the interferon-inducing
capacity decreased at the same rate as that of infectivity. There was
no effect on hemagglutination titers,, indicating that the protein coat
of the virus was unaffected by the treatment. These results confirmed
the significance of viral nucleic acid in the process of induction but did
not rule out the possibility that viral protein(s) may also be essential
for interferon induction.
Burke, Skehel and Low (1967) studied the early stages of inter
feron induction. The system employed was infection of chick cells
with SFV for one hour at 36C followed by shifting the temperature to
42C. Under these conditions interferon is produced but the virus
does not replicate. It was found that during the incubation period at
36 C some ribonuclease resistant RNA was synthesized in the infected
cells (Skehel and Burke, 1968b), and it was suggested that formation
of a double stranded, replicative form of viral RNA may be the first
step in induction of interferon formation. Once again, the role of


viral protein(s), if any, could not be ascertained.
22
Double stranded polyribonucleotides have been reported to induce
interferon production in vivo as well as in vitro but the role of single
stranded polyribonucleotides in interferon induction has not been
clearly established (discussed on pages 9.10, andll ). However, it has
been reported that nucleic acid preparations release preformed inter
feron (Finkelstein, Bausek., Merigan, 1968; Youngner, 1968), unlike
the viral-induced interferon which is synthesized de novo. Therefore,
it is a distinct possibility that induction for the two types of responses
may not be achieved through the same process.
Lockart et al. (1968) employed temperature sensitive mutants of
Sindbis virus in CE cells for interferon induction. The authors con
cluded that input viral RNA and the replication of viral RNA are not
sufficient for interferon induction but the induction event requires some
viral protein(s) or the process for which these proteins are necessary.
Similarly, Dianzani (1969) reported that NDV-infected mouse cells
synthesize interferon specific messenger RNA in the presence of
protein synthesis inhibitors. Under these conditions, viral replicative
events were prevented, thereby indicating that input parental viral
RNA or viral protein(s), or both, were the inducer for the synthesis
of interferon specific messenger RNA.
The information available at present does not resolve whether
single stranded RNA is sufficient to induce interferon production or
double stranded RNA is the necessary form. In addition, the role of
viral protein(s) in the process of induction is not well understood.


MATERIALS AND METHODS
Materials
Virus Strains
Vaccinia virus (VV). The N. Y. 914 strain, isolated by Dr. G. E.
Gifford from commercial lymph vaccine, was employed.
Semliki Forest virus (SFV). Kumba strain was obtained from
Dr. J. Porterfield, National Institute for Medical Research, London,
England.
Vesicular stomatitis virus (VSV). Indiana strain was kindly sup
plied by Dr. Samuel Baron, National Institute of Health, Bethesda,
Maryland.
Newcastle disease virus (NDV). Hertz strain was also obtained
from Dr. J. Porterfield, The Roakin strain was obtained from the
Research Reference Reagent Branch of the National Institute of
Allergy and Infectious Diseases and the Cincinnati strain was received
from Mr. M. Fruitstone, Department of Microbiology, University of
Florida.
Cell Cultures
Primary chick embryo cell cultures. Chick embryo (CE) cell
cultures were prepared as described in Methods.
Mouse L cell cultures. The continuous L cells (strain 929) were
23


kindly supplied by Mr. M. Fruitstone.
24
Media
Balanced salt solution. Gey's balanced salt solution (BSS) was
employed in the growth and maintenance media for CE cell cultures.
Growth medium. The growth medium for CE cell cultures con
sisted of BSS, 5% calf serum, 0.1% sodium bicarbonate, 0.1% lactal-
bumin hydrolysate (Nutritional Biochemicals) and 0. 1% proteose pep
tone (Difco). For L cell cultures, Eagle's minimum essential medium
(MEM) with 10% calf serum was used.
Maintenance medium. The maintenance medium (MM) for CE
cell cultures consisted of Gey's BSS with 0. 1% lactalbumin hydrolysate,
0.1% proteose peptone, 0.1% yeast extract (Difco "yeastolate") and
about 0.1% sodium bicarbonate. For determination of RNA synthesis
in cell cultures, a modified maintenance medium (MMM) was employed
in which yeast extract was omitted and 2. 5% calf serum was added in
some cases.
Overlay medium. The overlay medium used for plaque assay con
sisted of either 1% methyl cellulose in MEM with 10% calf serum, or
chick embryo cell culture growth medium containing 5% calf serum
and 0. 5% agar (Ion agar No. 2, "Oxoid" division of Oxo, Limited).
Reagents
Rabbit reticulocyte buffer (RSB). RSB was prepared according
to Penman et al. (1963). The composition of this buffer was 0. 01 Tris-
(hydroxymethyl)aminomethane(Tris), 0. 01 M potassium chloride and


25
0. 0015 M magnesium chloride with pH adjusted to 7.2 by the addition
of 0. 5 M hydrochloric acid.
Phosphate-chloride buffer. This buffer was made as described
by Petermann and Pavlovic (1963) and consisted of 0. 001 M potassium
phosphate and 0. 005 M magnesium chloride, pH 6. 8.
Acetate buffer. This buffer was prepared according to the method
of Friedman (1968) and consisted of 0. 1 M sodium chloride, 0. 01 M
sodium acetate and 0. 0005 M magnesium chloride with pH adjusted
to 5. 1 by addition of 0. 5 M hydrochloric acid.
Sucrose solutions. Ribonuclease-free sucrose (Mann Research
Laboratories, New York) was employed for all the sucrose solutions.
The following sucrose solutions were used:
a) 1. 8 M sucrose solution in RSB
b) 0. 5 M sucrose solution in RSB
c) 0. 3 M sucrose solution in phosphate--chloride
buffer
d) 25% (w/v) and 8% (w/v) solutions of sucrose in RSB, pH 7. 2
e) 30% (w/v) and 15% (w/v) sucrose solutions in 0.1 M potassium
chloride, 0. 01 M tris(hydroxymethyl)aminomethane (Tris)
and 0. 001 M ethylendiaminetetracetate (EDTA) buffer, pH 7. 1
f) 25% (w/v) and 8% (w/v) sucrose solutions in 0. 01 M tris, 0. 4 M
sodium chloride buffer, pH 7. 2. In some experiments, the
molarity of sodium chloride was varied at 0.2, 0.3, 0.4 and .
0. 6 M in tris buffer, sucrose solutions.


26
Ribonuclease solution. Ribonuclease A (Worthington Biochemical
Corporation, Freehold, New Jersey) was dissolved in 0. 1 M KC1,
0. 01 M Tris, 0. 001 M EDTA buffer, pH 7.1, to a final concentration
of 2 Op g/ml
Antibiotics. To all media was added 250 units/ml of potassium
penicillin G and 100 pg/ml of streptomycin sulphate.
Radioisotope Uridine-5-H was obtained from New England
Nuclear Corporation, Boston, Mass. The specific activity of the iso
tope preparations were either 7 ^/mM or 28.1 ^"/mM.
Scintillation fluid. The radioactive samples were diluted in
scintillation fluid containing 4 g of 2, 5-bis-2-(5-tert-Butylbenzox-
azolyl) thiophene (BBOT) (scintillation grade, Packard), 500 ml
toluene (Fisher Scientific Co. ) and 500 ml of methanol (Fisher
Scientific Company).
Sodium deoxycholate solution. A stock solution of 10%. (w/v)
sodium deoxycholate was prepared in acetate buffer, pH 5.1.
Phenol. Phenol (Fisher Scientific Co. ) was distilled and hydroxy-
quinoline was added to a concentration of 0. 1%. The distilled
phenol was saturated with 10 x acetate buffer, pH 5. 1, before use.
Bentonite suspension. Bentonite powder (Fisher Scientific Co. )
was processed according to the method of Petermann and Pavlovic
(1963). The bentonite concentration in coarse suspension was 73 mg/mL
Actinomycin D. This reagent was a gift from Merck, Sharpe and
Dohme, Rahway, New Jersey. Stock solution contained 100 pg/ml of


27
actinomycin D and was kept in the dark at -20 C.
Semliki Forest virus antiserum. SFV antiserum was kindly
supplied by Dr. S. T. Toy. The antiserum at a final dilution of 1:20
was capable of neutralizing approximately 99% of virus in 60 minutes
at 37 C.
Methods
Cell cultures
Chick embryo cell cultures. The method of Lindenmann and
Gifford (1963a) was followed with some modifications. Chick embryos,
10 to 11 days old, were decapitated, eviscerated and washed once with
Gey's BSS. The embryos were minced by forcing through a "Luer Lok"
syringe into a "Blico" trypsinizing flask. Trypsinization of the
tissues was carried out for 30 minutes at 37C with continuous stir
ring in 20 volumes of 0. 02% (w/v) trypsin (Grand Island Biological
Co. Long Island, New York) in Gey's BSS without calcium and mag
nesium. Large, undigested tissue fragments were removed by fil
tering the cell suspension through a gauze filter. The trypsin was
removed by centrifuging the cell suspension for 20 minutes at 200xg
at 5C. The cells were resuspended in growth medium and recentri
fuged for 20 minutes at 200xg and suspended again in growth medium;.
The cells were then passed through a coarse sintered glass filter
using negative pressure and dispensed either at a concentration of
12 x 10^ cells in a volume of 5 ml into plaque bottles (2-ounce, screw-
capped, square, soft glass bottles having a rectangular side of 3 x 6


cm) or 1 x 10^ cells into 32-ounce glass prescription bottles (rectan
gular side 17 x 7 cm). A cell monolayer was usually formed within
24-48 hours of incubation at 37C.
Mouse L cells (Strain 9^9). The cell cultures were maintained
and propagated in Eagle's MEM with 10% calf serum. The cell mono-
layers were maintained in 32-ouncc bottles. For passaging the cells,
the growth medium was decanted and the cell monolayer was washed
with Gey's BSS and 10 ml of 0. 02% (w/v) trypsin was used to dislodge
the cells from glass surface. After an incubation period of 10 to 15
minutes at room temperature, the cells were removed from the glass
and counted in a hemacytometer chamber. To form a monolayer,
2-ounce plaque bottles received 1-2 x 10^ cells and 32-ounce bottles
were plated with 8-10 x 10^ cells.
Growth, Purification and Assay of Viruses
Vaccinia virus
Preparation. Vaccinia virus was propagated in lL-to 12-day-old
chick embryos by inoculating 200 plaque forming units (pfu) of virus
in 0. 2 ml volume on the chorio-allantoic membrane and incubating
the eggs at 37C for 46 to 48 hours. The eggs were chilled at 5C
for several hours, the infected membranes were removed, frozen at
-60C and homogenized with a mortar and pestle with sterile car
borundum employing MM as a diluent. The suspension was then cen
trifuged at 800xg for 30 minutes to remove coarse particles. The
supernatent fluid was dispensed into glass ampules which were sealed


29
and stored at -60C.
Assay. Vaccinia virus was titrated by the method of Lindenmann
and Gifford (1963a). Growth medium was decanted from the 2-ounce
plaque bottles and different dilutions of virus in maintenance medium
were dispensed in a volume of 2 ml per bottle. Normally, four bot
tles were employed for each dilution of virus tested. The bottles were
incubated undisturbed on a flat, level surface at 37C for 46 to 48 hours.
After the incubation period, the medium was decanted from the bot
tles and the monolayers were stained with 0.1% crystal violet for
3 to 5 minutes, washed several times in running tap water and in
verted to dry. The plaque were counted after enlarging the mono
layer's image 6 to 7 times with a photographic enlarger.
Semliki Forest virus
Preparation. Semliki Forest virus was propagated in the brains
of 24-to 48-hour-old mice. The newborn ICR strain of mice were
intracerebrally inoculated with 0. 02 ml of stock virus suspension di
luted 1:100 in maintenance medium. The infected brains were har
vested 24 hours after inoculation and a 10% (w/v) suspension of brains
in maintenance medium was made using a tissue homogenizer with a
teflon pestle. The suspension was centrifuged at 800xg for 20 minutes
to remove the coarser particles. The virus suspension was dispensed
in glass ampules which were sealed and stored at -60C.
Assay. The SFV was assayed on primary chick embryo cell
monolayers employing either the agar overlay or methyl cellulose


30
techniques. The growth medium was decanted from the bottles and
virus dilutions in 0. 2 ml volume were added to each plaque bottle.
The virus was allowed to adsorb for 60 minutes at room temperature
with frequent rocking of the bottles to distribute the virus evenly on
the monolayers. The bottles were drained and overlaid with 3 ml of
0. 5% molten agar in growth medium with 5% calf scrum maintained
at 42C. After the agar solidified, the bottles were transferred to a
37C incubator for 46 to 48 hours. In the methyl cellulose overlay
method, each bottle received 4. 5 ml of 1% methyl cellulose in MEM
with 10% calf serum after the adsorption of virus. Subsequent steps
were similar to the agar overlay technique. After the incubation
period, the agar or methyl cellulose was decanted gently from the
cells and discarded, the bottles were stained, and plaques were
counted as in the vaccinia virus assay (vide supra). The virus prep
aration, assayed either by methyl cellulose or agar overlay method,
displayed no significant difference in titer.
Newcastle disease virus
Preparation. Newcastle disease virus was propagated in 11-to 12-
day-old embryonated eggs by inoculating 200 pfu in 0.1 ml volume into
the allantoic cavity. After 44 to 48 hours of incubation at 37C, the
eggs were chilled and the allantoic fluid collected and centrifuged at
800xg for 15 minutes at 5C to remove the coarser particles. The
virus preparation was stored at -60C in sealed glass ampules.
Assay. The NDV was titrated either by agar or methyl cellulose


31
overlay technique using chick embryo cultures. With the agar overlay,
the cell cultures were incubated for 46 to 48 hours; and with methyl
cellulose, the incubation period was 72 hours. The other details of
the assay system are the same as for SFV (vide supra).
Vesicular stomatitis virus
Preparation. Vesicular stomatitis virus was prepared by inocu
lating the allantoic cavity of 11-to 12-day-old chick embryos with 200
pfu in 0. 1 ml. The incubation period was 36 to 40 hours. The other
details of production and assay were similar as described above for
NDV (vide supra). Cultures were incubated for 48 hours before staining.
Assay for Neutralizing Antibodies Against SFV
A plaque inhibition test was performed for the detection of anti
bodies against Semliki Forest virus in rabbit antiserum. The method
consisted of employing a constant amount of antiserum with several
different dilutions of virus. The various dilutions of Semliki Forest
virus in 0. 5 ml were mixed with 0. 5 ml of antiserum and incubated
at 37C for 60 minutes. Thereafter, the virus-antibody complex was
centrifuged at 800xg for 15 minutes. The supernatent fluid was used
for Semliki Forest virus assay (vide supra). The virus preparation,
incubated with normal rabbit serum, served as a control. The batch
of Semliki Forest virus antiserum employed neutralized nearly 99%
of SFV. For example, it reduced the titer of SFV from 8 x 10 pfu/ml
to 9 x 10^ pfu/ml.
Production and Assay of Interferon


Chick interferon
32
Production. The method employed for the production of interferon
was that of Ruiz-Gomez and Isaacs (1963a) with some modifications.
The growth medium was decanted from 32-ounce, 48-hour-old chick
embryo cell monolayers. The monolayers contained nearly 35 x 10^
cells per bottle. The cells were infected with SFV at an input multi
plicity of about 0. 1 pfu/cell, and to each bottle was added 30 ml of
maintenance medium. The bottles were usually incubated for 24 hours
at 37C. The medium containing interferon was harvested and heated
at 65C for 30 minutes to inactivate the virus. Some batches of inter
feron preparations were centrifuged at 120, OOOxg for 3 hours to re
move most of the inactivated virus particles. The interferon prepa
rations were stored at -20C.
Assay. The procedure followed for the assay of chick interferon
was that of Lindenmann and Gifford (1963b). Growth medium was de
canted from CE cell monolayers and then various interferon dilutions
and vaccinia virus (usually 200 pfu) were dispensed in a final volume
of 2 ml per bottle. Control cultures received the same amount of
vaccinia virus in MM without interferon. Usually 4 bottles were em
ployed for each dilution of interferon. The cultures were incubated
for 46 to 48 hours, stained with crystal violet and plaques enumerated
as described in the vaccinia virus assay system (vide supra). The
plaque depressing dose¡-Q, the amount of interferon preparation in
microliters which depressed the plaque number to 50% of the control,


33
was calculated according to the method of Lindenmann and Gifford
(1963b).
L cell interferon
Production. The mouse interferon was produced in L cell mono-
£
layers in 32-ounce bottles containing 30-35 x 10 cells per bottle. The
growth medium was removed and cells were infected with NDV at a
multiplicity of about 100 pfu/cell. The virus was allowed to adsorb
at room temperature for 60 minutes with frequent rocking of the
bottles. Thereafter, the cell cultures were washed with Gey's BSS
and 50 ml of Eagle's MEM with 10% calf serum was added to each
bottle. The cultures were incubated at 37C for a period of 24 hours.
The medium containing interferon was then harvested, pooled and
centrifuged at 800xg for 20 minutes to remove cellular debris. The
interferon preparation was dialyzed against 100 volumes of 0. 001 N
HC1 buffer at pH 2 for 5 days with two changes of buffer during this
period and finally against two changes of Gey's BSS for a further
12-to 24-hours to restore the pH to neutrality. In some experiments,
this preparation was centrifuged for 3 hours at 120, OOOxg to sediment
most of the virus particles. The supernatent fluid was distributed
into glass ampules, sealed and stored at -20C.
Assay. The interferon preparations were assayed on 24 hour-old
mouse L cell monolayers in 2-ounce plaque bottles. The various di
lutions of interferon were made in Eagle's MEM with 10% calf serum.
The medium was drained from the bottles and 2 ml of each dilution


34
was added to each of 4 plaque bottles. After overnight incubation at
37C, the interferon dilutions were removed and the cell sheets were
washed twice with 5 ml of MM. VSV in 0. Z ml volumes containing
100 to 200 pfu was added to each bottle. The VSV assay and calcula
tion of PDD ^ units have been previously described (page 32).
Heat Inactivation of SFV
The kinetics of inactivation of SFV was studied. The stock SFV
suspension was thawed and incubated at 37C. At various time
intervals, an aliquot was removed and frozen at -60C. All the sam
ples were assayed for their residual virus titer as described under
the assay of SFV (vide supra). The titer of partially inactivated virus
was not appreciably changed by one freeze and thaw cycle. Figure 1
shows a representative study of the inactivation of SFV at 37C. The
decrease in virus titer with respect to time of incubation at 37C was
exponential. The rate of inactivation of Semliki Forest virus was one
log (90%) decrease in virus titer per 3. 2 hours of incubation at 37C.
10
When the stock virus, consisting of 10% infected mouse brain suspen
sion, was centrifuged at 15, OOOxg for 15 minutes before incubation at
37C, the rate of inactivation was similar.- Four lots of stock virus,
prepared in a similar manner, were found to be inactivated at a rate
of one log in 3. 2 hours +10 minutes.
e10
Incorporation of Uridine into RNA by Chick Embryo Cells
Measurement of uridine incorporation into CE cell cultures was
. 3
achieved by treating the cells at different time periods with H-uridine.


35
Figure 1. Heat inactivation of Semliki Forest virus at 37C. Aliquots
we,re removed at various times during incubation and
assayed for residual virus.


36
Plaque bottles, containing approximately 4 x 10^ cells, were em
ployed. Growth medium was decanted and the monolayers were washed
twice with 2 ml of modified maintenance medium (MMM). The cell
cultures after the appropriate treatment (infected or noninfected,
with or without actinomycin D, with or without interferon treatment)
were supplied with 2 ml of MMM and incubated at 37C. At different
time intervals, 0. 3 ml MMM containing 20 pC of H-uridine and
_5
3. 75 x 10 M each of thymidine and deoxycytidine was added to each
bottle. Presence of thymidine and deoxycytidine prevented the in
corporation of uridine into DNA. The yeast extract was omitted from
the maintenance medium because it contains nucleotides which com
pete with uridine for incorporation into RNA. The cell cultures were
replaced in the incubator at 37C and bottles were gently rocked every
3
10 minutes to facilitate the even distribution of H-uridine on the cell
monolayer. To stop the incorporation, 0.1 ml volume of cold uridine
(5 x 10 M) was added and bottles were immediately placed into an
ice bath. The medium was drained from the bottles and the cell mono-
layers were washed three times with cold 5% perchloric acid (PCA).
The cold extraction was done twice more with 5 ml and 10 ml of cold
5% PCA for 5 and 10 minutes respectively. The radioactivity re
maining in the last cold PCA extraction was within background levels.
Ribonucleic acid was hydrolyzed and extracted in 2 ml of 5% PCA by
heating the cell cultures for 30 minutes at 80C in a water bath.
Radioactivity Measurement
For uridine incorporation studies, 0.2 ml of hot PCA extract


was placed into 20 ml screw-capped, glass vials (Packard) con
taining 10 ml of scintillation fluid. Samples were counted twice in a
Packard "Tri-carb" liquid scintillation spectrometer for 10 minutes
and values were expressed as counts per minute.
Uridine Uptake
An experiment was performed to measure the, optimum incorpo
ration of uridine.into RNA by CE cells employing different concen
trations of uridine. The experimental procedure was the same as
described above. Figure 2 shows the incorporation of H-uridine at
various concentrations when a pulse labelling period of 30 minutes
was employed. The incorporation of H-uridine into cellular RNA in
creased almost linearly up to about 20 p C concentrations, but there
was no further increase in incorporation when higher concentrations
were employed. Uridine incorporation was also determined when
the cell cultures were exposed to uridine for different time periods.
In this experiment, the cell cultures received 15 P C of H-uridine,
but the pulse labelling period was 15, 30, 45 and 60 minutes. Fig
ure 3 shows the results of such an experiment. The rate of incor
poration was linear throughout the period of labelling.
An experiment was performed to determine the effect of various
3
concentrations of calf serum in MMM on H-uridine incorporation in
CE cell cultures. The cell cultures were exposed to 20 P C for 30
minutes. It is apparent from Figure 4 that incorporation of H-uri
dine into cellular RNA was considerably increased in the presence of


CPM x 1,000 / CELL CULTURE
IOjO-i
acH
ao-:
40 H
2.0 H
o
o
Figure 2.
i 1 I I ¡ I
5 10 15 20 25 30
CONC. (/iC) OF URIDINE
3
H-uridine incorporation in uninfected chick embryo cell
cultures exposed to various concentrations of JH-uridine
and incubated at 37C for 30 minutes.


39
O -¡ i l i i
O 15 30 45 60
MINUTES AT 37C
3
Figure 3. H-uridine incorporation in uninfected chick embryo
cell cultures exposed to 15 PC of ^H-uridine for
various periods of time.


CPM x 1,000 / CELL CULTURE
40
12.0-1
10.5-
9.0-
75-
with 2 /g/ml of actinomycin D
without actinomycin D
Figure 4. Effect of calf serum on H-uridlne incorporation of
uninfected chick embryo cell cultures with or with
out actinomycin D. The cell cultures were exposed
to 20 PC of ^H-uridine for 30 minutes.


41
calf serum. The optimum uptake of uridine was found when cell
monolayers were maintained in MMM with 2% calf serum. There
was no significant increase in uridine incorporation when a higher
concentration of calf serum was present in MMM. The data for the
uridine incorporation in presence of actinomycin D will be discussed
in the next section.
Since in most of the experiments the CE cell monolayers were
maintained in MMM for 12 hours or longer, an attempt was made to
determine the extent of uridine incorporation into RNA in cell cultures
which had been incubated at 37C for various time periods. As seen
in Figure 5, maximum incorporation of uridine occurs when the cells
have been incubated for 4 hours. After 6 hours of incubation at 37C,
there was a slight decrease in uridine incorporation at each successive
interval of measurement. After 12 hours of incubation, uridine in
corporation was reduced by 15 to 20% relative to that at 4 hours.
Inhibition of RNA Synthesis by Actinomycin D in CE Cell Cultures
The effect of actinomycin D on cellular RNA synthesis was de-
termined by measuring H-uridine incorporation in CE cell cultures
exposed to different concentrations of the drug in 2 ml of MMM.
Actinomycin was permitted to remain and RNA synthesis was mea
sured at various intervals. As seen in Figures 6 and 7, actinomycin
D concentrations of 0. 5 pg/ml or higher inhibited more than 95% of
the cellular RNA synthesis. In another experiment, actinomycin D
(2 p g) was added to each bottle in 0. 1 ml volume with 0. 2 ml of MMM.


CPM x 1,000 / CELL CULTURE
42
5X5-T
C 5 MMM 2.5% CS
X'"w ...V..VI
45-
4.0-
35-
30
25-
20
0
2
1 1 1 1 1
4 6 S 10 12 14
TliV.L ... hOURS
Figure 5. H-uridine incorporation, with or without calf (CS)
serum in uninfected chick embryo cell cultures. The
o
cell cultures were incubated at 37 C for various
periods and then exposed to 20 PC of H-uridine for
30 minutes.


PERCENT OF CONTROL
lOO-i
90-
80-
70-
60-i
50
I
40-
Figure 6. Inhibition of H-uridine incorporation in uninfected chick
embryo cell cultures with various concentrations of
actinomycin D. The cell cultures were exposed to 20 l1 C
of ^H-uridine for 30 minutes at the indicated times.


44
, 3
Figure 7. Inhibition of H-uridine incorporation in uninfe-cted chick
embryo cell cultures with various concentrations of
actinomycin D. The values plotted were obtained from
Figure 6 at 10 hours after the exposure to actinomycin D.
I


45
The bottles were incubated at room temperature for 60 minutes and
rocked gently every 10 minutes to ensure even distribution of acti-
nomycin D on cell mpnolayer. After the incubation period, each bot
tle was washed twice with MMM and then supplied with 2 ml of MMM
per bottle. Further incubation of these bottles was carried out at
37C. A 30-minute period of labelling with 20 PC of ^H-urid Lne was
made at various times thereafter. Under these conditions, more
than 95% of the cellular RNA synthesis was inhibited for nearly 12
hours. However, a gradual recovery of cell monolayers from the
effect of actinomycin D was observed after 12 to 15 hours of incubation.
In the presence of 2. 5% calf serum the uptake of uridine was con
siderably stimulated, but the percentage inhibition of cellular RNA
synthesis by actinomycin D (2 pg/ml) was more than 95% of that of
control cell cultures (Figure 4).
Isolation and Purification of Chick Ribosomes
The crude preparation of chick embryo ribosomes was made
according to the method of Wettstein, Staehelin and Noll (1963) with
several modifications. Ten-day-old chick embryos were decapitated,
eviscerated and chilled in crushed ice. All subsequent steps were
carried out at 4 to 5 unless otherwise noted. The embryos were
forced through a "Luer Lok" syringe. The minced tissue was washed
twice with 0. 15 M NaCl and twice with RSB. Thereafter, the minced
embryos were mixed with 3 volumes of RSB and homogenized with
5 strokes of a motor-driven teflon homogenizer. Under these .


conditions, most of the cells were broken, but their nuclei remained
intact when examined by light microscopy. The homogenate was
centrifuged at 800xg for 10 minutes to remove unbroken cells and
other cellular debris. To this supernatant fluid was added 150 mg
of coarse bentonite and 10% (w/v) sodium deoxycholate to a final con
centration of 0.2%. The suspension was again homogenized with 2-3
strokes and centrifuged at 20, OOOxg for 15 minutes to remove large
particles, such as mitochondria and bentonite. The post-mitochon
drial fraction in a 4 ml volume was gently added as a top layer in
10 ml centrifuge tubes previously filled with 3 ml of 0. 5 M sucrose
in RSB layered over 3 ml of 1. 8 M sucrose solution in RSB.
The samples were then centrifuged for 3 hours at 151, OOOxg in
the Beckman Model L centrifuge. The pellets were rinsed and sus
pended in RS buffer. The suspension was homogenized with 2 to 3
strokes of a motor-driven teflon homogenizer and clarified at low
speed (800xg for 10 minutes). This ribosomal preparation was
stored at -60C. The whole procedure, from the death of chick
embryos to the freezing step, took 4 to 4. 5 hours.
Purification of ribosomes :
The purification of ribosomes was achieved by layering 1 to 2 ml
of crude preparation on a 26 ml linear, 8 to 25% sucrose gradient.
Sucrose gradients were prepared from a standard sucrose gradient
maker. The linearity of a typical sucrose gradient, represented by
refractive index, is shown in Figure 8. The gradient tubes were


REFRACTIVE INDEX
47
Figure 8.
3750-.
c
Linearity of sucrose gradient as measured by refractive
index of each fraction with an Abbe refractometer.
¡
j
i


48
placed in prechilled swinging buckets of SW 25.1 Beckman rotor and
centrifuged at 24, 000 rpm for 8 hours. After centrifugation, the
bottom of the tube was punctured and 1 ml fractions were
by allowing the gradient to drip through a needle. Accuracy of volume
in each fraction was achieved by putting mineral oil on the top of the
gradient in an air-tight system through a hypodermic needle. Optical
density was determined for each fraction with the Beckman DU spec
trometer at 258 m p and in some cases also at 280 m p Figure 9
shows a typical profile for these gradients. The ratio of 280:258 was
1:8 as expected for ribosomes. The profile also showed the contami
nation of crude ribosomal preparation with a heavy and light contami
nant which appeared at the bottom and the top of the tube, respectively.
Initially, the S values for chick embryo ribosomes were estimated
according to the method of O'Brien and Kalf (1967). The crude ribo
somal preparation was layered on a 5 to 20% linear sucrose gradient
in 0. 05 M KC1 0. 005 M MgCl^ 0. 001 M Tris pH 7. 6 and centri
fuged for 13 hours at 15, 000 rpm in the Spinco SW 25. 1 rotor. Under
these conditions, the chick embryo ribosomal peak was found in a
similar position as determined for rat liver ribosomes by O'Brien
and Kalf (1967) and was designated 80.S.
Separation of ribosomal subunits
The separation of ribosomal subunits was achieved by the method
of Fenwick (1968) with some modifications. The purified chick em
bryo ribosomes were obtained by the method described above. The


OPTICAL DENSITY (258 m/j.)
49
0.8 -(
0.7-
0.6-1
Figure 9. Sucrose gradient analysis of chick embryo ribosomes.
One ml of crude ribosomal preparation was layered
on 8 to 25% linear sucrose gradient and then centrifuged
at 24, 000 rpm for 8 hours. The other details have been
described under Materials and Methods.
I \
I


purified preparation was pressure dialyzed against 100 volumes of
0.01 M NaCl 0..QLM THs HC1, pH 7. 2 buffer for 4 hours to reduce
the volume and to remove the sucrose. One tenth volume of 4 M
NaCl was added to the dialyzed sample and incubated for 5 minutes
at 4C according to the method of Fenwick (1968). One ml of the
NaCl treated sample was layered on 26 mi linear gradient of 8 to 25%
sucrose and centrifuged for 12 hours at 24, 000 rpm as described
above. Figure 10 shows the sedimentation profile of such a gradient.
There was very little dissociation of 80 S ribosomes into 60 S and
40 S subunits. The incubation of 4 M NaCl-treated ribosomal prepa
ration for 15 or 30 minutes instead of 5 minutes did not change the
sedimentation pattern significantly. In another experiment, the NaCl
concentration of ribosomal preparations was raised to 0. 2 M, 0. 3M,
0. 4M or 0. 6M. The samples were incubated for 15 minutes at 4C
and were centrifuged in gradients containing the same concentration
of NaCl in a buffer (0. 01M Tris, pH 7. 2) for 12 hours at 24, 000 rpm.
Figure 11 shows the sedimentation pattern of ribosomes in such an
experiment. The dissociation of chick ribosomes into their subunits
was directly proportional to the NaCl concentration. However, a
sizeable portion of the ribosomes remained undissociated even in
presence of a 0. 6 M NaCl concentration. The results were strik
ingly different from that of Fenwick (1968) who reported complete
dissociation of HeLa cell ribosomes into their subunits in presence
of 0. 2M or more concentration of NaCl. Therefore, it appears that


OPTICAL DENSITY (258 m^)
51
0.50-1
0.45
J\
040-
5 mlnufes incubation
o~o 15 minutes incubation
c-& 30 minutes incubation
0.35-,
0.30-
Q25-1
0.20-
0.15
1
0.10-
0.05 H
i
5
l
10
15
i
20
25
30
FRACTION NUMBER
Figure 10. Separation of chick embryo ribosomes into their sub-units.
The purified ribosomal preparation was mixed with l/lOth
volume of 4 M sodium chloride and incubated at 4C for
5, 15 or 30 minutes and then centrifuged at 24,000 rpm in a
8 to 25% linear sucrose gradient for 12 hours.


52
Figure 11. Separation of chick embryo ribosomes into their sub
units. The NaCl concentration of ribosomal preparation
was raised to 0. 2 M, 0. 3 M, 0. 4 M or 0. 6 M and in
cubated at 4C for 15 minutes. The samples were then
centrifuged in a 8 to 25% sucrose gradient containing the
same concentration of NaCl as that of sample and
centrifuged at 24, 000 rpm for 12 hours.


53
chick ribosomes were more difficult to dissociate into their component
subunits than were those of HeLa cells.
Mouse liver ribosomes. The mouse liver ribosomes were iso
lated according to the method of Petermann and Pavlovic (1963) with
some modifications. Adult, female mice were fasted overnight. They
were anesthetized, usually in groups of five, with ether. The livers
were removed and chilled in crushed ice. The livers were minced
with a scissors and the tissue was forced through a "Luer Lok" syringe.
The minced tissue was suspended in 5 volumes of 0. 3M sucrose solu
tion and 150 mg of coarse bentonite preparation was added. The sus
pension was centrifuged at 800xg for 10 minutes and to the supernatant
fluid was added 10% (w/v) sodium deoxycholate solution to bring the
concentration to 0. 2%. The suspension was homogenized with 2 to 3
strokes and centrifuged at 10, OOOxg for 10 minutes. The pH of the
supernatant was rapidly brought to 8 using 0. IN sodium hydroxide and
centrifugation at 20, OOOxg for 15 minutes was used to remove most of
the bentonite. Thereafter, all the steps in mouse liver ribosome
isolation and purification were the same as for the chick ribosomes
(vide supra).
*
Detection of Double Stranded, Replicative Intermediate Form of SFV
An attempt was made to detect the double stranded, replicative
intermediate form of Semliki Forest virus in CE cell cultures at
different times following infection. The method of Friedman (1968)
was followed with some modifications. CE cell monolayers, con
taining nearly 4 x 10^ cells, were infected with a multiplicity of


54
10 pfu/cell of SFV in a volume of 0.2 ml. Each bottle also received
4 P g of actinomycin D in 0. 1 ml. The control cells received 4 pg of
actinomycin D in 0. 1 ml and 0. 2 ml of MMM with 2. 5% calf serum.
The virus was allowed to adsorb for 60 minutes at 4C, the bottles
were rocked every 15 minutes to ensure even distribution of virus.
After the incubation period, 2 ml of cold MMM with 2. 5% calf serum
was added to each bottle and the cell cultures were left overnight at
4C to synchronize the infection. The following morning, the plaque
bottles were transferred to a 37C incubator for one hour. Then the
cell monolayers were washed twice with MMM, supplied with 2 ml of
MMM containing 2. 5% calf serum for each bottle and returned to the
37C incubator.
The time when cell cultures were first transferred to the 37C
incubator from 4C was considered as zero time for viral replication
and RNA synthesis cycles.
At various periods of time following the infection, 20 p C of
^H-uridine in 0. 1 ml volume was added to each bottle, and cell mono-
layers were further incubated for an additional 45 minutes at 37C.
The medium from the cell cultures was then removed, and cells
were washed three times with 5 ml of chilled phosphate buffered
saline, and twice with 2 ml of 0.1 M NaCl 0. 01 M sodium acetate
buffer, pH 5. 1. All subsequent steps were carried out in an ice bath
unless otherwise stated. To each bottle was added 1 ml of acetate
buffer, pH 5. 1, and cells were scraped off the: glass by the use of


a rubber policeman. Usually cells from 4 bottles of each group (in
fected with live virus, treated with heat-inactivated virus, or control,
untreated cells) were pooled and 15 mg of coarse bentonite suspension
and 0. 5 ml of 10% SDS solution were added immediately. The RNA
extraction was carried out by adding equal volume of distilled phenol
urd shaking hie mixture ior 3 to 5 minutes at room temperature. The
emulsion was broken by centrifuging at 800xg for 15 minutes and the
aqueous layer was separated and extracted again with an equal volume
of phenol. Finally, the two phenol phases were mixed and the re
maining RNA extracted with 5 ml of acetate buffer, pH 5.1. The
aqueous phase of each phenol extraction was combined and centri
fuged at 20, OOOxg for 15 minutes to remove most of the coarse ben
tonite. Two volumes of cold, absolute ethanol containing 2% potas
sium acetate were added to the supernatant. The suspension was kept
overnight at -20C and RNA was collected by centrifuging the sus
pension at 15, OOOxg for 15 minutes. The precipitate was washed once
with 70% ethanol containing 2% potassium acetate, and RNA was again
pelleted by centrifuging at 15, OOOxg for 15 minutes. The RNA pre
cipitate was dissolved in 0. 1 M KC1 0. 01 M Tris 0. 001 M EDTA
buffer, pH 7. 1 and clarified by centrifugation at 10, OOOxg for 15
minutes. Carrier RNA from L cells was either added during the
first phenol extraction period or just before the sucrose gradient
analysis of the extracted RNA.
For sucrose gradient analysis, 1 ml (4.927 to 7.126 OD ,
260


56
units) of extracted RNA was gently layered on a 15 to 30% linear, su
crose gradient prepared in 0. 1 M KC1, 0. 01 M Tris and 0. 001 M EDTA,
buffered at pH 7.1. The gradient tubes were placed in pre-chilled
buckets of SW 25. 1 Beckman-Spinco rotor and centrifuged for 20
hours at-22, 000 rpm. After centrifugation, one ml fractions were
collected as described under the purification of chick ribosomes.
Optical density at 260 m p was determined for each fraction and 0. 1
volume of each fraction was employed for the radioactivity mea
surements .
Ribonuclease Sensitivity of Viral RNA Isolated from Infected Cells
The viral RNA structure, isolated from CE cells infected with
SFV, was tested for its ribonuclease (RNAse) resistance to detect
the presence of double stranded replicative forms. The RNAse
sensitivity test was performed according to the method of Friedman
(1968). RNAse treatment was carried out either before or after the
sucrose gradient analysis of RNA extracted from infected cells. In
the latter case, the sucrose gradient fractions containing the radio
activity peak were pooled, pressure dialyzed against 100 volumes of
0. 1 M KC1, 0. 01 M Tris HC1 and 0. 001 M EDTA, buffered at pH 7. 1,
for 6 hours to reduce the volume and remove the sucrose from the
sample. To 0. 9 ml of RNA sample was added 0. 1 ml of RNAse solu- .
tion (20 P g/ml) in 0. 1 M KC1, 0. 01 M Tris, and 0. 001 M EDTA
buffer, pH 7.1, to bring the final concentration of the enzyme to
2 P g/ml in the reaction mixture, which then was incubated for 10


minutes at 37C. To stop the RNAse activity, 100 mg of coarse
bentonite preparation was added just after the incubation period. The
RNA was then iinmediately extracted with phenol. Carrier RNA (L
cell ribosomal RNA) was added, and sucrose gradient analysis
was performed as before.
The ribonuclease solution, at the concentration of 2 p g/ml, was
able to completely degrade two OD ^ units of chick ribosomal RNA
when incubated for 10 minutes at 3 7C.


RESULTS
Production of Interferon at Different Multiplicities of Infection
The effect of multiplicity of infection of Semliki Forest virus on
interferon production was studied. Figure 12 represents the 24-hour
yields of interferon in chick embryo cell cultures infected with dif
ferent input multiplicities of the virus. The best yield of interferon
was obtained with an input multiplicity of 0. 1 pfu/cell. When the in
put multiplicity of infection employed was increased to 1 pfu/cell or
more, the 24-hour interferon yields were much reduced. The reasons
for the multiplicity effect were further studied since it should provide
insight into conditions for maximal interferon production as well as
possible reasons for the variation in interferon yields.
Production of Interferon as Function of Time Following Infection
To further delineate the differences in interferon yield as a
function of multiplicity of infection, an experiment was designed to
study the production of interferon in cell cultures at various times
after infection with two different multiplicities of Semliki Forest
virus. The results are shown in Figure 13. With a multiplicity of
infection of 0.1 pfu/cell, there was a significant increase in interferon
production beyond 12 hours after infection. In other experiments,
with a multiplicity of 0.1 pfu/cell, the increase in interferon pro
duction continued beyond 18 hours following infection (e. g., see
58


59
Figure 12. Interferon yields at 24 hours in chick embryo cell
cultures infected with various input multiplicities
of Semliki Forest virus.


60
Figure 13.
Kinetics of interferon production in chick embryo cell
cultures infected with Semliki Forest virus at multi
plicities of 10 or 0. 1 pfu/cell.


Figui'e 18). However, the interferon yield was reduced by 36 hours
after infection. When cell cultures were infected with 10 pfu/cell
of the virus, interferon production nearly ceased by 8 hours after
infection (Figure 13 and 17, and Table Z). In this experiment
(Figure .13), the 24 hour yield of interferon in cell cultures infected
with 10 pfu/cell was nearly 30% of that obtained with 0.1 pfu/cell.
This difference usually varied between 14 to 20% in other experiments
(Table 2). This data indicated that infection of all the cells (as would
be the case with a multiplicity of 10 pfu/cell) resulted in early termi
nation of interferon systhesis. This possibility was further studied.
RNA Synthesis in the Infected Cells
Since interferon production represents the synthesis of induced
cellular protein, the viruses which rapidly shut off host macromolecu
lar synthesis would not be expected to be good interferon inducers.
Thus, viruses like poliovirus, Mengovirus and vesicular stomatitis
virus, which are known to inhibit cellular RNA synthesis (Holland,
1963; Baltimore, Franklin and Callender, 1963; wagner and Huang,
1966), have also been reported to be poor inducers of interferon in
cell cultures ( Wagner and Huang. 1966; Burke, 1966). Therefore,
the effect of Semliki Forest virus on RNA synthesis of infected cells,
at a multiplicity which apparently inhibited interferon production,
was studied. Actinomycin D (2 Pg/ml) was employed to determine
viral specific RNA synthesis in the infected cells. Figure 14 shows
RNA synthesis in chick embryo cell monolayers infected with a


62
TABLE 2
THE EFFECT OF TWO MULTIPLICITIES OF
VIRUS ON INTERFERON PRODUCTION
Experiment
Number
|
Multiplicity
of Infection
(pfu/cell)
Interferon Yield, PDD Units/ml
D 0
Hours After Infection
S 12 24
% Inhibition
at 10 PFU*
1
0.1
21. 9
36
78. 4
10
23. 6
25. 2
24. 8
68. 4
2
0. 1
12.1
37. 2
86. 6

10
14. 3
16
16. 2
81. 2
3
0. 1
9. 7
33. 2
65. 6
10
9.2
10. 1
9. 5
85. 5
* Percent inhibition compared to yield of interferon at a
multiplicity of 0.1 at 24 hours after infection.


63
Figure 14. RNA synthesis in chick embryo cell cultures infected
with Semliki Forest virus at a multiplicity of 10 pfu/cell
with or without actinomycin D. Actinomycin D sensitive
RNA was plotted as the difference between total RNA
synthesis and actinomycin D resistant RNA synthesis.


64
multiplicity of 10 pfu/cell of virus with or without actinomycin D.
There was an initial increase in total RNA synthesis which reached
a peak at 5 hours after infection. In different experiments, this
increase varied between 40 to 80% as compared to the control unin
fected cells. This early increase was followed by a rapid decline
in total RNA synthesis in the infected cells and by 12 hours following
infection, it was approximately 14% of that of the control cells. In
the presence of actinomycin D, RNA synthesis, presumably of viral
origin (referred to as actinomycin resistant RNA synthesis in Figure
14) started after a lag period of 2 to 3 hours and continued increasing
until 8 hours following the infection and declined thereafter. The
cellular RNA synthesis, plotted as the difference between the total
RNA and actinomycin D resistant RNA synthesis at each point, showed
a slight increase up to 5 hours after infection in some experiments
while this increase was not observed in other experiments. However,
cellular RNA (actinomycin sensitive) synthesis in the infected cells
was always lower than that of the uninfected cells. After 4 to 5
hours of infection, there was a very rapid inhibition of cellular RNA
synthesis and after 8 hours practically all the RNA synthesized was
actinomycin D resistant. Thus, interferon synthesis (Figure 13) is
terminated at about the time when cellular RNA synthesis ceases
almost completely and at the time of maximal viral RNA synthesis.
Growth Curve of Semliki Forest Virus
Replication of Semliki Forest virus was studied to correlate
viral specific RNA synthesis with the production of infectious viral


65
progeny. Figure 15 shows a growth curve of Semliki Forest virus
and the accompanying viral specific RNA synthesis in chick embryo
cell cultures infected with a multiplicity of 10 pfu/cell. The progeny
virus begins to appear after a lag period of 5 hours. Virus matura
tion occurred between 6 to 10 hours and was essentially completed by
10 hours following infection.
he maximal rate of viral specific
RNA synthesis was observed at approximately 8 hours following
infection. Thus, there was a lag period of 2 to 3 hours between the
synthesis of viral specific RNA and the appearance of infectious
progeny virus in cell cultures. In the presence of 2 p g/ml of acti-
nomycin D, which suppressed nearly 95% of cellular RNA synthesis,
the virus yield was nearly twice that of cell cultures without actino-
mycin D. These results confirmed the finding of Taylor (1964) that
actinomycin does not inhibit replication of SFV.
The data, so far presented, indicates that in chick embryo cell
cultures infected with a multiplicity of 10 pfu/cell, interferon pro
duction ceases at the time when progeny virus synthesis was maximal
and cellular RNA synthesis was severely inhibited. Thus the cessa
tion of interferon synthesis in the infected cells is most probably
due to cell death.
The continued synthesis of interferon beyond 8 hours, when low
multiplicity was employed, accounts for the higher yields of inter
feron obtained. Two possible explanations for this continued syn
thesis were considered: 1) interferon induction is due to "inactive"


66
Figure 15. Correlation of viral RNA synthesis with growth of
Semliki Forest virus (with or without actinomycin D)
in chick embryo ceil cultures at a multiplicity of
10 pfu/cell.
}.
!


67
virus particles in the virus preparation which do not inhibit host
macromolecular synthesis and thereby permit interferon synthesis
for longer periods of time, or 2) low multiplicity of infection leads
to several cycles of virus infection an4 therefore, prolonged inter
feron synthesis.
Interferon Production by Inactive Virus
The first possibility that interferon production is induced by the
nonreplicating virus particles present in Semliki Forest virus prepa
rations was considered. It is not possible to physically separate the
noninfectious particles from the infectious particles in Semliki Forest
virus preparations. Since Semliki Forest virus is readily inactivated
at 37C, many of the noninfectious particles found may be due to
inactivation during production and preparation of the virus stock.
Therefore, we chose to increase the number of inactive particles by
incubating the virus preparation at 37C and subsequently determine
the effect of such a virus preparation on the induction of interferon
synthesis in CE cells. The loss of infectivity followed first order
kinetics (Figure 1) and less than 5 pfu/ml remained after 24 hours of
incubation.
Figure 16 shows interferon production in chick embryo cells as
a function of time after induction. When cells were exposed to in
activated virus, equivalent to 0.1 pfu/cell before inactivation, the
24 hours' yield of interferon was much less than the amount induced
by an equivalent amount of live virus. Under these conditions, the


68
E
oo
Z3
O
LA
O
O
Cl-
IO
o r
25
HOURS AFTER i MFECTI ON
Figure 16. Kinetics of interferon production in chick embryo cell
cultures exposed to heat inactivated Semliki Forest
virus. The cell cultures received the equivalent of
either 10 or 0.1 piu/cell of the virus.


69
heat inactivated virus induced small amounts of interferon up to 8 to
12 hours after incubation with no further increase in synthesis there
after. A higher yield of interferon was obtained when cells were ex
posed to heat inactivated virus equivalent to 10 pfu/cell. However,
the total yield of interferon, in this case, was obtained within 12 hours
after the exposure of the cells. Thus the continued synthesis of inter
feron for 24 hours by cells infected with low multiplicity of the virus
is apparently not due to inactive particles present in the virus prepa
rations. However, the data indicates that inactive virus particles
do prolong interferon synthesis for some period of time as compared
to cells infected with live virus. The kinetics of interferon production
in cell cultures exposed to live virus at a multiplicity of 10 pfu/cell or
an equivalent amount of the heat inactivated virus were studied (Figure
17). With live virus, interferon synthesis was again nearly completed
by 8 hours following infection, while the production of interferon con
tinued for 12 hours in cell cultures induced by heat inactivated Semliki
Forest virus. The continued synthesis of interferon for 4 additional
hours explains the increased yield of interferon with inactive virus.
The maximum yield of interferon with the live virus was nearly 26%
of that induced by the heat inactivated virus. Similarly, Burke and
Walters (1966) observed that interferon production was essentially
completed in 10 hours when chick embryo cells were infected by Sem
liki Forest virus first at 36C for 1 hour and then at 42C. Under
these conditions, infectious virus particles were not synthesized but
interferon production was, apparently, not affected. The reason for


INTERFERON YIELD, PDD
60
Live Virus (m = lOpfu/cell)
£
oo
50
30-
20-
10-
x x Heat Inactivated Virus (m = IOpfu equivalent/cell)
0 | i i M . 1
0 4 8 12 16 20
HOURS AFTER INFECTION
Figure 17. Kinetics of interferon production with heat inactivated
and live Semliki Forest virus with cell cultures.


the cessation of interferon synthesis by inactive virus at 12 hours in
our study is unknown. In another experiment (Figure 24),it was ob
served that the heat inactivated virus, equivalent to an original mul
tiplicity of 10 pfu/cell, did not inhibit cellular RNA synthesis of the
induced chick embryo cells up to 12 hours after exposure. At 15 hour
cellular RNA synthesis was 86% of the controls and decreased to 69%
at 18 hours after exposure (not shown in the figure). The late inhibi
tion could have been due to the replication of the small amount of
residual virus in the inactivated virus preparation. Termination of
interferon synthesis by 12 hours after exposure to the heat inactivated
virus, even though cellular RNA synthesis was apparently normal,
conforms to the general observation that interferon synthesis stops
at varying times after induction. Cessation of interferon production
may be due to the inhibition of interferon production by interferon
(Vilcek and Rada, 1962; Cantell and Paucker, 1963; Friedman, 1966b).
However, it is difficult to explain why interferon stops its own syn
thesis in 12 hours in chick embryo cell induced by SFV but does not
affect the synthesis for 40 hours in chick embryo cells induced by
ultraviolet irradiated influenza virus (Burke, 1966). Alternatively,
some cellular control mechanism may be responsible for the termi
nation of interferon production but evidence for such a mechanism
is lacking. The cell may also be able to destroy the inducer, thereby
preventing the continued induction.
Interferon Production in Presence of Immune Serum
When chick embryo cell cultures are infected with SFV at 0.1


pfu/cell, less than 10% of the cells are initially infected. Since in
fected cells produce interferon for a maximum of 10 hours, the con
tinuous synthesis of interferon for 24 hours under these conditions
is most probably due to the subsequent infection of the remaining
cell population by the progeny virus.
To test this possibility, the kinetics of interferon production in
chick embryo cell cultures infected with 0.1 pfu/cell of Semliki
Forest virus, with or without the addition of SFV antiserum after
virus adsorption, were studied. The progeny virus produced by the
initially infected cells should be neutralized by the immune serum
present and thus would be unable to infect the remaining cells. The
cell monolayers were infected at the desired multiplicity in 0. 2 ml
volume and incubated for one hour at room temperature. After the
incubation period, each cell culture was washed twice with 5 ml
of Gey's balanced salt solution, supplied with 2 ml of maintenance
medium and incubated at 37C. Following 2. 5 hours of incubation,
0.1 ml of immune serum was added to some of the infected cell
cultures. Control cell cultures received 0.1 ml of normal rabbit
serum. The other details were the same as described under
Materials and Methods. Figure 18 shows the results of such an ex
periment. In presence of the immune serum, the total yield of
interferon was obtained within 12 hours of infection. In cell cultures
containing normal rabbit serum in place of immune serum, the
production of interferon was roughly linear for 24 hours following
the infection and was similar to that shown in Figure 13. However,


73
HOURS AFTER INFECTION
igure 18. Kinetics of interferon production in presence of immune
serum prepared against Semliki Forest virus. Details
have been described in the text.


74
the amount of interferon produced for the first 8 hours was similar
in both the cases. The results indicate that continuous interferon
synthesis for 24 hours in cells infected with 0.1 pfu/cell multiplicity
of the virus is due to the subsequent infection of the cells by the
progeny virus.
Interferon Production in Actinomycin D Pretreated Cells
Several investigators have suggested that there are two different
mechanisms responsible for the appearance of extracellular inter
feron following the addition of various inducers. These are, i. e. de
novo synthesis versus the release of preformed interferon (Ho and
Kono, 1965; Ho, Postic and Ke, 1968; Finkelstein, Bausek and
Merigan, 1968). Infection by viruses leads to de novo interferon
synthesis in the induced cells (Burke, 1966; Finkelstein, Bausek
and Merigan, 1968; Paucker, 1969). Actinomycin D inhibits viral
induced interferon synthesis but had no effect on interferon produced
in response to endotoxin, poly rl/rC and pyran, a carboxy copolymer
(Finkelstein, Bausek and Merigan, 1968) except when very high con
centrations were employed. Therefore, interferon production was
studied in cells pretreated with actinomycin D to gain information
regarding the type of interferon detected following the addition of
heat inactivated Semliki Forest virus to cell cultures. The procedure
of Gifford and Heller (1963) was essentially followed. Chick embryo
cell cultures were exposed to 0. 6 p. g/ml of actinomycin D in main
tenance medium at 37C for 4 hours. Thereafter, the maintenance
medium was removed and cell cultures were washed twice with fresh


75
maintenance medium. Control cell cultures were treated in an
identical manner except that actinomycin D was not added. The cell
cultures were then exposed to the same amount of live or heat inac
tivated Semliki Forest virus. Other details were the same as de
scribed in Materials and Methods.
As shown in Table 3, induction of interferon production by live
or heat inactivated Semliki Forest virus in chick embryo cell cul
tures was almost completely inhibited when the cells were pre
treated with actinomycin D. The interpretation placed on these re
sults is that interferon production is induced by the live as well as
heat inactivated Semliki Forest virus. Actinomycin D, by selectively
inhibiting cellular RNA synthesis, prevented the formation of inter
feron specific messenger RNA and thus indirectly prevented inter
feron production.
Viral Specific RNA Synthesis in the Induced Cells
It has been suggested that RNA viruses induce interferon pro
duction by synthesizing a double stranded form of RNA which then
acts as the inducing agent (Skehel and Burke, 1968b; Hilleman, 1969;
Colby and Chamberlin, 1969). Since heat inactivated virus, though
unable to replicate but induces more interferon than live virus, the
possibility was investigated as to whether heat inactivated Semliki
Forest virus undergoes a double stranded, replicative form as a
prerequisite for interferon production or if the single stranded viral
RNA is in itself the inducing agent. Live virus infection, at a mul
tiplicity of 10 pfu/cell, induces much lower yields of interferon but


76
TABLE 3
INTERFERON PRODUCTION IN PRESENCE
OR ABSENCE OF ACTINOMYCIN D
Virus
Preparation
Multiplicity
of Infection
pfu/cell
Presence or
Absence of
Actinomycin D
15 Hour Inter
feron Yields
(PDD^q Units/ml)
Heat Inactivated
Virus
10*

64. 5
Heat Inactivated
Virus
10*
+
3. 6
Live Virus
10

11.. 2
Live Virus
10
+
2. 0
* Equivalent amount of heat inactivated virus
Note: The virus titer at the end of 15 hours incubation was
6 to 8 x 1C)3 pfu/ml in cell cultures exposed to heat
inactivated virus.


was employed as a control to detect the double stranded, replicative
intermediate form of RNA in the infected cells. Chick embryo cell
monolayers were infected with Semliki Forest virus in the presence
of 2 P g/ml of actinomycin D. After various periods of incubation at
37UC, 20 PC of H-uridine was added for an additional 45 minutes
and the RNA was extracted as described in Materials and Methods.
Sucrose gradient analysis of viral specific RNA
The extracted RNA was gently layered on a 15 to 30% linear su
crose gradient and centrifuged at 22, 000 rp.m for 20 hours in a SW 25
rotor. The gradient fractions were collected in 1 ml aliquots and the
optical density measured at 260 mp and radioactivity measured in
the spectrometer. Ribosomal RNA from L cells was employed for
the estimation of S value of viral specific RNA. Sucrose gradient
analysis of the extracted RNA two hours after infection showed a peak
^H-uridine in the 26 S region with counts extending into ranges of
lower S values. When an aliquot of extracted RNA was incubated
with ribonuclease (2 P g/ml) for 10 minutes at 37C before sedimen
tation, another peak of radioactivity sedimenting in the 20 S region
was revealed (Figure 19). When RNA from the infected cells was
extracted 4 or 6 hours after the infection, an additional radioactive
RNA peak sedimenting at the 45 S region, as shown in Figures 20 and
21, was also obtained. These results are similar to those of
Friedman, Levy and Carter (1966) and Sonnabend, Martin and Mees
(1967) who reported that viral specific RNA from the infected cells
can be resolved into three components. They also reported that


78
Fraction No.
Figure 19. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
infected for 2 hours with Semliki Forest virus.
2
H-uridine (20 PC) was added for 45 minutes, and
RNA was extracted. L cell RNA was employed as
carrier RNA.
Optical Density (260mu)


C PM
79
Figure 20. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
infected for 4 hours with Semliki Forest virus.
JH-uridine (20 PC) was added for 45 minutes, and
RNA was then extracted. L cell RNA was employed
as carrier RNA.
i
Optical Density (260mp)


80
Figure 21. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
infected for 6 hours with Semliki Forest virus.
O
"H-uridine (20 pC) was added for 45 minutes, and
RNA was then extracted. L cell RNA was employed
as carrier RNA.
Optical Density (260mpi)


81
the RNA peak sedimenting at 45 S was infectious and corresponded to
the RNA that can be extracted from purified virus particles. The
second peak of radioactive RNA sedimenting at 26 S was not infec
tious but the base composition of this component was similar to that
of 45 S RNA and the RNA extracted from virus particles. The 20 S
RNA is ribonuclease resistant and thus presumably the double stranded
form of the RNA.
The RNA was also extracted at 2, 4 and 6 hours after exposure
of the cell cultures to heat inactivated Semliki Forest virus equiva
lent to 10 pfu/cell. Sucrose gradient analysis of these RNA extracts
did not reveal peak of radioactivity at any of the three regions (Figure
22 and 23) indicating that viral specific RNA was not synthesized in
the cells exposed to the heat inactivated virus. These cell cultures
had a titer of 5. 3 x 10^ pfu/ml when assayed for infectious virus at
12 hours after infection. The titer of live virus under similar con-
7 8
ditions usually varied between 8 x 10 to 2 x 10 pfu/ml.
Pulse-labelling method
Viral specific RNA synthesis in chick embryo cells exposed to
live as well as heat inactivated Semliki Forest virus was also
studied by the H-uridine pulse labelling method. The induced cells,
at various periods after the induction, were exposed to 20 p C of
H-uridine for 45 minutes and RNA was extracted with perchloric
acid as described in Materials and Methods. Chick embryo cell
monolayers infected with live virus, at a multiplicity of 10 pfu/cell,
showed the synthesis of viral specific RNA which progressively


82
Fraction No.
Figure 22. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
exposed for 2 hours with heat inactivated Semliki
Forest virus. H-uridine (20 hC) was added for
45 minutes and RNA was then extracted.
RNA was employed as carrier RNA.
L cell


83
Figure 23. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
exposed to heat inactivated Semliki Forest virus
for 4 or 6 hours. ^H-uridine (20 PC) was added
for 45 minutes, and RNA was then extracted. L
cell RNA was employed as carrier RNA.


84
increased with respect to lime after the infection. Most of the RNA
synthesized in the infected cells from 9 hours onward was viral
specific (Figure 24). However, no viral specific RNA synthesis
was detected when cells were exposed to the heat inactivated virus
equivalent to 10 pfu/cell. Virus directed RNA synthesis also could
not be detected when cells were exposed to the heat inactivated virus
3
for 7 hours in the presence of 20 p. C of H-uridine.
Association of Interferon with Ribosomes
The antiviral activity of interferon seems to depend upon the de
novo synthesis of another protein in the exposed cells (Taylor, 1964;
Lockart, 1964; Levine, 1966). This new protein, often referred to as
translation inhibitory protein (TIP), apparently attaches to the ribo
somes and renders these ribosomes incapable of translating the viral
messenger RNA (Marcus and Salb, 1966; Carter and Levy, 1967).
However, it is not known whether TIP alone, or in conjunction with
interferon, produces the change in ribosomal function. The study
of ribosomes from cells exposed to interferon alone or to interferon
in the presence of actinomycin D cannot answer this question since
in the former case the two factors cannot be distinguished from one
another and the latter case indicates that interferon alone is not
able to mediate the antiviral action in the cells. However, if inter
feron does play a direct role in the maintenance of virus resistant
state in the cells, it is reasonable to assume that it might combine
with ribosomes for the expression of antiviral activity.


Uridine Uptake (percent of control)
85
o Uninfected Cells and
Actinomycin D(2mg/ml)
Figure 24. RNA synthesis in chick embryo cell cultures exposed
to heat inactivated or live Semliki Forest virus in
presence or absence of actinomycin D.


86
To investigate this possibility, an experiment was performed in
which chick embryo and mouse liver ribosomes were examined for
their ability to combine with a chick interferon preparation which
had been partially purified by centrifugation at 105,000xg for 3 hours.
Chick embryo ribosomes (5 OD units) were mixed with 10 PDD
units of chick interferon and incubated for 30 minutes in a 37C water
bath. After incubation, the ribosomes were removed by centrifuga
tion at 105, OOOxg for 3 hours and the supernatant fluid was assayed
for residual interferon activity. Similar experiments were performed
employing chick interferon with mouse liver ribosomes, and also
using L cell interferon with mouse liver ribosomes or with chick
embryo ribosomes. As presented in Table 4, the results show that
almost all of the interferon was recovered after incubation with ribo
somes indicating that chick interferon did not bind specifically to
chick embryo ribosomes or nonspecifically to mouse liver ribosomes.
These studies were extended to include L cell interferon which did
not combine wi,th either mouse liver ribosomes or with chick embryo
ribosomes.
However, the possibility exists that interferon may bind to one
of the ribosomal sub-units but the attachment site is no longer avail
able when '60 S' and '40 S' sub-units combine to form the'80 S' ribo
somal unit. To test this possibility, chick embryo ribosomes were
separated into '60 S' and '40 S' sub-units as described under Materials
and Methods. Two OD units of chick embryo '40 S' ribosomal


TABLE 4
ASSOCIATION OF CHICK INTERFERON WITH MOUSE AND CHICK EMBRYO RIBOSOMES
Source of Interferon
Source of Ribosomes
PDD50 Units of
Interferon Added
PDD50 Units of
Interferon Recovered
Chick Embryo Cells
Chick Embryo (80S)
10
9. 7
Chick Embryo Cells
Mouse Liver (80S)
10
10. 4
L Cells
Mouse Liver (80S)
10
10. 3
L Cells
Chick Embryo (80S)
10
10. 1


sub-units were mixed with 10 PDDC interferon units, incubated at
D U
37C for one hour and ribosomes removed by centrifugation. Simi
larly, chick interferon was also incubated with '60 S' sub-unit as
well as with '80 S' ribosomes. As shown in Table 5, complete
recovery of interferon was made after incubation either with '60 S'
sub-unit or with '40 S' sub-unit. Thus the results indicate that
interferon does not combine with either of the ribosomal sub-units.


89
TABLE 5
ASSOCIATION OF CHICK INTERFERON WITH RIBOSOMAL SUBUNITS
Source of
Interferon
Chick
Ribosomes
PDD^q Units of
Interferon Added
PDDrg Units of
Interferon Recovered
Chick
60 S
10
9. 9
Chick
l
40 S
10
00
O'
Chick
80 S
10
1 0.1


DISCUSSION
Interferon Production
The dependence of interferon production, in various cell-virus
systems, on multiplicity of infection has been well documented in
the literature. In many cases, lower multiplicities of infection
induce maximum amounts of interferon while higher multiplicities of
infection result in reduced yields of interferon (De Mayer and De
Somer, 1962; Gifford, 1963; Aurelian and Roizman, 1965; Toy and
Gifford, 1967a). In the present study, the highest yield of interferon
was obtained when CE cell cultures were infected with SFV at a
multiplicity of about 0.1 pfu/cell. Multiplicities of 1 pfu/cell or more
of this virus resulted in considerably lower production of interferon.
These findings are in agreement with those of Toy and Gifford (1967a).
When two different multiplicities, i. e. 10 pfu/cell and 0. 1 pfu/
cell were employed, not only were the 24-hour yields different but
there were striking differences in the appearance of interferon in CE
cell cultures as a function of time after induction. The increase in
the yield of interferon between 8 to 24 hours after the infection was
roughly linear in cultures infected with a multiplicity of 0.1 pfu/cell.
The rate of appearance of interferon in culture medium during this
period varied between 0. 9 to 1. 1 PDD units/hour/10^ cells in
90


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J A 81,9(56,7< 2) )/25,'$ ,OOnOO :O ,, ,,,


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UNIVERSITY OF FLORIDA
3. Ill'll Wl II III
1262 08557 0561


STUDIES ON INTERFERON PRODUCTION
By
RAKESH MOHAN GOORHA
)
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA

ACKNOWLEDGMENTS
I acknowledge my deepest appreciation to Dr. G. E. Gifford
for his encouragement, supervision and guidance throughout the
period of this investigation and during the preparation of the manu¬
script. I also thank the members of my advisory committee,
Dr. H. E. Kaufman, Dr. L. W. Clem and Dr. P. Byvoet for
their suggestions and encouragement. I am grateful to Dr. P. A.
Small for critically reviewing the manuscript. I would like to
thank Dr. I. Rosen who helped me in many ways during my studies.
I would also like to thank Mrs. P. Jones for the generous
supply of cell cultures and other materials. I am also grateful
to Mr. M. Fruitstone, Mrs. B. Asch, Mrs. J. Curry and fellow
graduate students for their suggestions, aid and criticisms
during this investigation.
\
11

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS U
LIST OF TABLES iv
LIST OF FIGURES v
KEY TO ABBREVIATIONS viii
INTRODUCTION 1
REVIEW OF LITERATURE 4
Production of Interferon . . . . 12
MATERIALS AND METHODS . ; 23
Materials • 23
Methods . . . . J . 27
RESULTS 58
DISCUSSION 90
SUMMARY v 105
REFERENCES 107
VITA 114

LIST OF TABLES
Table Page
1. Comparison of Viral and Non-Viral
Induced Interferons. . 5
2. The Effect of Two Multiplicities of
Virus on Interferon Production ..... ¿2
3. Interferon Production in Presence
or Absence of Actinomycin D ..’... 76
4. Association of Chick Interferon with
Mouse and Chick Embryo Ribosomes .... 87
5. Association of Chick Interferon with
Ribosomal Subunits 89
iv

LIST OF FIGURES
Figure
1. Heat inactivation of Semliki Forest virus at 37°C.
Aliquots were removed at various times during
incubation and assayed for residual virus.
3
2. H-uridine incorporation in uninfected chick embryo
cell cultures exposed to various concentrations of
^H-uridine and incubated at 37°C for 30 minutes ..
3
3. H-uridine incorporation in uninfected chick embryo
cell cultures exposed to 15 l1 C of ^H-uridine for
various periods of time . ....
Q
4. Effect of calf serum on JH-uridine incorporation of
uninfected chick embryo cell cultures with or with¬
out actinomycin D. The cell cultures were exposed
to 20 pC of ^H-uridine for 30 minutes.
5. H-uridine incorporation, with or without calf (CS)
serum in uninfected chick embryo cell cultures. The
cell cultures were incubated at 37°C for various per-
iods and then exposed to 20 pC of H-uridine for
30 minutes ....... . . .
3
6. Inhibition of H-uridine incorporation in uninfected
chick embryo cell cultures with various concentra¬
tions of actinomycin D. The cell cultures were
exposed to 20 p C of H-uridine for 30 minutes at
the indicated times ... : . . .
3
7. Inhibition of H-uridine incorporation in uninfected
chick embryo cell cultures with various concentra¬
tions of actinomycin D. The values plotted were
obtained from Figure 6 at 10 hours after the expo¬
sure to actinomycin D.
Page
35
38
39
40
42
43
44
8. Linearity of sucrose gradient as measured by refrac¬
tive index of each fraction with an Abbe refractometer . 47
v

Figure
Page
9.Sucrose gradient analysis of chick embryo ribosomes.
One ml of crude ribosomal preparation was layered
on 8 to 25% linear sucrose gradient and then centri¬
fuged at 24, 000 rpm for 8 hours. The other details
have been described under Materials and Methods . . 49
10. Separation of chick embryo ribosomes into their
sub-units. The purified ribosomal preparation was
mixed with l/lOth volume of 4 M sodium chloride and
incubated at 4°C for 5, 15 or 30 minutes and then
centrifuged at 24, 000 rpmina8 to 25% linear sucrose
gradient for 12 hours . . . . . . ... 51
11. Separation of chick embryo ribosomes into their sub¬
units. The NaCl concentration of ribosomal prepara¬
tion was raised to 0. 2 M, 0. 3 M, 0. 4 M or 0. 6 M and
incubated at 4°C for 15 minutes. The samples were
then centrifuged ina 8 to 25% sucrose gradient containing
the same concentration of NaCl as that of sample and
centrifuged at 24, 000 rpm for 12 hours . . ... 52
12. Interferon yields at 24 hours in chick embryo cell
cultures infected with various input multiplicities
of Semliki Forest virus ... ..... 59
13. Kinetics of interferon production in chick embryo
cell cultures infected with Semliki Forest virus at
multiplicities of 10 or 0.1 pfu/cell. . . ... 60
14. RNA synthesis in chick embryo cell cultures in¬
fected with Semliki Forest virus at a multiplicity
of 10 pfu/cell with or without actinomycin D.
Actinomycin D sensitive RNA was plotted as the
difference between total RNA synthesis and
actinomycin D resistant RNA synthesis . ... 63
15. Correlation of viral RNA synthesis with growth of
Semliki Forest virus (with or without actinomycin
D) in chick embryo cell cultures at a multiplicity
of 10 pfu/cell. . . . . . . ... 66
16. Kinetics of interferon production in chick embryo
cell cultures exposed to heat inactivated Semliki
Forest virus. The cell cultures received the
equivalent of either 10 or 0. 1 pfu/cell of the virus . . 68
vi

Figure Page
17. Kinetics of interferon production with heat
inactivated and live Semliki Forest virus
with cell cultures ......... 70
18. Kinetics of interferon production in presence
of immune serum prepared against Semliki
Forest virus. Details have been described in
the text . . ......... 73
19. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
infected for 2 hours with Semliki Forest virus.
^H-uridine (20 P C) was added for 45 minutes, and
RNA was extracted. L cell RNA was employed
as carrier RNA ........
20. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
infected for 4 hours with Semliki Forest virus.
â– ^H-uridine (20 p C) was added for 45 minutes, and
RNA was then extracted. L cell RNA was employed
as carrier RNA . .......
21. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
infected for 6 hours with Semliki Forest virus.
•^H-uridine (20 pC) was added for 45 minutes, and
RNA was then extracted. L cell RNA was employed
as carrier RNA .... ....
22. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
exposed for 2 hours with heat inactivated Semliki
Forest virus. ^H-uridine (20 P C) was added for
45 minutes and RNA was then extracted. L cell
RNA was employed as carrier RNA ....
23. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
exposed to heat inactivated Semliki Forest virus
for 4 or 6 hours. ^H-uridine (20 pC) was added
for 45 minutes, and RNA was then extracted. L
cell RNA was employed as carrier RNA .
78
79
80
82
83
24. RNA synthesis in chick embryo cell cultures exposed
to heat inactivated or live Semliki Forest virus in
presence or absence of actinomycin D .85
vii

KEY TO ABBREVIATIONS
The following abbreviations were used in the text.
BSS
Balanced salt solution
CAM
Chorioallantoic membrane
CE
Chick embryo
CS
Calf serum
DNA
Deoxyribonucleic acid
FPA
Fluor ophenyalanine
MEM
Minimum essential medium
MM
Maintenance medium
MMM
Modified maintenance medium
m-RNA
Messenger ribonucleic acid
NDV
Newcastle disease virus
Pi
Post infection
PDD50
50 per cent plaque depressing dose
pfu
Plaque forming unit
rA/rU
Riboadenylic acid/ribouridylic acid
rl/rC
Riboinosinic acid/ribocytidylic acid
RNA
Ribonucleic acid
RNAse
Ribonuclease
RSB
Rabbit reticulocyte buffer
vm

SDS
S
TIP
VSV
w/v
Sodium dodecyl sulphate
Sedimentation coefficient
Translational inhibitory protein
Vesicular stomatitis virus
Weight per volume
ix

INTRODUCTION
One of the intriguing aspects of viral induced interferon production
is the role of multiplicity of infection. It has been known that high
multiplicities of certain viruses inhibit interferon production while
higher yields of interferon are obtained with lower multiplicities of
infection. (De Maeyer and De Somer, 1962; Gifford, 1963; Aurelian
and Roizman, 1965). Similar results were reported by Toy and Gifford
(1967a) who observed that maximal amount of interferon was obtained
in Semliki Forest virus (SFV) infected chick embryo (CE) cells at a
multiplicity of 0. 2 pfu/cell while multiplicity of 10 pfu/cell resulted in
marked reduction of interferon yield. An attempt was made to explain
this phenomenon by studying the effect of these two multiplicities of
infection on host cell macromolecular synthesis in the infected cells.
The study of RNA synthesis was suitable since cellular RNA could be
differentiated from viral RNA by the use of actinomycin D, an anti¬
biotic which is known to inhibit cellular RNA synthesis but has no
effect on Semliki Forest virus replication or its RNA synthesis. The
other factors considered in this study were: (1) effect of progeny virus
on interferon production, and (2) production of interferon by heat
inactivated virus.
The physico-chemical nature of the interferon inducers has, so
1

2
far, proved to be elusive. Recently, several investigators (Field et al. ,
1967; Vilcek et al. , 1968; Dianzani et al. , 1968) have reported that inter¬
feron production can be induced by complexes of polyriboinosinic acid
and polyribocytidylic acid (rl/rC) and other synthetic, double stranded
RNA preparations, in vivo as well as in vitro. However, the role of
single stranded RNA in interferon induction has been a subject of con¬
troversy. Field et al. (1967) reported that single stranded RNA did not
induce interferon production. These results have been confirmed by
Vilcek et al. (1968), Tytell et al. (1967) and Hilleman (1969). However,
Baron et al. (1968) and De Clercq and Merigan (1969a) found that certain
single stranded polyribonucleotide preparations such as polyriboinosinic
acid and polyribogunylic acid were able to induce small amounts of
interferon. Since heat inactivated SFV, though unable to replicate,
induces interferon production, the question that naturally presents itself
is: does the heat inactivated noninfectious SFV synthesize a double
stranded, replicative form when it induces interferon production in
CE cells or is the single stranded viral RNA in itself the inducing
agent? Part of the dissertation deals with an attempt to answer this
question.
The antiviral action of interferon in cells is due to the de novo
synthesis of another protein which apparently confers on ribosomes
the capability to distinguish between the cellular and viral messenger
RNA. These ribosomes are capable of forming polysomes with cellu¬
lar messenger RNA but bind poorly to viral messenger RNA and thus

the production of viral RNA directed proteins is inhibited (Marcus and
Salb, 1966; Carter and Levy, 1967). However, it is not known whether
this interferon induced protein acts alone or in conjunction with inter¬
feron. If interferon does play a direct role in the maintenance of a
virus resistant state in the cells, it is reasonable to assume that it
would also combine with ribosomes for the expression of antiviral
activity. To investigate this possibility, chick embryo and mouse
liver ribosomes and their subunits were exposed to various interferon
preparations and were examined for their capability to combine with
interferon.

REVIEW OF LITERATURE
Interferon is a term given to a group of antiviral proteins that can
be induced in vivo and in vitro by a wide variety of.agents. It was
first described by Isaacs and Lindenmann (1957) as a protective factor
against viral replication in chorio-allantoic membranes induced by
heat inactivated influenza virus. Since then, interferon has been in¬
duced by every major group of viruses and by a variety of nonviral
inducers of interferon-like substances. Thus, it has become evident
that production of interferon by cells is a general nonspecific response
to various stimuli. Interferons, in general, can be divided into two
broad classes. (1) Interferons whose synthesis is dependent upon RNA
and protein synthesis in the induced cells. Interferons produced in
response to viruses form a major group in this class. (2) Interferons
whose appearance after induction is not dependent upon RNA and pro¬
tein synthesis and seem to be released from certain cells (preformed
interferon). The other differences in the two classes of interferons
are summarized in Table 1. However, some inducers may invoke both
types of response and characterization of such preparations of inter¬
feron becomes difficult.
The inhibition of viral induced interferon production by actinomycin
D was first reported by Heller (1963) and Wagner (1963). This finding
4

TABLE 1
COMPARISON OF VIRAL AND NON-VIRAL INDUCED INTERFERONS
Characteristics
Viral
Non-viral
Reference
Kinetics of production (serum)
Late peak
Early peak
Youngner, J. , Hallum, J. , and
Stinebring, W. (1966)
Molecular weight (serum)
often > 50, 000
often < 50, 000
Ibid.
Sensitivity of Production:
Protein synthesis inhibitors
sensitive
not sensitive
Ho, M. , and Breinig, M. K.
(1965)
Actinomycin D
sensitive
not sensitive
Finkelstein, M. S. , Bausek,
G. H. , and Merigan, T. C. (1968)
Presence of spleen during
induction
not essential
es sential
De Somer, P. , and Billiau, A.
(1966)
Effect of BCG sensitization
on production
no influence
increased
production
Youngner, J. , Hallum, J. , and
Stinebring, W. (1966)
Elevated temperature
increased
production
no influence
Isaacs, A. (1961)--viral, De Some
P. , and Billiau, A. (1966)--non-
viral

was interpreted as evidence for the coding of interferon by the cellular
genome rather than by the inducer virus. A similar effect of other
DNA dependent RNA synthesis inhibitors like 4, 5, 6-trichloro-l- D-
ribofuranosyl benzimidazole and 8-azaguanine on interferon synthesis
has been reported by Walters, Burke and Skehel (1967). The other lines
of evidence which support this hypothesis are provided by the obser¬
vations that (1) interferons are species specific, and (2) DNA as well
as RNA viruses induced apparently identical interferons in chick cells
(Lampson et al. , 1965). Thus, it seems that in interferon induction by
viruses, a stimulus leads to cell directed RNA synthesis which then
controls the synthesis of the new protein, presumably different from the
other proteins of the cell.
The concept of viral induced interferon formation as a newly syn¬
thesized protein is supported by the work of Buchan and Burke (1965,
1966) who reported that 25 p g/ml of p-flurophenylalanine, in phenyl-
alanine-free medium, completely inhibited subsequent interferon for¬
mation in chick embryos exposed to ultraviolet-inactivated influenza
virus. This substance acts as an antagonist of phenylalanine and is
incorporated into proteins. Thus, the proteins might have a reduced
or no biological activity depending upon the importance of the phenyl¬
alanine residue for biological activity (Richmond, I960, 1963). Simi¬
larly, puromycin at low concentrations has also been found to com¬
pletely inhibit interferon production in various cell-virus systems.
It was reported by Wagner and Huang (1965) that once interferon

7
production has started in the Krebs-2 carcinoma cells-Newcastle
disease virus (NDV) system, the addition of puromycin resulted in the
immediate cessation of interferon production. Thus, the rate at
which interferon appears in cell culture fluids reflects the actual rate
of synthesis and there seems to be no build-up of the product in the
cells. Similar results have been obtained by Buchan and Burke (1965,
1966) employing chick embryos infected with ultraviolet-inactivated
virus.
This overall similarity of interferon production with Jacob and
Monod's operon model led Burke (1966) to propose a hypothetical scheme
in which virus invasion is presumably followed by uncoating and re¬
lease of an interferon inducer which induces the production of inter¬
feron specific messenger-RNA. This m-RNA then directs the syn¬
thesis of interferon. However, there is no well established case for
the operon model's counterpart in the mammalian system. In the inter¬
feron system, the exact nature of the inducer, the mechanism of inter¬
action between inducer and host genome, and the nature of the repres¬
sor molecule, if present, are largely unknown. Attempts to obtain
direct evidence for interferon specific messenger RNA have not been
successful (Burke and Low, 1965; Wagner and Huang, 1966). However,
Wagner and Huang (1965, 1966) reported that in the Krebs-2 carcinoma
cell-NDV system, practically all the interferon specific messenger
is synthesized within 6 hours after infection and is relatively stable
for at least 10 hours.

The discovery of substances other than viruses that can elicit
interferon production was first reported by Isaacs, Cox and Rotem
(1963). They found that chick, mouse and rabbit ribosomal RNA did
not induce interferon in homologous cells unless the RNA was first
made "foreign" by treatment with nitrous acid. Ribonucleic acid from
E. coli, turnip yellow mosaic virus and rat liver DNA though "foreign"
were not effective inducers of interferon in chick cells. Rotem, Cox
and Isaacs (1963) also showed that chick liver RNA inhibited the growth
of vaccinia virus in mouse cell cultures and mouse liver RNA inhibited
vaccinia virus in chick cell cultures.
Youngner and Stinebring (1964) reported the production of low
amount of interferon in chickens infected with Brucella abortus.
Stinebring and Youngner (1964) also found that intravenous injections
of endotoxin, Salmonella typhimurium, or Serratia marcescens caused
a rapid appearance of interferon in the blood stream of mice. Ho (1964)
also reported the production of interferon in rabbits injected with endo¬
toxin. Youngner and Stinebring (1965) found that endotoxin or Brucella
abortus induced interferon formation was unaffected by concentrations
of actinomycin and cyclohexamide which inhibited more than 97% of
RNA and protein synthesis, respectively, in mouse liver. They also
observed that pretreatment of animals with puromycin or cyclohexa¬
mide enhanced and prolonged the interferon response. Gifford (1965)
found that yeast RNA, hydrolysate of RNA, and mononucleotides in¬
hibited vaccinia and chikungunya virus in cell cultures but interferon

was not present in high enough titers to account for all the viral in¬
hibitory effects. Field et al. (1967) reported the formation of inter¬
feron in rabbits and their spleen cell cultures after induction by syn¬
thetic polyribonucleotides. Only double stranded polyribonucleotides,
such as polyinosinic acid/polycytidylic acid (rl/rC), were found to be
effective inducers while double stranded DNA polymers or single
stranded RNA polymers were unable to elicit an interferon response.
Similar results have also been obtained by Vilcek et al. (1968), Falcoff
and Bercoff (1968), Tytell et al. (1968), and Field et al. (1968).
However, it has also been claimed that single stranded polyribocy-
tidylic acid as well as polyriboinosinic acid can induce interferon for-
mation (Baron et al. , 1968; Levy et al. , 1968; Finkelstein, Bausek and
Merigan, 1968). It was then suggested that the inducing ability of
single stranded polyribonucleotides may be due to the contamination of
these preparations with double stranded polymers (Field et al. , 1968;
Tytell et al. , 1968). But De Clercq and Merigan (1969a) reportéd that
the single stranded homopolymers, polyriboguanylic acid, polyribo¬
inosinic acid and polyriboxanthylic acid, at neutral pH and polyribo-
adenylic acid and polyribocytidylic acid at acid pH can induce inter¬
feron production. The preparations were apparently free of double
stranded polymers and the inducibility characteristic was related to’
the stability of the secondary structure of homopolymers as indicated
by their high temperature of melting (Tm) values. However, triple
stranded complexes of polyribonucleotides, having higher Tm values,

10
were much less active than the double stranded polymers. It was post¬
ulated that differences in the inductive capability of various polyribo¬
nucleotides may be due to the differences in their affinity for the site
of initial interferon formation inside the cells, even though cells seem
to have similar permeabilities to various effective and ineffective RNA
polymers (Field, Tytell and Hilleman, 1969) and such polymers are
equally suceptible to nuclease degradation inside the cells (Colby and
Chamberlin, 1969). De Clercq, Eckstein and Merigan (1969) recently
reported that substitution of the phosphate group by a thiophosphate
group in an alternating copolymer, rA/rU, resulted in a 2-20 fold in¬
crease in its ability to induce interferon production. This increase in
inducibility was accompanied by a 10-100 fold increase in resistance to
pancreatic ribonuclease. It has been suggested (De Clercq and Merigan,
1969b) that the common structural requirement for polyribonucleotides
and other synthetic polyanion interferon inducers include (a) high
molecular weight, (b) a stable, primary and long, carbon to carbon
backbone, and (c) a regular and dense sequence of negative charges on
the backbone. It has been found by several investigators that polybasic
substances such as protamine sulphate, neomycin sulphate and DEAE
dextran significantly increase the yields of interferon in vitro as well
as in in vitro (Vilcek et al. , 1968; Falcoff and Bercoff, 1968) by poly¬
ribonucleotides. The mechanism of enhancement is not well under¬
stood although it has been postulated that enhancement is due to the
increased penetrability of the polyribonucleotides into the cells.

11
The effect of actinomycin D on polyribonucleotide induced inter¬
feron formation has been a subject of controversy. Vilcek et al. (1968)
observed that 2P g/ml of actinomycin D inhibited the production of
interferon in rabbit kidney cells induced by poly rl/rC. Similar re¬
sults were obtained by Falcoff and Bercoff (1968) in human leukocytes
and in amniotic membranes. However, Finkelstein, Bausek and Merigan
(1968) observed that the concentration of actinomycin D which suppressed
NDV-induced interferon production had no effect on the interferon for¬
mation induced by poly rl/rC, pyran, and endotoxin in human skin fibro¬
blast and mouse peritoneal macrophage cultures. It was also found that
actinomycin suppressed polyribonucleotide-induced interferon formation
only at concentrations which were cytotoxic. Vilcek, Rossman and
Varacalli (1969) observed that actinomycin D, if applied two hours or
later after the induction with poly rl/rC, does not affect the yields of
interferon in rabbit kidney cell cultures. The formation of polyribo¬
nucleotides-induced interferon is not inhibited by protein synthesis in¬
hibitors like p-flurophenylalanine and puromycin in vitro (Finkelstein,
Bausek and Merigan, 1968) or in vivo (Youngner and Hallum, 1968)
while viral-induced interferon synthesis was suppressed under similar
conditions.
Differences between the two classes of interferons (summarized in
Table 1 ) raises the question of whether or not the mechanism of induc¬
tion in both classes (preformed versus synthesized) is similar. The
question becomes more pertinent since the two responses also occur

in morphologically uniform population of célls like human skin fibro¬
blast indicating that the same cell is capable of responding in two
different ways. It has been postulated that there may be two differ¬
ent processes of interferon induction, i. e., de novo synthesis as well
as release of already formed interferon (Ho and Kono, 1965; Ho, Postic
and Ke, 1968; Youngner, 1968; Finkelatein, Bau§ek and Merigan, 1968).
It has also been suggested by Finkelstein, Bausek and Merigan (1968)
that both responses may involve de novo synthesis but that one requires
an additional step, e. g.t virus uncoating which is sénsitive to metabolic
inhibitors; or, that both responses may require de novo interferon syn¬
thesis and both have similar biological activity but possess different
sensitivities to metabolic inhibitors and are released or formed at dif¬
ferent rates. Youngner (1968) suggested that the two responses may
result in release of preformed interferon but the viral-induced type
may require an interferon activating enzyme whose synthesis is sup¬
pressed by metabolic inhibitors. However, this possibility is ex¬
cluded by the work of Fhucker (1969) who reported, on the basis of
radiolabelled pulse experiments, that interferon produced in L cells
by UV-unactivated NDV is essentially a product of de novo synthesis.
A definite explanation for the mechanism of the two types of responses
may be possible once radiolabelled amino acid incorporation studies
on polyribonucleotide induced-interferon become available.
Production of Interferon
Interferon is a product of the interaction of cell and inducer. Both

13
elements of interaction seem to be equally important determinants of
whether interferon is produced and how much. Production would also
be dependent upon the type of cellular environment in which this inter¬
action takes place.
Cellular Aspects of Interferon Production
Genetic control
The direct evidence for the genetic control of interferon production
by cells comes from the observation of Desmyter, Melnick and Rawls
(1968) that Vero cells, a line of African green monkey kidney cells, was
unable to produce interferon when infected with NDV, Sendai, Sindbis
or Rubella virus. This apparently qualitative defectiveness in inter¬
feron production has also been noted when Vero cells were exposed to
poly rl/rC with or without protamine sulphate or neomycin sulphate
(Schaffer and Lockart, 1969). The cell line, though incapable of pro¬
ducing interferon, is sensitive to the action of exogenous interferon.
There is no information in regard to the size of the locus or the num¬
ber of genes involved in this defectiveness. Cogniaux-Le Clerc, Levy
and Wagner (1966) reported that the loss of ability to produce inter¬
feron as a function of increasing UV dose showed first order kinetics
and it was suggested that a single site on cellular DNA controls the
formation of the interferon specific messenger RNA. Similar results
were obtained by Burke and Morrison (1966).
Types of cellular response
Endotoxin, when administered into intact animals, elicits significant

14
amounts of interferon but is unable to induce interferon in vitro except
in leukocytic cell cultures (Smith and Wagner, 1967). Lackovic et al.
(19o7) reported failure of L cells to produce interferon when treated
with mannan, a yeast polysaccharide, although a good response was
obtained with mouse peritoneal macrophage cultures. Similarly,
Finkelstein, Dausek and Merigan (1968) observed that pyran, a poly-
carboxylate polymer, and poly rl/rC induced interferon in mouse
peritoneal macrophages but not in L cells. Thus, it appears that
there are several types of cellular responses to inducers, with
aneuploid cell lines being most restrictive. The type of cellular re¬
sponse, at least in part, seems to depend upon the penetrability of
the inducer into the cell since L cells have been reported to produce
significant amount of interferon with poly rl/rC after prior treatment
with diethylaminoethyl (DEAE) dextran (Dianzani et al. , 1968). Other
factors which may contribute to variation in types of cellular responses
have not been elucidated.
Host macromolecular synthesis
The evidence available indicates that synthesis of interferon is a
latent cellular function, induced by viral infection or certain nonviral
agents. Thus, the role of cellular macromolecular synthesis in the
production of interferon becomes obvious. Several investigators have
reported that virulent viruses induce very low yields of interferon.
â–  I
Ruiz-Gomez and Isaacs (1963) reported that NDV, a cytopathic virus
in CE cells, grew in high titers but produced very little interferon.

15
However, when mouse embryo fibroblast or human thyroid cell cultures
were infected with the same virus, no cytopathology was observed and
high yields of interferon were obtained. Similar results were obtained
with vesicular stomatitis virus (VSV) (Wagner et al. , 1963), foot and
mouth disease virus (Sellers, 1963, 1964), polyoma virus (Friedman
and Rabson, 1964) and various arboviruses (Ruiz-Gomez and Isaacs,
1963). Virulent myxovirus has been reported to inhibit interferon syn¬
thesis induced by UV-inactivated (Lindenmann, I960) or avirulent
myxoviruses (Hermodsson, 1963). However, there is not always a
correlation between the virulence of virus and interferon production:
e. g. , some strains of influenza virus (Inglot, Kochman and Mastalerz,
1965; Link et al. , 1965a), Sindbis virus (Vilcek, 1964),vaccinia virus
(Link et al. , 1965b) and NDV (Baron, 1964) do not follow this pattern.
The mechanism by which certain viruses can inhibit interferon
production was investigated by Wagner and Huang (1966). They induced
interferon production in suspension cultures of Krebs-2 carcinoma
cells by employing an avirulent strain of NDV. Interferon was first
detected 3 to 4 hours after the infection and production increased al¬
most linearly, reaching a peak at 20 hours. However, interferon syn¬
thesis was terminated when cells were superinfected with VSV (50
pfu/cell) within 4 hours after infection with NDV. Vesicular stoma¬
titis virus does not induce interferon in this cell system. Krebs-2
carcinoma cells infected with VSV exhibited a rapid and almost immedi¬
ate decline in the rate of cellular RNA synthesis but NDV,. at the

16
multiplicity employed, had no appreciable effect for at least 3 hours
after infection. Therefore, it was postulated that VSV prevented inter¬
feron synthesis by inhibiting cellular RNA synthesis. Similar results
were reported by Aurelian and Roizman (1965) employing a strain of
herpes virus which caused an abortive infection in dog kidney cells.
Infection at a multiplicity of 100 pfu/cell led to the formation of viral
DNA and antigen without any production of interferon, whereas infec¬
tion at lower multiplicity (12 pfu/cell) resulted in the production of
interferon, but not virus. In the former case, virus infection caused
a rapid decline in cellular RNA synthesis and, hence, the cessation of
interferon production. At the lower multiplicity, virus induced inhi¬
bition of RNA synthesis did not occur until 5 to 6 hours after infection
and interferon production was thus permitted.
Bolognesi and Wilson (1966) have reported a rapid and profound
decline in the rate of cellular protein synthesis in NDV infected CE
cells. Most probably, marked inhibition of protein synthesis is the
reason for the lack of interferon production in this system. The in¬
hibition of protein synthesis can be ascribed to either reduction in
cellular RNA synthesis or inhibition of messenger RNA translation in
NDV infected CE cells. However, the relationship between interferon
production and the rate of cellular protein synthesis is complex;
Friedman (1966a) observed that interferon yields were reduced in SFV
infected CE cells under conditions when overall cell protein synthesis
was augmented and postulated that increased protein synthesis resulted

17
in the accumulation of an interferon repressor in the cells thereby
decreasing interferon yields.
Factors Influencing Interferon Production
All the conditions which favor the optimal production of interferon
are not yet known. However, there are some factors which have been
reported to influence interferon synthesis and are considered in this
section.
t
Priming
The priming effect is obtained when cells treated either with in¬
activated virus or with interferon respond by an enhanced production of
interferon on subsequent induction. Burke and Isaacs (1958) observed
that influenza virus could induce interferon formation in chick embryo
chorio-allantoic membrane only if the tissue had first been primed with
heat-inactivated virus. Similar results have been obtained with Eastern
equine encephalitis virus (Mahdy and Ho, 1964) and Sindbis virus (Ho
and Breinig, 1962). Isaacs and Burke (1958) also described the poten¬
tiating effect of interferon on subsequent interferon formation. However,
Vilcek and Rada (1962) and Paucker and Cantell (1963) reported that
pretreatment of cells with interferon inhibited the subsequent yields
of interferon when challenged with a viral inducer. Later it was found
(Lockart, 1963; Taylor, 1964; Friedman, 1966b) that pretreatment of
cells with interferon may indeed have both effects depending upon the
i
amount used and multiplicities of virus subsequently employed to
elicit interferon production. Levy, Buckler and Baron (1966) and

Friedman (1966b) showed that "priming" not only increased the yields
but interferon was synthesized more rapidly in the primed cells.
Friedman (1966b) has also shown that enhancement of interferon pro¬
duction is dependent upon active protein synthesis in the cells during
the priming period. However, synthesis of interferon specific mes¬
senger RNA was not detected during the period of priming. It was
also reported that interferon-cell interaction for priming could occur
at 4°C but would be expressed only if cells were further incubated at
37°C for several hours before challenging with virus.
Temperature
Interferon production can only occur within a certain range of
temperature as would be expected for any cellular metabolic process.
Its synthesis was inhibited when cells were incubated at 4°C (Isaacs,
1963). Ruiz-Gomez and Isaacs (1963a, b) observed that the optimum
temperature for interferon production was generally higher than that
for virus replication. For example, more interferon was induced by
chikungunya virus at 42°C, an unfavorable temperature for virus
replication, than at 39°C or 35°C. Burke, Skehel and Low (1967)
found that SFV induced optimal yields of interferon at 42°C when no
virus replication was detected. However, the cells have to be pre¬
incubated with virus at 37°C for some time to presumably allow its
adsorption and uncoating before shifting the temperature to 42°C.
Ruiz-Gomez and Isaacs (1963) also suggested thatthis increased inter¬
feron production at higher temperature may constitute a defense

19
mechanism which prevents the pathogenic effect of viruses during
febrile conditions. The results obtained by Ruiz-Gomez and Sosa-
Martinez (1965) tend to support this hypothesis. Mice inoculated with
Coxsackie B virus were kept at 4°C, 11°C and 25°C. Those kept at
4°C developed viremia with high titers in heart and liver and died
3 to 4 days after infection. Their livers had very little interferon
except on the first day after infection. However, the mice kept at
25°C survived, virus did not replicate in any tissue, and livers con¬
tained high titers of interferon.
The other aspect of this relationship was reported by Siegert, Shu
and Kholhage (1967) who found that production of interferon in rabbits
infected with myxovirus was accompanied by fever. Similarly,
Merigan (1968) reported that interferon inducing doses of pyran also
caused fever in man. However, this correlation was not observed
with measles vaccine which induced interferon production without
concomittant induction of fever in human beings. Conversely, bac¬
terial endotoxin or etiocholanalone induced fever without the production
of interferon. However, endotoxin which is capable of inducing inter¬
feron production in mice (Stinebring and Youngner, 1964) as well as
in rabbits (Ho, 1964) is also known to produce fever in rabbits.
Multiplicity of infection
In several instances of cell-virus interaction, the multiplicity of
infection seems to play a significant role in determining the amount
of interferon synthesized by the induced cells. De Maeyer and De Somer

20
(1962) reported that rat tumor cells infected with Sindbis virus pro¬
duced maximal amounts of interferon when a multiplicity of 0. 1 pfu/
cell was employed, and much lower yields were obtained at a multi¬
plicity of 10 pfu/cell. Gifford (1963) observed that chikungunya virus
best induced interferon production when the input multiplicity of in¬
fection was 0.1 pfu/cell while higher multiplicity reduced the yields of
interferon. The observations of Aurelian and Roizman (1965) in the
dog kidney cell-herpes virus system and Toy and Gifford (1967a) in
the SFV-CE system tend to support the concept that higher multiplicity
of infection often inhibits interferon production whereas more interferon
is produced with lower multiplicities.
Nature of Inducing Agent
The physico-chemical nature of the agents which induce interferon
has not been well elucidated. Isaacs (1961) suggested that the induc¬
tion of interferon by viruses might be due to their "foreign" nucleic
acid. This concept was in agreement with the observations that
viruses, composed of nucleic acid and protein only, could induce inter¬
feron production, but incomplete viruses containing less nucleic ácid
failed to induce.
Low yields of interferon have been obtained in various cell systems
by Rotem, Cox and Isaacs (1963) with heterologous RNA, and by Isaacs,
Cox and Rotem (1963) using nitrous acid treated homologous ribosomal
RNA. The few known properties of nucleic acid-induced viral inhibi¬
tors suggested identity with viral-induced interferon, but the physico¬
chemical characterization of these preparations has not been carried

21
out in detail. Moreover, several investigators were unable to confirm
these findings. However, the work of Skehel and Burke (1968a) supports
the hypothesis that complete viral nucleic acid is essential for the
induction of interferon production. These authors studied the effect
of hydroxylamine on SFV. Hydroxylamine has been extensively em¬
ployed to inactivate the infectivity of viruses by reacting with viral
nucleic acid; it has no effect on viral antigenicity (Schafer and Rott,
1962). The inactivation of SFV by 0. 2 M hydroxylamine at 25°C fol¬
lowed a pattern of first order kinetics, and the interferon-inducing
capacity decreased at the same rate as that of infectivity. There was
no effect on hemagglutination titers,, indicating that the protein coat
of the virus was unaffected by the treatment. These results confirmed
the significance of viral nucleic acid in the process of induction but did
not rule out the possibility that viral protein(s) may also be essential
for interferon induction.
Burke, Skehel and Low (1967) studied the early stages of inter¬
feron induction. The system employed was infection of chick cells
with SFV for one hour at 36°C followed by shifting the temperature to
42°C. Under these conditions interferon is produced but the virus
does not replicate. It was found that during the incubation period at
36 °C some ribonuclease resistant RNA was synthesized in the infected
cells (Skehel and Burke, 1968b), and it was suggested that formation
of a double stranded, replicative form of viral RNA may be the first
step in induction of interferon formation. Once again, the role of

viral protein(s), if any, could not be ascertained.
22
Double stranded polyribonucleotides have been reported to induce
interferon production in vivo as well as in vitro but the role of single
stranded polyribonucleotides in interferon induction has not been
clearly established (discussed on pages 9.10, andll ). However, it has
been reported that nucleic acid preparations release preformed inter¬
feron (Finkelstein, Bausek., Merigan, 1968; Youngner, 1968), unlike
the viral-induced interferon which is synthesized de novo. Therefore,
it is a distinct possibility that induction for the two types of responses
may not be achieved through the same process.
Lockart et al. (1968) employed temperature sensitive mutants of
Sindbis virus in CE cells for interferon induction. The authors con¬
cluded that input viral RNA and the replication of viral RNA are not
sufficient for interferon induction but the induction event requires some
viral protein(s) or the process for which these proteins are necessary.
Similarly, Dianzani (1969) reported that NDV-infected mouse cells
synthesize interferon specific messenger RNA in the presence of
protein synthesis inhibitors. Under these conditions, viral replicative
events were prevented, thereby indicating that input parental viral
RNA or viral protein(s), or both, were the inducer for the synthesis
of interferon specific messenger RNA.
The information available at present does not resolve whether
single stranded RNA is sufficient to induce interferon production or
double stranded RNA is the necessary form. In addition, the role of
viral protein(s) in the process of induction is not well understood.

MATERIALS AND METHODS
Materials
Virus Strains
Vaccinia virus (VV). The N. Y. 914 strain, isolated by Dr. G. E.
Gifford from commercial lymph vaccine, was employed.
Semliki Forest virus (SFV). Kumba strain was obtained from
Dr. J. Porterfield, National Institute for Medical Research, London,
England.
Vesicular stomatitis virus (VSV). Indiana strain was kindly sup¬
plied by Dr. Samuel Baron, National Institute of Health, Bethesda,
Maryland.
Newcastle disease virus (NDV). Hertz strain was also obtained
from Dr. J. Porterfield, The Roakin strain was obtained from the
Research Reference Reagent Branch of the National Institute of
Allergy and Infectious Diseases and the Cincinnati strain was received
from Mr. M. Fruitstone, Department of Microbiology, University of
Florida.
Cell Cultures
Primary chick embryo cell cultures. Chick embryo (CE) cell
cultures were prepared as described in Methods.
Mouse L cell cultures. The continuous L cells (strain 929) were
23

kindly supplied by Mr. M. Fruitstone.
24
Media
Balanced salt solution. Gey's balanced salt solution (BSS) was
employed in the growth and maintenance media for CE cell cultures.
Growth medium. The growth medium for CE cell cultures con¬
sisted of BSS, 5% calf serum, 0.1% sodium bicarbonate, 0.1% lactal-
bumin hydrolysate (Nutritional Biochemicals) and 0. 1% proteose pep¬
tone (Difco). For L cell cultures, Eagle's minimum essential medium
(MEM) with 10% calf serum was used.
Maintenance medium. The maintenance medium (MM) for CE
cell cultures consisted of Gey's BSS with 0. 1% lactalbumin hydrolysate,
0.1% proteose peptone, 0.1% yeast extract (Difco "yeastolate") and
about 0.1% sodium bicarbonate. For determination of RNA synthesis
in cell cultures, a modified maintenance medium (MMM) was employed
in which yeast extract was omitted and 2. 5% calf serum was added in
some cases.
Overlay medium. The overlay medium used for plaque assay con¬
sisted of either 1% methyl cellulose in MEM with 10% calf serum, or
chick embryo cell culture growth medium containing 5% calf serum
and 0. 5% agar (Ion agar No. 2, "Oxoid" division of Oxo, Limited).
Reagents
Rabbit reticulocyte buffer (RSB). RSB was prepared according
to Penman et al. (1963). The composition of this buffer was 0. 01 Tris-
(hydroxymethyl)aminomethane(Tris), 0. 01 M potassium chloride and

25
0. 0015 M magnesium chloride with pH adjusted to 7.2 by the addition
of 0. 5 M hydrochloric acid.
Phosphate-chloride buffer. This buffer was made as described
by Petermann and Pavlovic (1963) and consisted of 0. 001 M potassium
phosphate and 0. 005 M magnesium chloride, pH 6. 8.
Acetate buffer. This bvitfer was prepared according to the method
of Friedman (1968) and consisted of 0. 1 M sodium chloride, 0. 01 M
sodium acetate and 0. 0005 M magnesium chloride with pH adjusted
to 5. 1 by addition of 0. 5 M hydrochloric acid.
Sucrose solutions. Ribonuclease-free sucrose (Mann Research
Laboratories, New York) was employed for all the sucrose solutions.
The following sucrose solutions were used:
a) 1. 8 M sucrose solution in RSB
b) 0. 5 M sucrose solution in RSB
c) 0. 3 M sucrose solution in phosphate--chloride
buffer
d) 25% (w/v) and 8% (w/v) solutions of sucrose in RSB, pH 7. 2
e) 30% (w/v) and 15% (w/v) sucrose solutions in 0.1 M potassium
chloride, 0. 01 M tris(hydroxymethyl)aminomethane (Tris)
and 0. 001 M ethylendiaminetetracetate (EDTA) buffer, pH 7.1
f) 25% (w/v) and 8% (w/v) sucrose solutions in 0. 01 M tris, 0. 4 M
sodium chloride buffer, pH 7. 2. In some experiments, the
molarity of sodium chloride was varied at 0.2, 0. 3, 0.4 and .
0. 6 M in tris buffer, sucrose solutions.

26
Ribonuclease solution. Ribonuclease A (Worthington Biochemical
Corporation, Freehold, New Jersey) was dissolved in 0. 1 M KC1,
0. 01 M Tris, 0. 001 M EDTA buffer, pH 7.1, to a final concentration
of 2 Op g/ml
Antibiotics. To all media was added 250 units/ml of potassium
penicillin G and 100 pg/ml of streptomycin sulphate.
Radioisotope . Uridine-5-H was obtained from New England
Nuclear Corporation, Boston, Mass. The specific activity of the iso¬
tope preparations were either 7 ^/mM or 28.1 ^"/mM.
Scintillation fluid. The radioactive samples were diluted in
scintillation fluid containing 4 g of 2, 5-bis-2-(5-tert-Butylbenzox-
azolyl) thiophene (BBOT) (scintillation grade, Packard), 500 ml
toluene (Fisher Scientific Co. ) and 500 ml of methanol (Fisher
Scientific Company).
Sodium deoxycholate solution. A stock solution of 10%. (w/v)
sodium deoxycholate was prepared in acetate buffer, pH 5.1.
Phenol. Phenol (Fisher Scientific Co. ) was distilled and hydroxy-
quinoline was added to a concentration of 0. 1%. The distilled
phenol was saturated with 10 x acetate buffer, pH 5. 1, before use.
Bentonite suspension. Bentonite powder (Fisher Scientific Co. )
was processed according to the method of Petermann and Pavlovic
(1963). The bentonite concentration in coarse suspension was 73 mg/mL
Actinomycin D. This reagent was a gift from Merck, Sharpe and
Dohme, Rahway, New Jersey. Stock solution contained 100 pg/ml of

27
actinomycin D and was kept in the dark at -20° C.
\
Semliki Forest virus antiserum. SFV antiserum was kindly
supplied by Dr. S. T. Toy. The antiserum at a final dilution of 1:20
was capable of neutralizing approximately 99% of virus in 60 minutes
at 37° C.
Methods
Cell cultures
Chick embryo cell cultures. The method of Lindenmann and
Gifford (1963a) was followed with some modifications. Chick embryos,
10 to 11 days old, were decapitated, eviscerated and washed once with
Gey's BSS. The embryos were minced by forcing through a "Luer Lok"
syringe into a "Bélico" trypsinizing flask. Trypsinization of the
tissues was carried out for 30 minutes at 37°C with continuous stir¬
ring in 20 volumes of 0. 02% (w/v) trypsin (Grand Island Biological
Co. , Long Island, New York) in Gey's BSS without calcium and mag¬
nesium. Large, undigested tissue fragments were removed by fil¬
tering the cell suspension through a gauze filter. The trypsin was
removed by centrifuging the cell suspension for 20 minutes at 200xg
at 5°C. The cells were resuspended in growth medium and recentri¬
fuged for 20 minutes at 200xg and suspended again in growth medium:.
The cells were then passed through a coarse sintered glass filter
using negative pressure and dispensed either at a concentration of
12 x 10^ cells in a volume of 5 ml into plaque bottles (2-ounce, screw-
capped, square, soft glass bottles having a rectangular side of 3 x 6

cm) or 1 x 10^ cells into 32-ounce glass prescription bottles (rectan¬
gular side 17 x 7 cm). A cell monolayer was usually formed within
24-48 hours of incubation at 37°C.
Mouse L cells (Strain 9^9). The cell cultures were maintained
and propagated in Eagle's MEM with 10% calf serum. The cell mono-
layers were maintained in 32-ouncc bottles. For passaging the cells,
the growth medium was decanted and the cell monolayer was washed
with Gey's BSS and 10 ml of 0. 02% (w/v) trypsin was used to dislodge
the cells from glass surface. After an incubation period of 10 to 15
minutes at room temperature, the cells were removed from the glass
and counted in a hemacytometer chamber. To form a monolayer,
2-ounce plaque bottles received 1-2 x 10^ cells and 32-ounce bottles
were plated with 8-10 x 10^ cells.
Growth, Purification and Assay of Viruses
Vaccinia virus
Preparation. Vaccinia virus was propagated in lL-to 12-day-old
chick embryos by inoculating 200 plaque forming units (pfu) of virus
in 0. 2 ml volume on the chorio-allantoic membrane and incubating
the eggs at 37°C for 46 to 48 hours. The eggs were chilled at 5°C
for several hours, the infected membranes were removed, frozen at
-60°C and homogenized with a mortar and pestle with sterile car¬
borundum employing MM as a diluent. The suspension was then cen¬
trifuged at 800xg for 30 minutes to remove coarse particles. The
supernatent fluid was dispensed into glass ampules which were sealed

29
and stored at -60®C.
Assay. Vaccinia virus was titrated by the method of Lindenmann
and Gifford (1963a). Growth medium was decanted from the 2-ounce
plaque bottles and different dilutions of virus in maintenance medium
were dispensed in a volume of 2 ml per bottle. Normally, four bot¬
tles were employed for each dilution of virus tested. The bottles were
incubated undisturbed on a flat, level surface at 37°C for 46 to 48 hours.
After the incubation period, the medium was decanted from the bot¬
tles and the monolayers were stained with 0.1% crystal violet for
3 to 5 minutes, washed several times in running tap water and in¬
verted to dry. The plaque were counted after enlarging the mono¬
layer's image 6 to 7 times with a photographic enlarger.
Semliki Forest virus
Preparation. Semliki Forest virus was propagated in the brains
of 24-to 48-hour-old mice. The newborn ICR strain of mice were
intracerebrally inoculated with 0. 02 ml of stock virus suspension di¬
luted 1:100 in maintenance medium. The infected brains were har¬
vested 24 hours after inoculation and a 10% (w/v) suspension of brains
in maintenance medium was made using a tissue homogenizer with a
teflon pestle. The suspension was centrifuged at 800xg for 20 minutes
to remove the coarser particles. The virus suspension was dispensed
in glass ampules which were sealed and stored at -60°C.
Assay. The SFV was assayed on primary chick embryo cell
monolayers employing either the agar overlay or methyl cellulose

30
techniques. The growth medium was decanted from the bottles and
virus dilutions in 0. 2 ml volume were added to each plaque bottle.
The virus was allowed to adsorb for 60 minutes at room temperature
with frequent rocking of the bottles to distribute the virus evenly on
the monolayers. The bottles were drained and overlaid with 3 ml of
0. 5% molten agar in growth medium with 5% calf serum maintained
at 42°C. After the agar solidified, the bottles were transferred to a
37°C incubator for 46 to 48 hours. In the methyl cellulose overlay
method, each bottle received 4. 5 ml of 1% methyl cellulose in MEM
with 10% calf serum after the adsorption of virus. Subsequent steps
were similar to the agar overlay technique. After the incubation
period, the agar or methyl cellulose was decanted gently from the
cells and discarded, the bottles were stained, and plaques were
counted as in the vaccinia virus assay (vide supra). The virus prep¬
aration, assayed either by methyl cellulose or agar overlay method,
displayed no significant difference in titer.
Newcastle disease virus
Preparation. Newcastle disease virus was propagated in 11-to 12-
day-old embryonated eggs by inoculating 200 pfu in 0.1 ml volume into
the allantoic cavity. After 44 to 48 hours of incubation at 37°C, the
eggs were chilled and the allantoic fluid collected and centrifuged at
800xg for 15 minutes at 5°C to remove the coarser particles. The
virus preparation was stored at -60°C in sealed glass ampules.
Assay. The NDV was titrated either by agar or methyl cellulose

31
overlay technique using chick embryo cultures. With the agar overlay,
the cell cultures were incubated for 46 to 48 hours; and with methyl
cellulose, the incubation period was 72 hours. The other details of
the assay system are the same as for SFV (vide supra).
Vesicular stomatitis virus
Preparation. Vesicular stomatitis virus was prepared by inocu¬
lating the allantoic cavity of 11-to 12-day-old chick embryos with 200
pfu in 0. 1 ml. The incubation period was 36 to 40 hours. The other
details of production and assay were similar as described above for
NDV (vide supra). Cultures were incubated for 48 hours before staining.
Assay for Neutralizing Antibodies Against SFV
A plaque inhibition test was performed for the detection of anti¬
bodies against Semliki Forest virus in rabbit antiserum. The method
consisted of employing a constant amount of antiserum with several
different dilutions of virus. The various dilutions of Semliki Forest
virus in 0. 5 ml were mixed with 0. 5 ml of antiserum and incubated
at 37°C for 60 minutes. Thereafter, the virus-antibody complex was
centrifuged at 800xg for 15 minutes. The supernatent fluid was used
for Semliki Forest virus assay (vide supra). The virus preparation,
incubated with normal rabbit serum, served as a control. The batch
of Semliki Forest virus antiserum employed neutralized nearly 99%
of SFV. For example, it reduced the titer of SFV from 8 x 10 pfu/ml
to 9 x 10^ pfu/ml.
Production and Assay of Interferon

Chick interferon
32
Production. The method employed for the production of interferon
was that of Ruiz-Gomez and Isaacs (1963a) with some modifications.
The growth medium was decanted from 32-ounce, 48-hour-old chick
embryo cell monolayers. The monolayers contained nearly 35 x 10^
cells per bottle. The cells were infected with SFV at an input multi¬
plicity of about 0. 1 pfu/cell, and to each bottle was added 30 ml of
maintenance medium. The bottles were usually incubated for 24 hours
at 37°C. The medium containing interferon was harvested and heated
at 65°C for 30 minutes to inactivate the virus. Some batches of inter¬
feron preparations were centrifuged at 120, OOOxg for 3 hours to re¬
move most of the inactivated virus particles. The interferon prepa¬
rations were stored at -20°C.
Assay. The procedure followed for the assay of chick interferon
was that of Lindenmann and Gifford (1963b). Growth medium was de¬
canted from CE cell monolayers and then various interferon dilutions
and vaccinia virus (usually 200 pfu) were dispensed in a final volume
of 2 ml per bottle. Control cultures received the same amount of
vaccinia virus in MM without interferon. Usually 4 bottles were em¬
ployed for each dilution of interferon. The cultures were incubated
for 46 to 48 hours, stained with crystal violet and plaques enumerated
as described in the vaccinia virus assay system (vide supra). The
plaque depressing dose^Q, the amount of interferon preparation in
microliters which depressed the plaque number to 50% of the control,

33
was calculated according to the method of Lindenmann and Gifford
(1963b).
L cell interferon
Production. The mouse interferon was produced in L cell mono-
£
layers in 32-ounce bottles containing 30-35 x 10 cells per bottle. The
growth medium was removed and cells were infected with NDV at a
multiplicity of about 100 pfu/cell. The virus was allowed to adsorb
at room temperature for 60 minutes with frequent rocking of the
bottles. Thereafter, the cell cultures were washed with Gey's BSS
and 50 ml of Eagle's MEM with 10% calf serum was added to each
bottle. The cultures were incubated at 37°C for a period of 24 hours.
The medium containing interferon was then harvested, pooled and
centrifuged at 800xg for 20 minutes to remove cellular debris. The
interferon preparation was dialyzed against 100 volumes of 0. 001 N
HC1 buffer at pH 2 for 5 days with two changes of buffer during this
period and finally against two changes of Gey's BSS for a further
12-to 24-hours to restore the pH to neutrality. In some experiments,
this preparation was centrifuged for 3 hours at 120, OOOxg to sediment
most of the virus particles. The supernatent fluid was distributed
into glass ampules, sealed and stored at -20°C.
Assay. The interferon preparations were assayed on 24 hour-old
mouse L cell monolayers in 2-ounce plaque bottles. The various di¬
lutions of interferon were made in Eagle's MEM with 10% calf serum.
The medium was drained from the bottles and 2 ml of each dilution

34
was added to each of 4 plaque bottles. After overnight incubation at
37°C, the interferon dilutions were removed and the cell sheets were
washed twice with 5 ml of MM. VSV in 0. Z ml volumes containing
100 to 200 pfu was added to each bottle. The VSV assay and calcula¬
tion of PDD ^ units have been previously described (page 32).
Heat Inactivation of SFV
The kinetics of inactivation of SFV was studied. The stock SFV
suspension was thawed and incubated at 37°C. At various time
intervals, an aliquot was removed and frozen at -60°C. All the sam¬
ples were assayed for their residual virus titer as described under
the assay of SFV (vide supra). The titer of partially inactivated virus
was not appreciably changed by one freeze and thaw cycle. Figure 1
shows a representative study of the inactivation of SFV at 37°C. The
decrease in virus titer with respect to time of incubation at 37°C was
exponential. The rate of inactivation of Semliki Forest virus was one
log (90%) decrease in virus titer per 3. 2 hours of incubation at 37°C.
10
When the stock virus, consisting of 10% infected mouse brain suspen¬
sion, was centrifuged at 15, OOOxg for 15 minutes before incubation at
37°C, the rate of inactivation was similar.- Four lots of stock virus,
prepared in a similar manner, were found to be inactivated at a rate
of one log in 3. 2 hours +10 minutes.
e10
Incorporation of Uridine into RNA by Chick Embryo Cells
Measurement of uridine incorporation into CE cell cultures was
. 3
achieved by treating the cells at different time periods with H-uridine.

35
Figure 1. Heat inactivation of Semliki Forest virus at 37°C. Aliquots
we,re removed at various times during incubation and
assayed for residual virus.

36
Plaque bottles, containing approximately 4 x 10^ cells, were em¬
ployed. Growth medium was decanted and the monolayers were washed
twice with 2 ml of modified maintenance medium (MMM). The cell
cultures after the appropriate treatment (infected or noninfected,
with or without actinomycin D, with or without interferon treatment)
were supplied with 2 ml of MMM and incubated at 37°C. At different
time intervals, 0. 3 ml MMM containing 20 pC of H-uridine and
_5
3. 75 x 10 M each of thymidine and deoxycytidine was added to each
bottle. Presence of thymidine and deoxycytidine prevented the in¬
corporation of uridine into DNA. The yeast extract was omitted from
the maintenance medium because it contains nucleotides which com¬
pete with uridine for incorporation into RNA. The cell cultures were
replaced in the incubator at 37°C and bottles were gently rocked every
3
10 minutes to facilitate the even distribution of H-uridine on the cell
monolayer. To stop the incorporation, 0.1 ml volume of cold uridine
(5 x 10 M) was added and bottles were immediately placed into an
ice bath. The medium was drained from the bottles and the cell mono-
layers were washed three times with cold 5% perchloric acid (PCA).
The cold extraction was done twice more with 5 ml and 10 ml of cold
5% PCA for 5 and 10 minutes respectively. The radioactivity re¬
maining in the last cold PCA extraction was within background levels.
Ribonucleic acid was hydrolyzed and extracted in 2 ml of 5% PCA by
heating the cell cultures for 30 minutes at 80°C in a water bath.
Radioactivity Measurement
For uridine incorporation studies, 0.2 ml of hot PCA extract

was placed into 20 ml screw-capped, glass vials (Packard) con¬
taining 10 ml of scintillation fluid. Samples were counted twice in a
Packard "Tri-carb" liquid scintillation spectrometer for 10 minutes
and values were expressed as counts per minute.
Uridine Uptake
An experiment was performed to measure the, optimum incorpo¬
ration of uridine.into RNA by CE cells employing different concen¬
trations of uridine. The experimental procedure was the same as
described above. Figure 2 shows the incorporation of H-uridine at
various concentrations when a pulse labelling period of 30 minutes
was employed. The incorporation of H-uridine into cellular RNA in
creased almost linearly up to about 20 p C concentrations, but there
was no further increase in incorporation when higher concentrations
were employed. Uridine incorporation was also determined when
the cell cultures were exposed to uridine for different time periods.
In this experiment, the cell cultures received 15 P C of H-uridine,
but the pulse labelling period was 15, 30, 45 and 60 minutes. Fig¬
ure 3 shows the results of such an experiment. The rate of incor¬
poration was linear throughout the period of labelling.
An experiment was performed to determine the effect of various
3
concentrations of calf serum in MMM on H-uridine incorporation in
CE cell cultures. The cell cultures were exposed to 20 P C for 30
minutes. It is apparent from Figure 4 that incorporation of H-uri¬
dine into cellular RNA was considerably increased in the presence of

38
u i ¡ I I I ! I
0 5 10 15 20 25 30
CONC. (¿¿C) OF URIDINE
3
Figure 2. H-uridine incorporation in uninfected chick embryo cell
cultures exposed to various concentrations of ^H-uridine
and incubated at 37°C for 30 minutes.
I

39
O i i ¡ i i
O 15 30 45 60
MINUTES AT 37°C
3
H-undine incorporation in uninfected chick embryo
cell cultures exposed to 15 PC of ^H-uridine for
various periods of time.
Figure 3.

CPM x 1,000 / CELL CULTURE
40
12.0 —i
10.5-
9.0-
75-
with 2 /¿g/ml of actinomycin D
without actinomycin D
Figure 4. Effect of calf serum on H-uridlne incorporation of
uninfected chick embryo cell cultures with or with¬
out actinomycin D. The cell cultures were exposed
to 20 PC of ^H-uridine for 30 minutes.

41
calí serum. The optimum uptake of uridine was found when cell
monolayers were maintained in MMM with 2% calf serum. There
was no significant increase in uridine incorporation when a higher
concentration of calf serum was present in MMM. The data for the
uridine incorporation in presence of actinomycin D will be discussed
in the next section.
Since in most of the experiments the CE cell monolayers were
maintained in MMM for 12 hours or longer, an attempt was made to
determine the extent of uridine incorporation into RNA in cell cultures
which had been incubated at 37°C for various time periods. As seen
in Figure 5, maximum incorporation of uridine occurs when the cells
have been incubated for 4 hours. After 6 hours of incubation at 37°C,
there was a slight decrease in uridine incorporation at each successive
interval of measurement. After 12 hours of incubation, uridine in¬
corporation was reduced by 15 to 20% relative to that at 4 hours.
Inhibition of RNA Synthesis by Actinomycin D in CE Cell Cultures
The effect of actinomycin D on cellular RNA synthesis was de-
termined by measuring H-uridine incorporation in CE cell cultures
exposed to different concentrations of the drug in 2 ml of MMM.
Actinomycin was permitted to remain and RNA synthesis was mea¬
sured at various intervals. As seen in Figures 6 and 7, actinomycin
D concentrations of 0. 5 pg/ml or higher inhibited more than 95% of
the cellular RNA synthesis. In another experiment, actinomycin D
(2 p g) was added to each bottle in 0. 1 ml volume with 0. 2 ml of MMM.

CPM x 1,000 / CELL CULTLHE
42
5X5-T
C— 5 MMM * 2.5% CS
^ ^ tniVtiVi
45-
35-
30
25-
20—
0
2
1 1 1 ! 1 1
4 6 S 10 12 14
TI.V.L ... hOURS
Figure 5. H-uridine incorporation, with or without calf (CS)
serum in uninfected chick embryo cell cultures. The
o
cell cultures were incubated at 37 C for various
periods and then exposed to 20 PC of H-uridine for
30 minutes.
!

PERCENT OF CONTROL
lOO-i
43
90-
80-
70 4
60 -f
50 —
I
40-
Figure 6. Inhibition of H-uridine incorporation in uninfected chick
embryo cell cultures with various concentrations of
actinomycin D. The cell cultures were exposed to 20 U C
of ^H-uridine for 30 minutes at the indicated times.

44
. 3
Figure 7. Inhibition of H-uridine incorporation in uninfe-cted chick
embryo cell cultures with various concentrations of
actinomycin D. The values plotted were obtained from
Figure 6 at 10 hours after the exposure to actinomycin D.
I

45
The bottles were incubated at room temperature for 60 minutes and
rocked gently every 10 minutes to ensure even distribution of acti-
nomycin D on cell mpnolayer. After the incubation period, each bot¬
tle was washed twice with MMM and then supplied with 2 ml of MMM
per bottle. Further incubation of these bottles was carried out at
37°C. A 30-minute period of labelling with 20 PC of ^H-urid Lne was
made at various times thereafter. Under these conditions, more
than 95% of the cellular RNA synthesis was inhibited for nearly 12
hours. However, a gradual recovery of cell monolayers from the
effect of actinomycin D was observed after 12 to 15 hours of incubation.
In the presence of 2. 5% calf serum the uptake of uridine was con¬
siderably stimulated, but the percentage inhibition of cellular RNA
synthesis by actinomycin D (2 pg/ml) was more than 95% of that of
control cell cultures (Figure 4).
Isolation and Purification of Chick Ribosomes
The crude preparation of chick embryo ribosomes was made
according to the method of Wettstein, Staehelin and Noll (1963) with
several modifications. Ten-day-old chick embryos were decapitated,
eviscerated and chilled in crushed ice. All subsequent steps were
carried out at 4° to 5° unless otherwise noted. The embryos were
forced through a "Luer Lok" syringe. The minced tissue was washed
twice with 0. 15 M NaCl and twice with RSB. Thereafter, the minced
embryos were mixed with 3 volumes of RSB and homogenized with
5 strokes of a motor-driven teflon homogenizer. Under these

conditions, most of the cells were broken, but their nuclei remained
intact when examined by light microscopy. The homogenate was
centrifuged at 800xg for 10 minutes to remove unbroken cells and
other cellular debris. To this supernatant fluid was added 150 mg
of coarse bentonite and 10% (w/v) sodium deoxycholate to a final con¬
centration of 0.2%. The suspension was again homogenized with 2-3
strokes and centrifuged at 20, OOOxg for 15 minutes to remove large
particles, such as mitochondria and bentonite. The post-mitochon¬
drial fraction in a 4 ml volume was gently added as a top layer in
10 ml centrifuge tubes previously filled with 3 ml of 0. 5 M sucrose
in RSB layered over 3 ml of 1. 8 M sucrose solution in RSB.
The samples were then centrifuged for 3 hours at 151, OOOxg in
the Beckman Model L centrifuge. The pellets were rinsed and sus¬
pended in RS buffer. The suspension was homogenized with 2 to 3
strokes of a motor-driven teflon homogenizer and clarified at low
speed (800xg for 10 minutes). This ribosomal preparation was
stored at -60°C. The whole procedure, from the death of chick
embryos to the freezing step, took 4 to 4. 5 hours.
Purification of ribosomes
The purification of ribosomes was achieved by layering 1 to 2 ml
of crude preparation on a 26 ml linear, 8 to 25% sucrose gradient.
Sucrose gradients were prepared from a standard sucrose gradient
maker. The linearity of a typical sucrose gradient, represented by
refractive index, is shown in Figure 8. The gradient tubes were

REFRACTIVE INDEX
47
Figure 8.
3750-.
c
Linearity of sucrose gradient as measured by refractive
index of each fraction with an Abbe refractometer.
¡
j ' ‘
i

48
placed in prechilled swinging buckets of SW 25.1 Beckman rotor and
centrifuged at 24, 000 rpm for 8 hours. After centrifugation, the
bottom of the tube was punctured and 1 ml fractions were
by allowing the gradient to drip through a needle. Accuracy of volume
in each fraction was achieved by putting mineral oil on the top of the
gradient in an air-tight system through a hypodermic needle. Optical
density was determined for each fraction with the Beckman DU spec¬
trometer at 258 m p and in some cases also at 280 m p . Figure 9
shows a typical profile for these gradients. The ratio of 280:258 was
1:8 as expected for ribosomes. The profile also showed the contami¬
nation of crude ribosomal preparation with a heavy and light contami¬
nant which appeared at the bottom and the top of the tube, respectively.
Initially, the S values for chick embryo ribosomes were estimated
according to the method of O'Brien and Kalf (1967). The crude ribo¬
somal preparation was layered on a 5 to 20% linear sucrose gradient
in 0. 05 M KC1 - 0. 005 M MgCl^ - 0. 001 M Tris pH 7. 6 and centri¬
fuged for 13 hours at 15, 000 rpm in the Spinco SW 25. 1 rotor. Under
these conditions, the chick embryo ribosomal peak was found in a
similar position as determined for rat liver ribosomes by O'Brien
and Kalf (1967) and was designated 80.S.
Separation of ribosomal subunits
The separation of ribosomal subunits was achieved by the method
of Fenwick (1968) with some modifications. The purified chick em¬
bryo ribosomes were obtained by the method described above. The

OPTICAL DENSITY (258 m/j.)
49
0.8
0.7-
0.6-1
Figure 9. Sucrose gradient analysis of chick embryo ribosomes.
One ml of crude ribosomal preparation was layered
on 8 to 25% linear sucrose gradient and then centrifuged
at 24, 000 rpm for 8 hours. The other details have been
described under Materials and Methods.
j \
'

purified preparation was pressure dialyzed against 100 volumes of
0.01 M NaCl - 0..QLM THs HC1, pH 7. 2 buffer for 4 hours to reduce
the volume and to remove the sucrose. One tenth volume of 4 M
NaCl was added to the dialyzed sample and incubated for 5 minutes
at 4°C according to the method of Fenwick (1968). One ml of the
NaCl treated sample was layered on 26 ml linear gradient of 8 to 25%
sucrose and centrifuged for 12 hours at 24, 000 rpm as described
above. Figure 10 shows the sedimentation profile of such a gradient.
There was very little dissociation of 80 S ribosomes into 60 S and
40 S subunits. The incubation of 4 M NaCl-treated ribosomal prepa¬
ration for 15 or 30 minutes instead of 5 minutes did not change the
sedimentation pattern significantly. In another experiment, the NaCl
concentration of ribosomal preparations was raised to 0. 2 M, 0. 3M,
0. 4M or 0. 6M. The samples were incubated for 15 minutes at 4°C
and were centrifuged in gradients containing the same concentration
of NaCl in a buffer (0. 01M Tris, pH 7. 2) for 12 hours at 24, 000 rpm.
Figure 11 shows the sedimentation pattern of ribosomes in such an
experiment. The dissociation of chick ribosomes into their subunits
was directly proportional to the NaCl concentration. However, a
sizeable portion of the ribosomes remained undissociated even in
presence of a 0. 6 M NaCl concentration. The results were strik¬
ingly different from that of Fenwick (1968) who reported complete
dissociation of HeLa cell ribosomes into their subunits in presence
of 0. 2M or more concentration of NaCl. Therefore, it appears that

OPTICAL DENSITY (258 m^)
51
0.50-,
1 ! I ! ! 1 I
0 5 10 15 20 25 30
FRACTION NUMBER
Figure 10. Separation of chick embryo ribosomes into their sub-units.
The purified ribosomal preparation was mixed with l/lOth
volume of 4 M sodium chloride and incubated at 4°C for
5, 15 or 30 minutes and then centrifuged at 24,000 rpm in a
8 to 25% linear sucrose gradient for 12 hours.

52
Figure 11. Separation of chick embryo ribosomes into their sub¬
units. The NaCl concentration of ribosomal preparation
was raised to 0. 2 M, 0. 3 M, 0. 4 M or 0. 6 M and in¬
cubated at 4°c for 15 minutes. The samples were then
centrifuged in a 8 to 25% sucrose gradient containing the
same concentration of NaCl as that of sample and
centrifuged at 24, 000 rpm for 12 hours.
\
I • '

53
chick ribosomes were more difficult to dissociate into their component
subunits than were those of HeLa cells.
Mouse liver ribosomes. The mouse liver ribosomes were iso¬
lated according to the method of Petermann and Pavlovic (1963) with
some modifications. Adult, female mice were fasted overnight. They
were anesthetized, usually in groups of five, with ether. The livers
were removed and chilled in crushed ice. The livers were minced
with a scissors and the tissue was forced through a "Luer Lok" syringe.
The minced tissue was suspended in 5 volumes of 0. 3M sucrose solu¬
tion and 150 mg of coarse bentonite preparation was added. The sus¬
pension was centrifuged at 800xg for 10 minutes and to the supernatant
fluid was added 10% (w/v) sodium deoxycholate solution to bring the
concentration to 0. 2%. The suspension was homogenized with 2 to 3
strokes and centrifuged at 10, OOOxg for 10 minutes. The pH of the
supernatant was rapidly brought to 8 using 0. IN sodium hydroxide and
centrifugation at 20, OOOxg for 15 minutes was used to remove most of
the bentonite. Thereafter, all the steps in mouse liver ribosome
isolation and purification were the same as for the chick ribosomes
(vide supra).
*
Detection of Double Stranded, Replicative Intermediate Form of SFV
An attempt was made to detect the double stranded, replicative
intermediate form of Semliki Forest virus in CE cell cultures at
different times following infection. The method of Friedman (1968)
was followed with some modifications. CE cell monolayers, con¬
taining nearly 4 x 10^ cells, were infected with a multiplicity of

54
10 pfu/cell of SFV in a volume of 0.2 ml. Each bottle also received
4 P g of actinomycin D in 0. 1 ml. The control cells received 4 pg of
actinomycin D in 0. 1 ml and 0. 2 ml of MMM with 2. 5% calf serum.
The virus was allowed to adsorb for 60 minutes at 4°C, the bottles
were rocked every 15 minutes to ensure even distribution of virus.
After the incubation period, 2 ml of cold MMM with 2. 5% calf serum
was added to each bottle and the cell cultures were left overnight at
4°C to synchronize the infection. The following morning, the plaque
bottles were transferred to a 37°C incubator for one hour. Then the
cell monolayers were washed twice with MMM, supplied with 2 ml of
MMM containing 2. 5% calf serum for each bottle and returned to the
37°C incubator.
The time when cell cultures were first transferred to the 37°C
incubator from 4°C was considered as zero time for viral replication
and RNA synthesis cycles.
At various periods of time following the infection, 20 p C of
^H-uridine in 0. 1 ml volume was added to each bottle, and cell mono-
layers were further incubated for an additional 45 minutes at 37°C.
The medium from the cell cultures was then removed, and cells
were washed three times with 5 ml of chilled phosphate buffered
saline, and twice with 2 ml of 0.1 M NaCl - 0. 01 M sodium acetate
buffer, pH 5. 1. All subsequent steps were carried out in an ice bath
unless otherwise stated. To each bottle was added 1 ml of acetate
buffer, pH 5. 1, and cells were scraped off the: glass by the use of

a rubber policeman. Usually cells from 4 bottles of each group (in¬
fected with live virus, treated with heat-inactivated virus, or control,
untreated cells) were pooled and 15 mg of coarse bentonite suspension
and 0. 5 ml of 10% SDS solution were added immediately. The RNA
extraction was carried out by adding equal volume of distilled phenol
r v.
o. .. . vu
shaking the mixture for 3 to 5 minutes at room temperature. The
emulsion was broken by centrifuging at 800xg for 15 minutes and the
aqueous layer was separated and extracted again with an equal volume
of phenol. Finally, the two phenol phases were mixed and the re¬
maining RNA extracted with 5 ml of acetate buffer, pH 5.1. The
aqueous phase of each phenol extraction was combined and centri¬
fuged at 20, OOOxg for 15 minutes to remove most of the coarse ben¬
tonite. Two volumes of cold, absolute ethanol containing 2% potas¬
sium acetate were added to the supernatant. The suspension was kept
overnight at -20°C and RNA was collected by centrifuging the sus¬
pension at 15, OOOxg for 15 minutes. The precipitate was washed once
with 70% ethanol containing 2% potassium acetate, and RNA was again
pelleted by centrifuging at 15, OOOxg for 15 minutes. The RNA pre¬
cipitate was dissolved in 0. 1 M KC1 - 0. 01 M Tris - 0. 001 M EDTA
buffer, pH 7. 1 and clarified by centrifugation at 10, OOOxg for 15
minutes. Carrier RNA from L cells was either added during the
first phenol extraction period or just before the sucrose gradient
analysis of the extracted RNA.
For sucrose gradient analysis, 1 ml (4.927 to 7.126 OD ,
260

56
units) of extracted RNA was gently layered on a 15 to 30% linear, su¬
crose gradient prepared in 0. 1 M KC1, 0. 01 M Tris and 0.001 M EDTA,
buffered at pH 7.1. The gradient tubes were placed in pre-chilled
buckets of SW 25. 1 Beckman-Spinco rotor and centrifuged for 20
hours at-22, 000 rpm. After centrifugation, one ml fractions were
collected as described under the purification of chick ribosomes.
Optical density at 260 m p was determined for each fraction and 0. 1
volume of each fraction was employed for the radioactivity mea¬
surements .
Ribonuclease Sensitivity of Viral RNA Isolated from Infected Cells
The viral RNA structure, isolated from CE cells infected with
SFV, was tested for its ribonuclease (RNAse) resistance to detect
the presence of double stranded replicative forms. The RNAse
sensitivity test was performed according to the method of Friedman
(1968). RNAse treatment was carried out either before or after the
sucrose gradient analysis of RNA extracted from infected cells. In
the latter case, the sucrose gradient fractions containing the radio¬
activity peak were pooled, pressure dialyzed against 100 volumes of
0. 1 M KC1, 0. 01 M Tris HC1 and 0. 001 M EDTA, buffered at pH 7. 1,
for 6 hours to reduce the volume and remove the sucrose from the
sample. To 0. 9 ml of RNA sample was added 0. 1 ml of RNAse solu- .
tion (20 P g/ml) in 0. 1 M KC1, 0. 01 M Tris, and 0. 001 M EDTA
buffer, pH 7.1, to bring the final concentration of the enzyme to
2 P g/ml in the reaction mixture, which then was incubated for 10

minutes at 37°C. To stop the RNAse activity, 100 mg of coarse
bentonite preparation was added just after the incubation period. The
RNA was then iinmediately extracted with phenol. Carrier RNA (L
cell ribosomal RNA) was added, and sucrose gradient analysis
was performed as before.
The ribonuclease solution, at the concentration of 2 p g/ml, was
able to completely degrade two OD_^q units of chick ribosomal RNA
when incubated for 10 minutes at 3 7°C.

RESULTS
Production of Interferon at Different Multiplicities of Infection
The effect of multiplicity of infection of Semliki Forest virus on
interferon production was studied. Figure 12 represents the 24-hour
yields of interferon in chick embryo cell cultures infected with dif¬
ferent input multiplicities of the virus. The best yield of interferon
was obtained with an input multiplicity of 0. 1 pfu/cell. When the in¬
put multiplicity of infection employed was increased to 1 pfu/cell or
more, the 24-hour interferon yields were much reduced. The reasons
for the multiplicity effect were further studied since it should provide
insight into conditions for maximal interferon production as well as
possible reasons for the variation in interferon yields.
Production of Interferon as Function of Time Following Infection
To further delineate the differences in interferon yield as a
function of multiplicity of infection, an experiment was designed to
study the production of interferon in cell cultures at various times
after infection with two different multiplicities of Semliki Forest
virus. The results are shown in Figure 13. With a multiplicity of
infection of 0.1 pfu/cell, there was a significant increase in interferon
production beyond 12 hours after infection. In other experiments,
with a multiplicity of 0.1 pfu/cell, the increase in interferon pro¬
duction continued beyond 18 hours following infection (e. g., see
58

59
Figure 12. Interferon yields at 24 hours in chick embryo cell
cultures infected with various input multiplicities
of Semliki Forest virus.

60
Figure 13. Kinetics of interferon production in chick embryo cell
cultures infected with Semliki Forest virus at multi¬
plicities of 10 or 0. 1 pfu/cell.

Figure 18). However, the interferon yield was reduced by 36 hours
after infection. When cell cultures were infected with 10 pfu/cell
of the virus, interferon production nearly ceased by 8 hours after
infection (Figure 13 and 17, and Table Z). In this experiment
(Figure .13), the 24 hour yield of interferon in cell cultures infected
with 10 pfu/cell was nearly 30% of that obtained with 0.1 pfu/cell.
This difference usually varied between 14 to 20% in other experiments
(Table 2). This data indicated that infection of all the cells (as would
be the case with a multiplicity of 10 pfu/cell) resulted in early termi¬
nation of interferon systhesis. This possibility was further studied.
RNA Synthesis in the Infected Cells
Since interferon production represents the synthesis of induced
cellular protein, the viruses which rapidly shut off host macromolecu
lar synthesis would not be expected to be good interferon inducers.
Thus, viruses like poliovirus, Mengovirus and vesicular stomatitis
virus, which are known to inhibit cellular RNA synthesis (Holland,
1963; Baltimore, Franklin and Callender, 1963; wagner and Huang,
1966), have also been reported to be poor inducers of interferon in
cell cultures ( Wagner and Huang. 1966; Burke, 1966). Therefore,
the effect of Semliki Forest virus on RNA synthesis of infected cells,
at a multiplicity which apparently inhibited interferon production,
was studied. Actinomycin D (2 Pg/ml) was employed to determine
viral specific RNA synthesis in the infected cells. Figure 14 shows
RNA synthesis in chick embryo cell monolayers infected with a

62
TABLE 2
THE EFFECT OF TWO MULTIPLICITIES OF
VIRUS ON INTERFERON PRODUCTION
Experiment
Number
|
Multiplicity
of Infection
(pfu/cell)
Interferon Yield, PDD Units/ml
D 0
Hours After Infection
S 12 â–  24
% Inhibition
at 10 PFU*
1
0.1
21. 9
36
78. 4
10
23. 6
25. 2
24. 8
68. 4
2
0. 1
12.1
37. 2
86. 6
â– 
10
14. 3
16
16. 2
81. 2
3
0. 1
9. 7
33. 2
65. 6
10
9.2
10. 1
9. 5
85. 5
* Percent inhibition compared to yield of interferon at a
multiplicity of 0.1 at 24 hours after infection.

63
Figure 14. RNA synthesis in chick embryo cell cultures infected
with Semliki Forest virus at a multiplicity of 10 pfu/cell
with or without actinomycin D. Actinomycin D sensitive
RNA was plotted as the difference between total RNA
synthesis and actinomycin D resistant RNA synthesis.

64
multiplicity of 10 pfu/cell of virus with or without actinomycin D.
There was an initial increase in total RNA synthesis which reached
a peak at 5 hours after infection. In different experiments, this
increase varied between 40 to 80% as compared to the control unin¬
fected cells. This early increase was followed by a rapid decline
in total RNA synthesis in the infected cells and by 12 hours following
infection, it was approximately 14% of that of the control cells. In
the presence of actinomycin D, RNA synthesis, presumably of viral
origin (referred to as actinomycin resistant RNA synthesis in Figure
14) started after a lag period of 2 to 3 hours and continued increasing
until 8 hours following the infection and declined thereafter. The
cellular RNA synthesis, plotted as the difference between the total
RNA and actinomycin D resistant RNA synthesis at each point, showed
a slight increase up to 5 hours after infection in some experiments
while this increase was not observed in other experiments. However,
cellular RNA (actinomycin sensitive) synthesis in the infected cells
was always lower than that of the uninfected cells. After 4 to 5
hours of infection, there was a very rapid inhibition of cellular RNA
synthesis and after 8 hours practically all the RNA synthesized was
actinomycin D resistant. Thus, interferon synthesis (Figure 13) is
terminated at about the time when cellular RNA synthesis ceases
almost completely and at the time of maximal viral RNA synthesis.
Growth Curve of Semliki Forest Virus
Replication of Semliki Forest virus was studied to correlate
viral specific RNA synthesis with the production of infectious viral

65
progeny. Figure 15 shows a growth curve of Semliki Forest virus
and the accompanying viral specific RNA synthesis in chick embryo
cell cultures infected with a multiplicity of 10 pfu/cell. The progeny
virus begins to appear after a lag period of 5 hours. Virus matura¬
tion occurred between 6 to 10 hours and was essentially completed by
10 hours following infection.
’he maximal rate of viral specific
RNA synthesis was observed at approximately 8 hours following
infection. Thus, there was a lag period of 2 to 3 hours between the
synthesis of viral specific RNA and the appearance of infectious
progeny virus in cell cultures. In the presence of 2 p g/ml of acti-
nomycin D, which suppressed nearly 95% of cellular RNA synthesis,
the virus yield was nearly twice that of cell cultures without actino-
mycin D. These results confirmed the finding of Taylor (1964) that
actinomycin does not inhibit replication of SFV.
The data, so far presented, indicates that in chick embryo cell
cultures infected with a multiplicity of 10 pfu/cell, interferon pro¬
duction ceases at the time when progeny virus synthesis was maximal
and cellular RNA synthesis was severely inhibited. Thus the cessa¬
tion of interferon synthesis in the infected cells is most probably
due to cell death.
The continued synthesis of interferon beyond 8 hours, when low
multiplicity was employed, accounts for the higher yields of inter¬
feron obtained. Two possible explanations for this continued syn¬
thesis were considered: 1) interferon induction is due to "inactive"

66
Figure 15. Correlation of viral RNA synthesis with growth of
Semliki Forest virus (with or without actinomycin D)
in chick embryo ceil cultures at a multiplicity of
10 pfu/cell.
t

67
virus particles in the virus preparation which do not inhibit host
macromolecular synthesis and thereby permit interferon synthesis
for longer periods of time, or 2) low multiplicity of infection leads
to several cycles of virus infection an4 therefore, prolonged inter¬
feron synthesis.
Interferon Production by Inactive Virus
The first possibility that interferon production is induced by the
nonreplicating virus particles present in Semliki Forest virus prepa¬
rations was considered. It is not possible to physically separate the
noninfectious particles from the infectious particles in Semliki Forest
virus preparations. Since Semliki Forest virus is readily inactivated
at 37°C, many of the noninfectious particles found may be due to
inactivation during production and preparation of the virus stock.
Therefore, we chose to increase the number of inactive particles by
incubating the virus preparation at 37°C and subsequently determine
the effect of such a virus preparation on the induction of interferon
synthesis in CE cells. The loss of infectivity followed first order
kinetics (Figure 1) and less than 5 pfu/ml remained after 24 hours of
incubation.
Figure 16 shows interferon production in chick embryo cells as
a function of time after induction. When cells were exposed to in¬
activated virus, equivalent to 0.1 pfu/cell before inactivation, the
24 hours' yield of interferon was much less than the amount induced
by an equivalent amount of live virus. Under these conditions, the

68
E
oo
Z3
O
Lf\
O
O
Q_
5 10 15 20 25
HOURS AFTER INFECTION
Figure 16. Kiaelics of interferon production in chick embryo cell
cultures exposed to heat inactivated Sen.liki Forest
virus. The cell cultures received the equivalent of
either 10 or 0.1 piu/cell of the virus.

69
heat inactivated virus induced small amounts of interferon up to 8 to
12 hours after incubation with no further increase in synthesis there¬
after. A higher yield of interferon was obtained when cells were ex¬
posed to heat inactivated virus equivalent to 10 pfu/cell. However,
the total yield of interferon, in this case, was obtained within 12 hours
after the exposure of the cells. Thus the continued synthesis of inter¬
feron for 24 hours by cells infected with low multiplicity of the virus
is apparently not due to inactive particles present in the virus prepa¬
rations. However, the data indicates that inactive virus particles
do prolong interferon synthesis for some period of time as compared
to cells infected with live virus. The kinetics of interferon production
in cell cultures exposed to live virus at a multiplicity of 10 pfu/cell or
an equivalent amount of the heat inactivated virus vvere studied (Figure
17). With live virus, interferon synthesis was again nearly completed
by 8 hours following infection, while the production of interferon con¬
tinued for 12 hours in cell cultures induced by heat inactivated Semliki
Forest virus. The continued synthesis of interferon for 4 additional
hours explains the increased yield of interferon with inactive virus.
The maximum yield of interferon with the live virus was nearly 26%
of that induced by the heat inactivated virus. Similarly, Burke and
Walters (1966) observed that interferon production was essentially
completed in 10 hours when chick embryo cells were infected by Sem¬
liki Forest virus first at 36°C for 1 hour and then at 42°C. Under
these conditions, infectious virus particles were not synthesized but
interferon production was, apparently, not affected. The reason for

INTERFERON YIELD, PDD
60
Live Virus (m = lOpfu/cell)
£
oo
50
O
LT\
40
30-
20-
10-
x x Heat Inactivated Virus (m = IOpfu equivalent/cell)
0 . " | i i M , . . â– 
0 4 8 12 ¡6 20
HOURS AFTER INFECTION
Figure 17. Kinetics of interferon production with heat inactivated
and live Semliki Forest virus with cell cultures.

the cessation of interferon synthesis by inactive virus at 12 hours in
our study is unknown. In another experiment (Figure 24),it was ob¬
served that the heat inactivated virus, equivalent to an original mul¬
tiplicity of 10 pfu/cell, did not inhibit cellular RNA synthesis of the
induced chick embryo cells up to 12 hours after exposure. At 15 hour
cellular RNA synthesis was 86% of the controls and decreased to 69%
at 18 hours after exposure (not shown in the figure). The late inhibi¬
tion could have been due to the replication of the small amount of
residual virus in the inactivated virus preparation. Termination of
interferon synthesis by 12 hours after exposure to the heat inactivated
virus, even though cellular RNA synthesis was apparently normal,
conforms to the general observation that interferon synthesis stops
at varying times after induction. Cessation of interferon production
may be due to the inhibition of interferon production by interferon
(Vilcek and Rada, 1962; Cantell and Paucker, 1963; Friedman, 1966b).
However, it is difficult to explain why interferon stops its own syn¬
thesis in 12 hours in chick embryo cell induced by SFV but does not
affect the synthesis for 40 hours in chick embryo cells induced by
ultraviolet irradiated influenza virus (Burke, 1966). Alternatively,
some cellular control mechanism may be responsible for the termi¬
nation of interferon production but evidence for such a mechanism
is lacking. The cell may also be able to destroy the inducer, thereby
preventing the continued induction.
Interferon Production in Presence of Immune Serum
When chick embryo cell cultures are infected with SFV at 0.1

pfu/cell, less than 10% of the cells are initially infected. Since in¬
fected cells produce interferon for a maximum of 10 hours, the con¬
tinuous synthesis of interferon for 24 hours under these conditions
is most probably due to the subsequent infection of the remaining
cell population by the progeny virus.
To test this possibility, the kinetics of interferon production in
chick embryo cell cultures infected with 0.1 pfu/cell of Semliki
Forest virus, with or without the addition of SFV antiserum after
virus adsorption, were studied. The progeny virus produced by the
initially infected cells should be neutralized by the immune serum
present and thus would be unable to infect the remaining cells. The
cell monolayers were infected at the desired multiplicity in 0. 2 ml
volume and incubated for one hour at room temperature. After the
incubation period, each cell culture was washed twice with 5 ml
of Gey's balanced salt solution, supplied with 2 ml of maintenance
medium and incubated at 37°C. Following 2. 5 hours of incubation,
0.1 ml of immune serum was added to some of the infected cell
cultures. Control cell cultures received 0.1 ml of normal rabbit
serum. The other details were the same as described under
Materials and Methods. Figure 18 shows the results of such an ex¬
periment. In presence of the immune serum, the total yield of
interferon was obtained within 12 hours of infection. In cell cultures
containing normal rabbit serum in place of immune serum, the
production of interferon was roughly linear for 24 hours following
the infection and was similar to that shown in Figure 13. However,

INTERFERON YIELD, PDD,n UNITS/ml
HOURS AFTER INFECTION
Figure 18. Kinetics of interferon production in presence of immune
serum prepared against Semliki Forest virus. Details
have been described in the text.

74
the amount of interferon produced for the first 8 hours was similar
in both the cases. The results indicate that continuous interferon
synthesis for 24 hours in cells infected with 0.1 pfu/cell multiplicity
of the virus is due to the subsequent infection of the cells by the
progeny virus.
Interferon Production in Actinomycin D Pretreated Cells
Several investigators have suggested that there are two different
mechanisms responsible for the appearance of extracellular inter¬
feron following the addition of various inducers. These are, i. e. , de
novo synthesis versus the release of preformed interferon (Ho and
Kono, 1965; Ho, Postic and Ke, 1968; Finkelstein, Bausek and
Merigan, 1968). Infection by viruses leads to de novo interferon
synthesis in the induced cells (Burke, 1966; Finkelstein, Bausek
and Merigan, 1968; Paucker, 1969). Actinomycin D inhibits viral
induced interferon synthesis but had no effect on interferon produced
in response to endotoxin, poly rl/rC and pyran, a carboxy copolymer
(Finkelstein, Bausek and Merigan, 1968) except when very high con¬
centrations were employed. Therefore, interferon production was
studied in cells pretreated with actinomycin D to gain information
regarding the type of interferon detected following the addition of
heat inactivated Semliki Forest virus to cell cultures. The procedure
of Gifford and Heller (1963) was essentially followed. Chick embryo
cell cultures were exposed to 0. 6 p. g/ml of actinomycin D in main¬
tenance medium at 37°C for 4 hours. Thereafter, the maintenance
medium was removed and cell cultures were washed twice with fresh

75
maintenance medium. Control cell cultures were treated in an
identical manner except that actinomycin D was not added. The cell
cultures were then exposed to the same amount of live or heat inac¬
tivated Semliki Forest virus. Other details were the same as de¬
scribed in Materials and Methods.
As shown in Table 3, induction of interferon production by live
or heat inactivated Semliki Forest virus in chick embryo cell cul¬
tures was almost completely inhibited when the cells were pre¬
treated with actinomycin D. The interpretation placed on these re¬
sults is that interferon production is induced by the live as well as
heat inactivated Semliki Forest virus. Actinomycin D, by selectively
inhibiting cellular RNA synthesis, prevented the formation of inter¬
feron specific messenger RNA and thus indirectly prevented inter¬
feron production.
Viral Specific RNA Synthesis in the Induced Cells
It has been suggested that RNA viruses induce interferon pro¬
duction by synthesizing a double stranded form of RNA which then
acts as the inducing agent (Skehel and Burke, 1968b; Hilleman, 1969;
Colby and Chamberlin, 1969). Since heat inactivated virus, though
unable to replicate but induces more interferon than live virus, the
possibility was investigated as to whether heat inactivated Semliki
Forest virus undergoes a double stranded, replicative form as a
prerequisite for interferon production or if the single stranded viral
RNA is in itself the inducing agent. Live virus infection, at a mul¬
tiplicity of 10 pfu/cell, induces much lower yields of interferon but

76
TABLE 3
INTERFERON PRODUCTION IN PRESENCE
OR ABSENCE OF ACTINOMYCIN D
Virus
Preparation
Multiplicity
of Infection
pfu/cell
Presence or
Absence of
Actinomycin D
15 Hour Inter¬
feron Yields
(PDD^q Units/ml)
Heat Inactivated
Virus
10*
■—
64. 5
Heat Inactivated
Virus
10*
+
3. 6
Live Virus
10
—
11.. 2
Live Virus
10
+
2. 0
* Equivalent amount of heat inactivated virus
Note: The virus titer at the end of 15 hours incubation was
6 to 8 x 1C)3 pfu/ml in cell cultures exposed to heat
inactivated virus.

was employed as a control to detect the double stranded, replicative
intermediate form of RNA in the infected cells. Chick embryo cell
monolayers were infected with Semliki Forest virus in the presence
of 2 P g/ml of actinomycin D. After various periods of incubation at
37UC, 20 PC of H-uridine was added for an additional 45 minutes
and the RNA was extracted as described in Materials and Methods.
Sucrose gradient analysis of viral specific RNA
The extracted RNA was gently layered on a 15 to 30% linear su¬
crose gradient and centrifuged at 22, 000 rp.m for 20 hours in a SW 25
rotor. The gradient fractions were collected in 1 ml aliquots and the
optical density measured at 260 mp and radioactivity measured in
the spectrometer. Ribosomal RNA from L cells was employed for
the estimation of S value of viral specific RNA. Sucrose gradient
analysis of the extracted RNA two hours after infection showed a peak
^H-uridine in the 26 S region with counts extending into ranges of
lower S values. When an aliquot of extracted RNA was incubated
with ribonuclease (2 P g/ml) for 10 minutes at 37°C before sedimen¬
tation, another peak of radioactivity sedimenting in the 20 S region
was revealed (Figure 19). When RNA from the infected cells was
extracted 4 or 6 hours after the infection, an additional radioactive
RNA peak sedimenting at the 45 S region, as shown in Figures 20 and
21, was also obtained. These results are similar to those of
Friedman, Levy and Carter (1966) and Sonnabend, Martin and Mees
(1967) who reported that viral specific RNA from the infected cells
can be resolved into three components. They also reported that

Fraction No.
Figure 19. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
infected for 2 hours with Semliki Forest virus.
H-uridine (20 PC) was added for 45 minutes, and
RNA was extracted. L cell RNA was employed as
carrier RNA.
Optical Density (260mp)

C PM
79
Figure 20. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
infected for 4 hours with Semliki Forest virus.
JH-uridine (20 PC) was added for 45 minutes, and
RNA was then extracted. L cell RNA was employed
as carrier RNA.
i
Optical Density (260mp)

CPM
80
Figure 21. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
infected for 6 hours with Semliki Forest virus.
o
"’H-uridine (20 pC) was added for 45 minutes, and
RNA was then extracted. L cell RNA was employed
as carrier RNA.
.
Optical Density (260mpi)

81
the RNA peak sedimenting at 45 S was infectious and corresponded to
the RNA that can be extracted from purified virus particles. The
second peak of radioactive RNA sedimenting at 26 S was not infec¬
tious but the base composition of this component was similar to that
of 45 S RNA and the RNA extracted from virus particles. The 20 S
RNA is ribonuclease resistant and thus presumably the double stranded
form of the RNA.
The RNA was also extracted at 2, 4 and 6 hours after exposure
of the cell cultures to heat inactivated Semliki Forest virus equiva¬
lent to 10 pfu/cell. Sucrose gradient analysis of these RNA extracts
did not reveal peak of radioactivity at any of the three regions (Figure
22 and 23) indicating that viral specific RNA was not synthesized in
the cells exposed to the heat inactivated virus. These cell cultures
had a titer of 5. 3 x 10^ pfu/ml when assayed for infectious virus at
12 hours after infection. The titer of live virus under similar con-
ditions usually varied between 8 x 10 to 2 x 10 pfu/ml.
Pulse -labelling method
Viral specific RNA synthesis in chick embryo cells exposed to
live as well as heat inactivated Semliki Forest virus was also
studied by the H-uridine pulse labelling method. The induced cells,
at various periods after the induction, were exposed to 20 p C of
H-uridine for 45 minutes and RNA was extracted with perchloric
acid as described in Materials and Methods. Chick embryo cell
monolayers infected with live virus, at a multiplicity of 10 pfu/cell,
showed the synthesis of viral specific RNA which progressively

82
Fraction No.
E
CD
vO
CNJ
LO
C
CD
O
Figure 22. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
exposed for 2 hours with heat inactivated Semliki
Forest virus. H-uridine (20 PC) was added for
45 minutes and RNA was then extracted. L cell
RNA was employed as carrier RNA.

83
Figure 23. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
exposed to heat inactivated Semliki Forest virus
for 4 or 6 hours. ^H-uridine (20 PC) was added
for 45 minutes, and RNA was then extracted. L
cell RNA was employed as carrier RNA.

84
increased with respect to lime after the infection. Most of the RNA
synthesized in the infected cells from 9 hours onward was viral
specific (Figure 24). However, no viral specific RNA synthesis
was detected when cells were exposed to the heat inactivated virus
equivalent to 10 pfu/cell. Virus directed RNA synthesis also could
not be detected when cells were exposed to the heat inactivated virus
3
for 7 hours in the presence of 20 p. C of H-uridine.
Association of Interferon with Ribosomes
The antiviral activity of interferon seems to depend upon the de
novo synthesis of another protein in the exposed cells (Taylor, 1964;
Lockart, 1964; Levine, 1966). This new protein, often referred to as
translation inhibitory protein (TIP), apparently attaches to the ribo¬
somes and renders these ribosomes incapable of translating the viral
messenger RNA (Marcus and Salb, 1966; Carter and Levy, 1967).
However, it is not known whether TIP alone, or in conjunction with
interferon, produces the change in ribosomal function. The study
of ribosomes from cells exposed to interferon alone or to interferon
in the presence of actinomycin D cannot answer this question since
in the former case the two factors cannot be distinguished from one
another and the latter case indicates that interferon alone is not
able to mediate the antiviral action in the cells. However, if inter¬
feron does play a direct role in the maintenance of virus resistant
state in the cells, it is reasonable to assume that it might combine
with ribosomes for the expression of antiviral activity.

Uridine Uptake (percent of control)
85
© o Uninfected Cells and
Actinomycin D(2mg/ml)
Figure 24. RNA synthesis in chick embryo cell cultures exposed
to heat inactivated or live Semliki Forest virus in
presence or absence of actinomycin D.

86
To investigate this possibility, an experiment was performed in
which chick embryo and mouse liver ribosomes were examined for
their ability to combine with a chick interferon preparation which
had been partially purified by centrifugation at 105,000xg for 3 hours.
Chick embryo ribosomes (5 OD units) were mixed with 10 PDD
units of chick interferon and incubated for 30 minutes in a 37°C water
bath. After incubation, the ribosomes were removed by centrifuga¬
tion at 105, OOOxg for 3 hours and the supernatant fluid was assayed
for residual interferon activity. Similar experiments were performed
employing chick interferon with mouse liver ribosomes, and also
using L cell interferon with mouse liver ribosomes or with chick
embryo ribosomes. As presented in Table 4, the results show that
almost all of the interferon was recovered after incubation with ribo¬
somes indicating that chick interferon did not bind specifically to
chick embryo ribosomes or nonspecifically to mouse liver ribosomes.
These studies were extended to include L cell interferon which did
not combine with either mouse liver ribosomes or with chick embryo
ribosomes.
However, the possibility exists that interferon may bind to one
of the ribosomal sub-units but the attachment site is no longer avail¬
able when '60 S' and '40 S' sub-units combine to form the'80 S' ribo¬
somal unit. To test this possibility, chick embryo ribosomes were
separated into '60 S' and '40 S' sub-units as described under Materials
and Methods. Two OD units of chick embryo '40 S' ribosomal

TABLE 4
ASSOCIATION OF CHICK INTERFERON WITH MOUSE AND CHICK EMBRYO RIBOSOMES
Source of Interferon
Source of Ribosomes
PDD50 Units of
Interferon Added
PDD50 Units of
Interferon Recovered
Chick Embryo Cells
Chick Embryo (80S)
10
9. 7
Chick Embryo Cells
Mouse Liver (80S)
10
10. 4
L Cells
Mouse Liver (80S)
10
10. 3
L Cells
Chick Embryo (80S)
10
10. 1

sub-units were mixed with 10 PDDC„ interferon units, incubated at
D U
37°C for one hour and ribosomes removed by centrifugation. Simi¬
larly, chick interferon was also incubated with '60 S' sub-unit as
well as with '80 S' ribosomes. As shown in Table 5, complete
recovery of interferon was made after incubation either with '60 S'
sub-unit or with '40 S' sub-unit. Thus the results indicate that
interferon does not combine with either of the ribosomal sub-units.

89
TABLE 5
ASSOCIATION OF CHICK INTERFERON WITH RIBOSOMAL SUBUNITS
Source of
Interferon
Chick
Ribosomes
PDD^q Units of
Interferon Added
PDDjq Units of
Interferon Recovered
Chick
60 S
10
9. 9
Chick
l
40 S
10
00
O'
Chick
80 S
10
1 0.1

DISCUSSION
Interferon Production
The dependence of interferon production, in various cell-virus
systems, on multiplicity of infection has been well documented in
the literature. In many cases, lower multiplicities of infection
induce maximum amounts of interferon while higher multiplicities of
infection result in reduced yields of interferon (De Mayer and De
Somer, 1962; Gifford, 1963; Aurelian and Roizman, 1965; Toy and
Gifford, 1967a). In the present study, the highest yield of interferon
was obtained when CE cell cultures were infected with SFV at a
multiplicity of about 0.1 pfu/cell. Multiplicities of 1 pfu/cell or more
of this virus resulted in considerably lower production of interferon.
These findings are in agreement with those of Toy and Gifford (1967a).
When two different multiplicities, i. e. , 10 pfu/cell and 0. 1 pfu/
cell were employed, not only were the 24-hour yields different but
there were striking differences in the appearance of interferon in CE
cell cultures as a function of time after induction. The increase in
the yield of interferon between 8 to 24 hours after the infection was
roughly linear in cultures infected with a multiplicity of 0.1 pfu/cell.
The rate of appearance of interferon in culture medium during this
period varied between 0. 9 to 1. 1 PDD units/hour/10^ cells in
90

91
different experiments. A similar linear increase in interferon pro¬
duction has been reported by Heller (1963) and Toy and Gifford (1967b)
employing chikungunya virus at a multiplicity of 0.1 pfu/cell in CE
cell cultures. However, the total amount of interferon produced by
SFV during the 24-hour period, as well as its rate of appearance,
was lower than that of the chikungunya-CE cell system, When inter¬
feron was induced with SFV at 10 pfu/cell, the total yield of inter¬
feron varied between 14 to 30% of that induced by 0. 1 pfu/cell, and all
of the interferon was produced within 10 hours following the infection.
In the current study, when yields were measured 36 hours after
infection by SFV, there was some reduction in the amount of inter¬
feron as compared to the 24-hour yield. Similar results have been
reported by Toy and Gifford (1967b) in the chikungunya-CE cell sys¬
tem. They reported that the disappearance of interferon from the
medium was due to its adsorption by the cells since there was no
evidence for the presence of proteolytic enzyme(s) in the culture
medium to account for the loss. Moreover, the initial rate of dis¬
appearance of interferon was comparable to the rate of loss observed
when the interferon was added to the uninfected cell cultures. A
similar mechanism probably accounted for the decrease in yields
of interferon at the 36 hours post-infection period in cells infected
with SFV.
The mechanism of inhibition of interferon production by viruses
under certain conditions has not been well defined. However, in
several cell-virus systems, the production of interferon is dependent

92
on the continuation of macromolecular synthesis, especially RNA,
during the initial stages of viral infection. The necessity of RNA
synthesis also becomes apparent by considering the available data
which suggest that synthesis of interferon occurs de novo and requires
the formation of a new messenger RNA from information encoded
into the cellular DNA (reviewed by Burke, 1966),
Aurelian and Roizman (1965) reported that interferon production
was partially inhibited in dog kidney cultures infected with 100 pfu/cell
of herpes virus (MPdk~strain) which caused a rapid decline in cellular
RNA synthesis. However, in cell cultures infected with 12 pfu/cell of
this virus, the decrease in the cellular RNA synthesis was delayed
up to 5 to 6 hours after infection and 70% more interferon was pro¬
duced. The interpretation of this data is difficult since the RNA syn¬
thesis determinations included both viral messenger RNA formation
as well as cellular RNA synthesis and it is difficult to correlate
interferon production with the severity of the inhibition of cellular
RNA synthesis in the infected cells.
Wagner and Huang (1966) observed that interferon synthesis was
inhibited in NDV induced Kreb-2 cells when superinfected with viru¬
lent VSV at a multiplicity of 50 pfu/cell. Vesicular stomatitis virus
infection caused a rapid decline in the rate of cellular RNA synthesis.
Interferon synthesis was inhibited when Kreb-2 cells were superin¬
fected within 4 hours after NDV infection and not at 6 or 8 hours.
Although VSV caused considerable inhibition of cellular RNA syn¬
thesis, it is somewhat difficult to relate this effect to inhibition of

93
interferon synthesis, not only because of the complexities of the
dual infection but also because VSV inhibits cellular DNA and protein
synthesis.
In the present study, the differentiation between viral and cellu¬
lar RNA synthesis was achieved by employing actinomycin D which
selectively inhibits cellular RNA synthesis but has no inhibitory effect
on SFV replication or viral RNA synthesis (Taylor, 1964). The con¬
centration of actinomycin D (2 Ug/ml) employed has been reported to
have no effect on cellular DNA synthesis (Kirk, I960). Under the
conditions employed, i.e.,when interferon production was minimal
(10 pfu/cell of SFV), the total RNA synthe sis at 7 hours after infection
equalled that of control cells but cellular RNA synthesis was suppressed
to 30% of that of uninfected cells (Figure 14). Interferon production
ceased at a time when virus synthesis was near completion and cellu¬
lar RNA synthesis was declining at a rapid rate. These results are
in variance with those of Taylor (1965) who reported a nearly linear
increase in interferon production for 26 hours in chick embryo cells
infected with 10 pfu/cell of Semliki Forest virus. The author re¬
ported that the total RNA synthesis of infected cells was similar to
controls at 6 to 7 hours, at which time 70 to 90% of the RNA synthe¬
sized in the infected cells was of viral origin and indicated a pro¬
found inhibition of cellular RNA synthesis. The discrepancy between
Taylor's result and those reported here remained unexplained.
In the present studies, interferon synthesis was barely detect¬
able at 5 hours and continued up to 8 to 10 hours post infection

94
indicating that RNA synthesis was not shut off early enough to com¬
pletely prevent the interferon specific messenger RNA from being
synthesized. Thus, infected cells must be producing both virus and
interferon in the initial stages of infection. Termination of interferon
synthesis at about 8 hours after infection, even after the formation
of Interferon specific messenger RNA, apparently contradicts the
finding of Wagner (1965) and Wagner and Huang (1966). They found that
messenger RNA for interferon, in Newcastle disease virus induced
Kreb-2 carcinoma cells, is formed early in the induction period and
is relatively stable for nearly 10 hours. However, SFV is a cytopathic
virus and cell killing would terminate interferon synthesis even
though m-RNA was still present. NDV infection of Kreb-2 carcinoma
cells does not lead to cell death and thereby permits the continued
functioning of interferon specific messenger RNA. Furthermore,
the interferon specific messenger RNA induced by Semliki Forest
virus in chick embryo cells may be less stable. This possibility is
supported by the observation that interferon synthesis, induced by
the heat inactivated SFV, stopped within 12 hours after induction.
Similarly, Burke and Walters (1966) reported that interferon syn¬
thesis was completed in 10 hours in chick embryo cells induced by
Semliki Forest virus under restrictive conditions when virus replica¬
tion did not occur, but they used elevated temperatures which may
i
have other effects on the stability of interferon specific messenger RNA.
A prolonged synthesis of interferon in cells infected with SFV
at a multiplicity of 0.1 pfu/cell was observed and found to be due to

95
subsequent infection and induction of additional cells by progeny virus.
Interferon production stopped at 12 hours after the induction when
cells were infected with 0.1 pfu/cell and immune serum against Semliki
Forest virus was added before progeny virus appeared. In cell
cultures infected under identical conditions but receiving normal rab¬
bit serum, interferon production continued to increase beyond 18
hours after infection. The obvious explanation for such differences
in interferon production is that approximately 10% of the cells were
initially infected. The cells exposed to live virus produced infectious
virus particles and small amounts of interferon. After the first repli¬
cation cycle, the remaining cells were infected and induced further
synthesis of interferon which continued beyond 18 hours. However, in
cell cultures containing immune serum, the progeny virus was neu¬
tralized and the remaining cells were not infected and thus an earlier
termination of interferon synthesis was observed. Additional evidence
for this hypothesis is provided by the observation that cell cultures
exposed to heat inactivated virus equivalent to 0.1 pfu/cell induced
interferon production up to 7 to 8 hours and this yield was comparable
to the live virus at that time under identical conditions. However,
further synthesis of interferon does not occur because heat inactivated
virus is unable to replicate and thus the majority of the cells remain
uninfected and uninduced.
An intriguing question is why more interferon synthesized in
cell cultures infected at a lower multiplicity than with a high mul¬
tiplicity. The reason for the increased yield of interferon is not

96
obvious since the same number of cells were employed in both cases
and eventually all the cells would be infected and induced. At this
low multiplicity of infection, more than one cycle of virus replication
was required for the infection of all the cells which would result in
prolonged interferon synthesis but would not explain the increased
yield, However, it has been reported by several investigators
(Isaacs and Burke, 1958; Friedman, 1966b) that pretreatment of the
cells with interferon before the addition of virus enhances the sub¬
sequent production of interferon. Thus, at lower multiplicity of
infection, interferon produced in the first cycle could enhance the
production of interferon in cells infected during the subsequent
cycle of viral replication which may result in higher yield of inter¬
feron. The other possibility is that inactive virus particles in the
virus preparations also induce interferon production, and since such
virus particles may not inhibit host macromolecular synthesis, inter¬
feron production would be prolonged and enhanced. Therefore, the
effect of heat inactivated virus on interferon and host macromolecular
synthesis were studied.
Cell monolayers exposed to heat inactivated virus equivalent to
10 pfu/cell produced considerably higher amounts of interferon than
the cell cultures infected with an identical amount of live virus. The
total yield of interferon was obtained within 12 hours of the exposure
i
of the cell cultures to heat inactivated virus. This higher yield of
interferon with heat inactivated virus seems to be due to an enhanced
as well as prolonged synthesis of interferon. These effects may be

97
explained by the observation that heat inactivated virus does not shut ,
off cellular RNA synthesis for the first 15 hours after exposure of the
cells. However, even with heat inactivated virus, interferon syn¬
thesis is eventually terminated within 1Z hours. This may be due to
stability characteristics of interferon specific messenger RNA and/or
degfcruetien e£ the itidueer- in the cell. Interferon may algo be respen*
sible for the inhibition of its own synthesis. It has been reported
(Vilcek and Rada, 1962; Paucker and Cantell, 1963; Lockart, 1963;
Taylor, 1964) that large amounts of interferon in cell culture would
inhibit subsequent interferon production when such cells are exposed
to an interferon inducer. Thus, such a mechanism could account
for the termination of interferon synthesis in cells exposed to heat
inactivated virus.
The type of induction initiated by the heat inactivated virus seems
identical to that of live Semliki Forest virus since interferon produc¬
tion, in both cases, was almost completely inhibited by pretreatment
of cells with actinomycin D. Similar results have been reported by
Gifford and Heller (1963) who found that heat-inactivated virus, as
well as live chikungunya virus, induced interferon synthesis in
chick embryo cells which was inhibited by pretreatment of cells by
actinomycin D. These results suggest that heat inactivated as well
as live SFV elicit interferon production by inducing de novo protein
synthesis in chick embryo cells. The presence of actinomycin D
inhibited the formation of interferon specific m-RNA and thus pre¬
vented the production of interferon in these cells.

98
Several workers (Hilleman, 1969; Colby and Chamberlin, 1969)
have suggested that RNA viruses induce interferon production by
synthesizing double stranded, viral RNA which then acts as the
inducing agent. The supportive evidence for this suggestion comes
from the observation that double stranded, but not single stranded,
RNA from synthetic (Field et al. , 1967, 1968; Falcoff and Bercoff,
1968; Vilcek et al. , 1968) as well as from natural sources (Field et al. ,
1968; Hilleman, 1969; Falcoff and Falcoff, 1969) induce interferon pro¬
duction in various cell systems. However, in our experiments, viral
specific, double stranded RNA could not be detected in the cells in¬
duced by heat inactivated Semliki Forest virus. It is concluded,
therefore, that input, single stranded, viral RNA is able to induce
interferon production and the formation of double stranded RNA is
not essential for the induction event. Dianzani (1969) has reported
that in L cells, infected with Newcastle disease virus and simultane¬
ously treated with cyclohexamide or p-flurophenylalanine, interferon
was produced after the removal of protein synthesis inhibitor and the
addition of actinomycin D. It was concluded that in the presence of
protein synthesis inhibitor no viral replication occurred but inter¬
feron specific messenger RNA was synthesized; therefore, parental
viral RNA or protein(s) or both were the inducers for interferon syn¬
thesis. However, these results are apparently contrary to the find¬
ings of Skehel and Burke (1968b) who found that Semliki Forest virus
induced interferon production in chick embryo cell cultures at 4Z°C

99
provided the virus was allowed to first incubate with the cells for a
1 to 2 hour period at 36°C. Under these conditions, progeny virus
was not synthesized but during the cell-virus interaction at 36°C for
1 to 2 hours, the infected cells synthesized a mixture of double and
single stranded viral RNA and it was suggested that synthesis of the
double §tranded RNA by the virus may be the initial step required
for the induction of interferon synthesis. However, it is possible
that mechanisms of interferon induction by viruses and double stranded
RNA may not be the same. There are certain differences in the pro¬
cess of interferon production by these two groups of inducing agents
which support the concept that double stranded RNA preparations
release preformed interferon (Finkelstein, Bausek and Merigan, 1968;
Youngner and Hallum, 1968) whereas viral induced interferon produc¬
tion requires de novo protein synthesis (Wagner and Huang, 1965;
Burke, 1966; Paucker, 1969). The induction of interferon formation
by double stranded RNA from several bacterial and animal viruses
(Field et al. , 1968; Hilleman, 1969) apparently indicates that this may
be the process by which RNA viruses induce interferon production.
But the possibility exists that interferon induction by viruses include
both de novo synthesis as well as the release of preformed interferon
and that production of interferon by various double stranded RNA
preparations represent the latter response only.
Several investigators have found that certain single stranded RNA
preparations are capable of inducing interferon production (Baron et
al. , 1968; Finkelstein, Bausek and Merigan, 1968). It has been

100
reported by De Clercq and Merigan (1969a) that the single stranded
homopolymers, polyriboguanylic acid, polyriboinosinic acid and
polyriboxanthylic acid at neutral pH and polyriboadenylic acid and
polyribocytidylic acid at an acid pH can induce small amounts of
interferon. The inducibility characteristic was related to the stability
of the secondary structure of homopolymers as indicated by their
high temperature of melting (Tm) values. Therefore, the other pos¬
sible interpretation of our data is that the mechanism of interferon
induction by viruses and RNA preparations may be the same but single
stranded, viral RNA acts as an efficient inducing agent. The induci¬
bility characteristic of viral RNA may be related to one or more of
the physico-chemical properties of viruses. These may include
1) better penetrability of virion and thus its RNA into the cells;
2) probable protection of RNA by viral coat against ribonucleases;
3) a possible stable, secondary structure of viral RNA; and 4) an
inherent, higher affinity of viral RNA for a hypothetical receptor site
which recognizes the inducer and triggers on the mechanism for
interferon production inside the cell.
Lockart et al. (1968) employed temperature sensitive mutants of
% —— ——
Sindbis virus to investigate viral event(s) necessary for the induction
of interferon synthesis in chick embryo cells. Three RNA + strains
(able to synthesize viral RNA but not infectious virus particles at
42°C) produced interferon at 29°C but not at 42°C. Since the defect
in these temperature sensitive mutants apparently result from the
synthesis of viral proteins which are not functional at restrictive

101
temperatures, it was suggested that viral protein(s), or the process
for which they are necessary, is required for interferon production.
The possible role of viral protein(s) synthesis in the process of
interferon induction by heat inactivated Semliki Forest virus, can¬
not be excluded in our experiments. This is because the exact
mechanism of heat inactivation of viruses is not well understood.
The kinetic data obtained by Ginoza (1958) suggested that the spon¬
taneous decay of tobacco mosaic virus--ribonuclec acid (TMV-RNA)
is related to the inherent susceptibility of its phosphodiester bonds
to heat induced hydrolysis. However, the phosphodiester bonds of
single and double stranded DNA are stable under thermal treatment
at neutral pH but in either form is relatively sensitive to depurination
(Ginoza et al. , 1964). The TMV-RNA was found to be 30 times more
sensitive to heat inactivation at 37°C than single stranded DNA of
comparable size. On the basis of such observations, it has been
suggested (Ginoza, 1968) that the inactivation of viruses at lower
temperature is due to some simple covalent bond scission occurring
in the nucleic acid. It is possible, however, that part of the multi-
cistronic genome of the heat inactivated virus may remain functional
and act as its own messenger RNA so that viral protein(s) synthesis
in chick embryo cells exposed to heat inactivated Semliki Forest
virus can not be ruled out.
The results show that, under the experimental conditions em¬
ployed, interferon does not combine specifically with homologous

ribosomes or nonspecifically with heterologous ribosomes. The
failure to detect association could have been due to the presence of a
greater number of interferon molecules than ribosomes, especially
if only one interferon molecule could react with one ribosome. In
such a case, only a fraction of interferon molecules would attach to
the ribosomes and this small loss would not be detectable in the in-
13
terferon assay. However, the number of ribosomes (4.4 x 10
molecules/5 OD^gg units) far exceeded the number of interferon
molecules (9-0 x 10^/10 PDD^ units) in the experiments and thus
excluded such a possibility. The calculations for the number of
ribosome molecules were based on the extinction coefficient (É^°
20
135 + 3/cm at 260 mp ) and molecular weight (5. 0+ 0.2 x 10^) of
guinea pig liver ribosomes as described by Tashiro and Siekevitz
(1965). These values should be applicable for mouse and chick embryo
ribosomes since the S values of the ribosomes from all three animal
species are comparable. The calculations for interferon molecules
were based on the observation that each PDD unit protects nearly
2 x 10^ cells and according to Merigan, Winget and Dixon (1965), in
most purified preparations a maximum of 4, 500 molecules of inter¬
feron would be required to protect one cell.
These results suggest that interferon does not directly affect
the ribosomal function which is necessary for the viral resistant state
in the cell. Similarly, Heller (1968) observed that the presence of
interferon did not affect the rate of amino acid incorporation into a
cell-free protein synthesis system. It seems that the role of

103
interferon is to induce the synthesis of TIP in the exposed cells which
then combines alone with ribosomes so that these ribosomes are
capable of forming polysomes with cellular messenger RNA but bind
poorly to viral messenger RNA, thus inhibiting viral RNA directed
protein synthesis. However, it is possible that interferon does pos¬
sess the ability to combine with ribosomes and the failure to detect
such an association in the present experiments may be due to several
factors. These include 1) the temperature (37°C) and pH (7. 2) of the
mixture may not be suitable for the association of interferon with
ribosomes; 2) the interferon preparation might have some factor(s)
which inhibited the attachment of interferon to the ribosomes; 3) it
has been reported (Brown and Doty, 1968) that washing of ribosomes
with 1 M ammonium chloride solution results in the removal of 3
proteins .and one of these proteins is required for the attachment of
f-2 RNA to the ribosomes. It is possible, therefore, that during the
isolation and purification of the ribosomes in the present experiments,
some protein(s) might have been removed and the prior presence of
these protein(s) may be essential for the attachment of interferon to
the ribosomes; and 4) finally, interferon may attach only when TIP
is already bound to the ribosomes or that TIP interferon must
interact before the complex can bind with the ribosome. The direct
role of interferon in the maintenance of viral resistant state can be
definitely established only under the conditions in which purified TIP
and interferon could be added to the ribosomes, and to analyse the

affect of each factor alone and both combined in the interaction and
translation of viral and cellular messenger RNA in vitro.

SUMMARY
Interferon production in chick embryo cell cultures infected
with different multiplicities of Semiiki Forest virus was studied;
Input multiplicities of 1 pfu/cell or more resulted in low yields of
interferon while considerably higher yields were obtained when cells
were infected with an input multiplicity of 0.1 pfu/cell. At an input
multiplicity of 10 pfu/cell, interferon production stopped at 10 hours
after the infection whereas interferon synthesis continued beyond
18 hours following infection with 0. 1 pfu/cell. Prolonged interferon
synthesis with the lower multiplicity was shown to be due to con¬
tinuous induction by progeny virus. When cells were exposed to suf¬
ficient virus to infect each cell, much less interferon was found with
live virus than with inactivated virus. It was concluded that early
termination of interferon synthesis with higher multiplicities of in¬
fection was due to the rapid and profound inhibition of cellular RNA
synthesis of the infected cells. This conclusion was based on the
evidence that Semiiki Forest virus at a multiplicity of 10 pfu/cell
caused rapid inhibition of cellular RNA synthesis so that after 8 hours
practically all the RNA synthesized in the infected cell was of viral
origin. Chick embryo cell cultures exposed to 10 pfu/cell equivalent
amount of heat inactivated Semiiki Forest virus produced consider¬
ably higher amounts of interferon with no concomitant inhibition of
105

106
the cellular RNA synthesis up to 1Z hours following the induction.
It seems that the mechanism of appearance of interferon in chick
embryo cells exposed to heat inactivated as well as live Semliki Forest
virus is due to de novo synthesis of protein in the induced cells since
the presence of actinomycin D in cell cultures completely inhibited
the appearance of interferon in both cases. No double stranded viral
specific RNA synthesis could be detected in chick embryo cells ex¬
posed to 10 pfu/cell equivalent amount of heat inactivated Semliki
Forest virus. Therefore, it was concluded that single stranded input
viral RNA in itself is capable of inducing interferon production in the
induced cells. However, the role of viral protein(s), if any, in
interferon induction could not be ascertained.
Under the experimental conditions employed, we could not detect
the association of interferon with ribosomes of homologous or hetero¬
logous species. Binding of interferon could also not be detected with
40 S and 60 S ribosomal units of the homologous species. Therefore,
it seems likely that interferon does not play a direct role in the main¬
tenance of a viral resistant state in the cells.

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Memo 227.

VITA
Rakesh Mohan Goorha was born November 6, 1940,at Gwallior,
Madhya Pradesh, India. In April, 1961, he obtained the degree
of Bachelor of Veterinary Science and Animal Husbandry
(B. V. Sc. & A. H. ) from Vikram University, Ujjain (M. P. ). He
subsequently joined the P. A. College of Animal Sciences, Mukteswar,
and received the degree of Master of Veterinary Science (M. V. Sc. )
with a major in Bacteriology and Virology in September, 1963. From
October, 1963 to December, 1964, he worked as Senior Research
Fellow of the Council of Scientific and Industrial Research in the
Department of Microbiology, M. A. Medical College, New Delhi. In
1965 he worked at the Virus Research Center, Poona, as a Senior
Research Fellow of Indian Council of Medical Research. From
January, 1966, until the present time he has pursued his work towards
the degree of Doctor of Philosophy in the Department of Microbiology,
College of Medicine, University of Florida. He was supported by
NIH Training Grant 5TI-AI-0128-10 during this period.
114

This dissertation was prepared under the direction of the
chairman of the candidate's supervisory committee and has been
approved by all members of that committee. It was submitted to
the Dean of the College of Medicine and to the Graduate Council,
and was approved as partial fulfillment of the requirements for the
degree of Doetor of Philosophy,
December, 1969

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