Studies on interferon production

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Title:
Studies on interferon production
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ix, 114 leaves : ill. ; 29 cm.
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English
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Goorha, Rakesh Mohan, 1940-
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Subjects / Keywords:
Interferons   ( mesh )
Interferon Inducers   ( mesh )
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bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

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

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University of Florida
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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














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.















TABLE OF CONTENTS


Page


ACKNOWLEDGMENTS . .

LIST OF TABLES . .

LIST OF FIGURES . .

KEY TO ABBREVIATIONS . .

INTRODUCTION .... ...

REVIEW OF LITERATURE. ... .....

Production of Interferon . .

MATERIALS AND METHODS .........


Materials . .

Methods . .

RESULTS . .

DISCUSSION ... .

SUMMARY .. .. .

REFERENCES . .

VITA . ... .


23

S. 27

58

S. 90

* 105

. 107

. 114


ii

iv

v

viii

1

4

12

23








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 62

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














LIST OF FIGURES


Figure Page

1. Heat -inactivation of Semliki Forest virus at 370C.
Aliquots were removed at various times during
incubation and assayed for residual virus. 35

2. H-uridine incorporation in uninfected chick embryo
cell cultures exposed to various concentrations of
3H-uridine and incubated at 370C for 30 minutes .. 38

3. H-uridine incorporation in uninfected chick embryo
cell cultures exposed to 15 P C of 3H-uridine for
various periods of time 39

4. Effect of calf serum on 3H-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. 40

5. 3H-uridine incorporation, with or without calf (CS)
serum in uninfected chick embryo cell cultures. The
cell cultures were incubated at 370C for various per-
iods and then exposed to 20 pC of 3H-uridine for
30 minutes 42

6. Inhibition of 3H-uridine incorporation in uninfected
chick embryo cell cultures with various concentra-
tions of actinomycin P. The cell cultures were
exposed to 20 p C of H-uridine for 30 minutes at
the indicated times 43

7. Inhibition of 3H-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. 44

8. Linearity of sucrose gradient as measured by refrac-
tive index of each fraction with an Abbe refractometer. 47








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 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 1/10th volume of 4 M sodium chloride and
incubated at 40C for 5, 15 or 30 minutes and then
centrifuged at 24, 000 rpm'ina8 to 25% linear sucrose
gradient for 12 hours 51

11. Separation of chick embryo ribosomes into their sub-
units. The NaCI concentration of ribosomal prepara-
tion was raised to 0. 2 M, 0. 3 M, 0. 4 M or 0. 6 M and
incubated at 40C 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


Page








Figure


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.
3H-uridine (20 1 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.
3H-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.
3H-uridine (20 p C) 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. 3H-uridine (20 1 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. 3H-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


Page














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 Pbst infection

PDD50 50 per cent plaque depressing dose

pfu Plaque forming unit

rA/rU Riboadenylic acid/ribouridylic acid

rI/rC Riboinosinic acid/ribocytidylic acid

RNA Ribonucleic acid

RNAse Ribonuclease

RSB Rabbit reticulocyte buffer


viii








SDS Sodium dodecyl sulphate

S Sedimentation coefficient

TIP Translational inhibitory protein

VSV Vesicular stomatitis virus

W/V Weight per volume














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







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 (rI/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

































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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 i 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, 1960, 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








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 (rI/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) reported 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 susceptible 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 Z-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.








The effect of actinomycin D on polyribonucleotide induced inter-

feron formation has been a subject of controversy. Vilcek et al. (1968)

observed that 2 P g/ml of actinomycin D inhibited the production of

interferon in rabbit kidney cells induced by poly rI/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 rI/rC, pyran, and endotoxin in human skin fibro-

blast and mouse peritoneal macrophage cultures. It was also found that

actinomycin suppre s sed 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 rI/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 cells 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, 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., virus uncoating which is sensitive 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 Paucker (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 rI/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.

(1967) 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, Bausek and Merigan (1968) observed that pyran, a poly-

carboxylate polymer, and poly rI/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 rI/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, 1960) 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








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








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.

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

amount used and multiplicities of virus subsequently employed to

elicit interferon production. Levy, Buckler and Baron (1966) and







18
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 40C but would be expressed only if cells were further incubated at

370C 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 40C (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 420C, an unfavorable temperature for virus

replication, than at 390C or 350C. Burke, Skehel and Low (1967)

found that SFV induced optimal yields of interferon at 420C when no

virus replication was detected. However, the cells have to be pre-

incubated with virus at 370C for some time to presumably allow its

adsorption and uncoating before shifting the temperature to 420C.

Ruiz-Gomez and Isaacs (1963) also suggested thatthis increased inter-.

feron production at higher temperature may constitute a defense








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 40C, 11C and 250C. Those kept at

40C 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

250C 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







(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 acid

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 250C 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 biut 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 360C followed by shifting the temperature to

42C. Under these conditions interferon isproduced but the virus

does not replicate. It was found that during the incubation period at

36 0C 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.

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








kindly supplied by Mr. M. Fruitstone.

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








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--chlpride.

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 20p g/mL

Antibiotics. To all media was added 250 units/ml of potassium

penicillin G and 100 p g/ml of streptomycin sulphate.

Radioisotope Uridine-5-H3 was obtained from New England

Nuclear Corporation, Boston, Mass. The specific activity of the iso-

tope preparations were either 7 C/mM or 28.1 C/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/irL

Actinomycin D. This reagent was a gift from Merck, Sharpe and

Dohme, Rahway, New Jersey. Stock solution contained 100 pg/ml of








actinomycin D and was kept in the dark at -200 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 370 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 "Bellco" trypsinizing flask. Trypsinization of the

tissues was carried out for 30 minutes at 370C 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 50C. 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 108 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 370C.

Mouse L cells (Strain 929). The cell cultures were maintained

and propagated in Eagle's MEM with 10% calf serum. The cell mono-

layers were maintained in 32-ounce 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 106 cells and 32-ounce bottles

were plated with 8-10 x 106 cells.

Growth, Purification and Assay of Viruses

Vaccinia virus

Preparation. Vaccinia virus was propagated in 11-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 370C for 46 to 48 hours. The eggs were chilled at 5C

for several hours, the infected membranes were removed, frozen at

-600C 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








and stored at -600C.

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 370C 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 hiomogenizer 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 -600C.

Assay. The SFV was assayed on primary chick embryo cell

monolayers employing either the agar overlay or methyl cellulose








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 maint:* ,:od

at 420C. After the agar solidified, the bottles were transferred to a

370C 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 videe 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 370C, the

eggs were chilled and the allantoic fluid collected and centrifuged at

800xg for 15 minutes at 50C 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 videe 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 videe 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 370C 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 videe 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 107 pfu/ml

to 9 x 105 pfu/ml.

Productionand Assay of Interferon








Chick interferon

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 106

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 oi

maintenance medium. The bottles were usually incubated for 24 hours

at 370C. The medium containing interferon was harvested and heated

at 650C for 30 minutes to inactivate the virus. Some batches of inter-

feron preparations were centrifuged at 120, 000xg for 3 hours to re-

move most of the inactivated virus particles. The interferon prepa-

rations were stored at -200C.

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 videe supra). The

plaque depressing dose50, the amount of interferon preparation in

microliters which depressed the plaque number to 50% of the control,








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 andr cells w'ere 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 370C 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, 000xg to sediment

most of the virus particles. The supernatent fluid was distributed

into glass ampules, sealed and stored at -200C.

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








was added to each of 4 plaque bottles. After overnight incubation at

370C, the interferon dilutions were removed and the cell sheets were

washed twice with 5 ml of MM. VSV in 0. 2 ml volumes containing

100 to 200 pfu was added to each bottle. The VSV assay and calcula-

tion of PDD0 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 -600C. All the sam-

ples were assayed for their residual virus titer as described under

the assay of SFV videe 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 370C. The

decrease in virus titer with respect to time of incubation at 370C was

exponential. The rate of inactivation of Semliki Forest virus was one

log10 (90%) decrease in virus titer per 3.2 hours of incubation at 370C.

When the stock virus, consisting of 10% infected mouse brain suspen-

sion, was centrifuged at 15, 000xg for 15 minutes before incubation at

370C, 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 log10 in 3. 2 hours +10 minutes.

Incorporation of Uridine into RNA by Chick Embryo Cells

Measurement of uridine incorporation into CE cell cultures was

achieved by treating the cells at different time periods with 3H-uridine.























































HOURS AT 370C


Heat inactivation of Semliki Forest virus at 37C. Aliquots
were removed at various times during incubation and
assayed for residual virus.


Figure 1.








Plaque bottles, containing approximately 4 x 106 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 370C. At different

time intervals, 0.3 ml MMM containing 20 pC of 3H-uridine and
-5
3. 75 x 105 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 370C and bottles were gently rocked every

3
10 minutes to facilitate the even distribution of 3H-uridine on the cell

monolayer. To stop the incorporation, 0. 1 ml volume of cold uridine

(5 x 10-3 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% perchloricacid (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 800C 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 incO:po-

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 3H-uridine at

various concentrations when a pulse labelling period of 30 minutes

was employed. The incorporation of 3H-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 3H-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

concentrations of calf serum in MMM on 3H-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 3H-uri-

dine into cellular RNA was considerably increased in the presence of


















I0.0-


80
ao0-




6.0-




40D-




2.0-


-9-


CONC. (p.C) OF URIDINE


Figure 2.


3H-uridine incorporation in uninfected chick embryo cell
cultures exposed to various concentrations of 3H-uridine
and incubated at 370C for 30 minutes.









18.0



6.0



14.0-


12.0-



I0.0-



8.0-



6.0-



40-



20-


i I I I I
0 15 30 45 6(
MINUTES AT 370C


Figure 3.


3H-uridine incorporation in uninfected chick embryo
cell cultures exposed to 15 PC of 3H-uridine for
various periods of time.







40






12.0-


e--- with 2 /pg/ml of actinomycin D
10.5- --o without actinomycin 0







9.0



4.5J
.J
-j

N 6.0-
0



2 4.5-




3.0-




L5-



0 --- -----------------^.----

O 2 4 6 8 10
PERCENTAGE OF CALF SERUM IN MEDIUM

Figure 4. Effect of calf serum on 3H-uridine incorporation of
uninfected chick embryo cell cultures with or with-
out actinomycin D. The cell cultures were exposed
to O20 PC of 3H-uridine for 30 minutes.








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 370C 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 370C,

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 3H-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 ii g) was added to each bottle in 0. 1 ml volume with 0. 2 ml of MMM.













^--









?MM?2.5%CS










"w-----0I1,
^
o,^

s "


.'J 6


0 2 4
0 2 4


I 1
6 8
TIM: .., HOURS


0 12
10 12 14


Figure 5.


3H-uridine incorporation, with or without calf (CS)
serum in uninfected chick embryo cell cultures. The
cell cultures were incubated at 37 C for various
periods and then exposed to 20 PC of 3H-uridine for
30 minutes.


5.5-



w -




u
"4,





35-



























60



50-



40-


0.025o.g







0.05~g


A 0- 00.I/g



10 -1 -2 5 .


HOURS AFTER ADDITION


Figure 6.


Inhibition of 3H-uridine incorporation in uninfected chick
embryo cell cultures with various concentrations of
actinomycin D. The cell cultures were exposed to 20 P C
of 3H-uridine for 30 minutes at the indicated times.


.................. ....

























100-



I80-
So


S60-
z

40-



LIU
20-




0 02 0.4 06 08 1.0 12 1.4 1.6 Is 20
ACNINOMYCIN 0 (j.g / ml)


Figure 7. Inhibition of 3H-uridine incorporation in uninfected 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.









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 m9nolayer. 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

370C. A 30-minute period of labelling with 20 iPC of 3H-uridine 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 p g/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 40 to 50 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, 000xg 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, 000xg 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 -600C. 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







47

3750-








3700-














I.-


3600-























index of each fraction with an Abbe refractometer.








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 mp. 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 MgCl1 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










0.8-


0.7-




0.6-


0.5-




0.4-




0.3-


0.2-




0.1-


I-I


FRACTION NUMBER


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.








purified preparation was pressure dialyzed against 100 volumes of

0.01 M NaCI Q..(M Tris HC1, pH 7. 2 buffer for 4 hours to reduce

the volume and to remove the sucrose. One tenth volume of 4 M

NaC1 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

NaCI treated sample was layered on 26 ml linear gradient of 8 to 25%a

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 NaCI

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 NaC1 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 NaC1 concentration. However, a

sizeable portion of the ribosomes remained undissociated even in

presence of a 0. 6 M NaC1 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 NaC1. Therefore, it appears that













0--* 5 minutes incubation
o---0 15 minutes incubation
A--4 30 minutes incubation


OJ5-





on- b -----------------
I .o


0.10-
I 5. 0

o1 j


15 2(
FRACTION NUMBER


S 25 30


Figure 10.


Separation of chick embryo ribosomes into their sub-units.
The purified ribosomal preparation was mixed with 1/10th
volume of 4 M sodium chloride and incubated at 40C for
5, 15 or 30 minutes and then centrifuged at 24, 000 rpm in a
8 to 25% linear sucrose gradient for 12 hours.


0.50 -


PI


ri
I
Jo



6


I
< 11I


0.40


035-


S0.30-
co

- 025-
UJ
z
_J
2 0.20-
o






















































0 ;


o 5 10 15
FRACTION NUMBER


20 25 30


Figure 11.


Separation of chick embryo ribosomes into their sub-
units. The NaC1 concentration of ribosomal preparation
was raised to 0. 2 M, 0. 3 M, 0.4 M or 0. 6 Mand in-
cubated at 4C for 15 minutes. The samples were then
centrifuged in a 8 to 25% sucrose gradient containing the
same concentration of NaCI as that of sample and
centrifuged at 24, 000 rpm for 12 hours.


0.9-



Q8-



0.7-



" 0.6-


4-
0,5-



04-


v






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, 000xg for 10 minutes. The pH of the

supernatant was rapidly brought to 8 using 0. IN sodium hydroxide, and

centrifugation at 20, 000xg 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

videe 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 106 cells, were infected with a multiplicity of








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 ig 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 40C, 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

40C to synchronize the infection. The following morning, the plaque

bottles were transferred to a 370C 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

370C incubator.

The time when cell cultures were first transferred to the 370C

incubator from 40C was considered as zero time for viral replication

and RNA synthesis cycles.

At various periods of time following the infection, 20 -p C of

3H-uridine in 0. 1 ml volume was added to each bottle, and cell mono-

layers were further incubated for an additional 45 minutes at 370C.

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 NaC1 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

d; sh.a'4i.g Ahe mixtu:c for 3 to 5 rrli..s at room Lc-Patue.. 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, 000xg 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 -200C and RNA was collected by centrifuging the sus-

pension at 15, 000xg for 15 minutes. The precipitate was washed once

with 70% ethanol containing 2% potassium acetate, and RNA was again

pelleted by centrifuging at 15, 000xg for 15 minutes. The RNA pre-

cipitate was dissolved in 0.1 M KC1 0. 01 M Tris 0..00LM EDTA

buffer, pH 7. 1 and clarified by centrifugation at 10, 000xg 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, I ml (4.927 to 7.126 OD6
260-








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 EpTA,

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 KCI, 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 1 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 1i g/ml in the reaction mixture, which then was incubated for 10






57
minutes at 370C. To stop the RNAse activity, 100 mg of coarse

bentonite preparation was added just after the incubation period. The

RNA was then immediately 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 12 g/ml, was

able to completely degrade two OD units of chick ribosomal RNA
260
when incubated for 10 minutes at 37 0C.














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























































Log10 INPUT MULTIPLICITY


Figure 12.


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


1201


105-


E

2 90-

zO
D


S75'


60i

0




L. 45"

I-,-
>-








30




15
15-






















0--0 m =0.

0 ....O m= 10


h0O


I \---I-
I I I I
5 10 15 20


25 35
25 35


HOURS AFTER INFECTION


Figure 13. Kinetics
cultures
plicities


of interferon production in chick embryo cell
infected with Semliki Forest virus at multi-
of 10 or 0. 1 pfu/cell.


E

I-
z
=3
o
r,,

0-
-,J


z

I--


La
z
I-


80-




60-


40-


20-








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 2). 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








TABLE 2

THE EFFECT OF TWO MULTIPLICITIES OF
VIRUS ON INTERFERON PRODUCTION


Interferon Yield, PDD50 Units/ml
Multiplicity
Experiment of Infection Hours After Infection %/ Inhibition
Number (pfu/cell) 8 !3 24 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.









160-1


-.J

z 120-
0



I--



S80-
0
a-
o
0

LU



;40-
2 -
-CL


-


/

I /

/





I
/






000..-0 _
1'


-0 No Actinomycin

0-0 Actinomycin Resistant

. -0. Actinomycin Sensitive
RNA


I
I'
I
i
i


I I I
8 10
INFECTION


I 12
12


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.


S i
2


I I I
4
HOURS


6
AFTER








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 actinomycinn 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








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 folluwih, ilfction. The 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 u 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"





























E (













2





0
Figure 15.
S ,
















Figure 15.


HOURS AFTER INFECTION


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.








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 and, 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 370C, 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 370C 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


60 -


E
Ln









U-
z










0
ilr
L,



L-


LJ
>-



I-
2r


/


0..O rn 0.1
0--0 m= 10


40

It


20


Figure 16.


-I
S.... ...- --.......-----..................









5 10 15 20 25
HOURS AFTER iNFECTIO \

KineL cs of interfro n product i n chick cenryo cefl
cultures exposed to heat i.:czLvatc Sedli .i Fslest
v-rus. The cell cultures received the equivalent oi
either 10 or 0.1 piu/cell of the virus.


o-~----C







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 vere 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 360C for 1 hour and then at 420C. Under

these conditions, infectious virus particles were not synthesized but

interferon production was, apparently, not affected. The reason for



















o---o Live Virus (m=10pfu/cell)

x-- x Heat Inactivated Virus (m 10 pfu equivalent/cell)


0 0


8
HOURS


12 16
AFTER INFECTION


Figure 17.


Kinetics of interferon production with heat inactivated
and live Semliki Forest virus with cell cultures.


60


E

S50-
z




-J
-




- 30

LU


- 20-

z
---


---I r r








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 hours,

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 10o 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 370C. 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,











0-0 0.1 pfu/cell+normal serum

0--0 O.Ipfu cell+immune serum


Immune Serum added



/


5 10 15 20 25
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.


120




105-


E




t,-
z


I-J
LrU
C


U-
LJ,
>-

0

LJ
I-.








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 indupers. 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 rI/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 370C for 4 hours. Thereafter, the maintenance

medium was removed and cell cultures were washed twice with fresh








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, inducLion 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








TABLE 3

INTERFERON PRODUCTION IN PRESENCE
OR ABSENCE OF ACTINOMYCIN D


Multiplicity Presence or 15 Hour'Inter-
Virus of Infection Absence of feron Yields
Preparation pfu/cell Actinomycin D (PDDs0 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 103 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 1 g/ml of actinomycin D. After various periods of incubation at

370C, 20 PC of 3H-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 mr 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

3H-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 11 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 Mecs

(1967) who reported that viral specific RNA from the infected cells

can be resolved into three components. They also reported that


















'28'S


e--* Total Radio-activity in Infected Cells
e-** Ribonuclease Resistant Radio-activity
**-*.* Carrier RNA (OD 260)
e-* Total Radio-activity in Uninfected Cells


'18'S
s \



s :


'4'S
I.s







i"
j :




/
"I/a
: /


--
'.
E


o
0.8 -



a.

0
0.4


0 5 10 15 20 25 30
Fraction No.


Figure 19.


Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
infected for Z hours with Semliki Forest virus.
3H-uridine (20 1C) was added for 45 minutes, and
RNA was extracted. L cell RNA was employed as
carrier RNA.

















350-



300



250.



200,



150.



100.



50.


-1.0

E

0.8


w
-0.6 ,


0.4
-0.4 o


0 5 10 15 20 25
Fraction No.


Figure 20.


Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
infected for 4 hours with Semliki Forest virus.
3H-uridine (20 pC) was added for 45 minutes, and
RNA was then extracted. L cell RNA was employed
as carrier RNA.


*--o Total Radio-activity in Infected Cells
*- Ribonuclease Resistant Radio-activity
S*.......* Carrier RNA (00 260)
I *- Total Radio-activity in Uninfected
I Cells
'2 8'S

I -, '4'S
I I I \ .or

I I \ I
I I a

i i I ,I
41 \ '4 1


/ /

A

















'28'S
9


.---- Totol Radio-activity in Infected Cells
* --.* Ribonucleose Resistant Rodio-actiuty
*--- Carrier RNA (00 260)
e---. Total Radio-activity in Uninfected

4'S


0 5 b 15 2"0
Fraction No.


25 30


Figure 21.


Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
infected for 6 hours with Semliki Forest virus.
3H-uridine (20 pC) was added for 45 minutes, and
RNA was then extracted. L cell RNA was employed
as carrier RNA.


400o



350-



300O



250,



200.



150,



I00



504


06



.05
CA



0

403 "
a-
o


U' _






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 103 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 107 to 2 x 108 pfu/ml.

Pulse -labelling, method

Viral specific RNA synthesis in chick embryo cells exposed to

live as well as heat inactiyated Semliki Forest virus was also

studied by the 3H-uridine pulse labelling method. The induced cells,

at various periods after the induction, were exposed to 20 p C of

3H-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






















...,* Total Radio-activity
*--* Carrier RNA
--- Total Radio-activity of Control Cells


'2 8'S


10 15 20
Fraction No.


25 30


Figure 22. Sucrose gradient analysis of RNA extracted from
actinomycin D-treated chick embryo cell cultures
exposed for 2 h urs 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.


300




250.




200-



S150.




100




50.


1.0




-08 .
o
'0








o
-0.6
C
0)


-0.4




-C2





















e-. e Total Radio-activity of Infected Cells at 6hrs p.i.
*-.... Total Radio-activity of Infected Cells t 4hrs. p.i.
*--. Carrier RNA
*-- Total Radio-activity of Uninfected Cells


'28'S


'18'S


I I
5 10 15 20 25 30
Fraction No.


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. 3H-uridine (20 PC) was added
for 45 minutes, and RNA was then extracted. L
cell RNA was employed as carrier RNA.


300,


250.



200-




S150-



100.




50-


-1.2




-1.0



.0.8 E
.0
CD


.0.6 ,.



,0.4 .-
o








increased with respect to cime 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

for 7 hours in the presence of 20 1 C of 3H-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.















e-.-* Uninfected Cells and
Actinomycin D(2mg/ml)
e--o Live SFV and Actinomycin
D(2mg/ml)
e.--. Heat inactivated SFV and
Actinomycin D (2mg/ml)
*--* Live SFV
e...-.. Heat inactivated SFV


... H r.s ---- .--..--.- I. ..in---

2 4 6 8 10 12
Hours After Induction


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.


150-




- 125-
L.

--
. 100.
o

C


a 75.
0.






S--
S25
= 25








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 OD60 units) were mixed with 10 PDD50
Z 260 50

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, 000xg 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 260 units of chick embryo '40 S' ribosomal







87







0 ,
Co

o
0
o 4 0 '5


Do





U
( 0





0 o04
z o .
z1-





o
0 ul
k :
u a





SO o o o o
So o 0
F"' *UO"









0o 0 0
-' m $4







2 2


to
4 04








-- U)
o o I -4



z U U
0 (3 01 0




0 U) UU


*F i l 5
W VU U>I -






88
sub-units were mixed with 10 PDD50 interferon units, incubated at

370C 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.








TABLE 5

ASSOCIATION OF CHICK INTERFERON WITH RIBOSOMAL SUBUNITS



Source of Chick PDD50 Units of PDD50 Units of
Interferon Ribosomes Interferon Added Interferon Recovered

Chick 60 S 10 9.9

Chick 40 S 10 9.8

Chick 80S 10 10.1














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/106 cells in
50








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