A Genetic analysis of poliovirus RNA replication in vivo

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A Genetic analysis of poliovirus RNA replication in vivo
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Thesis:
Thesis (Ph.D.)--University of Florida, 1991.
Bibliography:
Bibliography: leaves 69-71.
Statement of Responsibility:
by Philip Schuyler Collis.
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Typescript.
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Vita.

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A GENETIC ANALYSIS OF
POLIOVIRUS RNA REPLICATION IN VIVO




















By

PHILIP SCHUYLER COLLIS


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

UNIVERSITY OF FLORIDA


1991
















TABLE OF CONTENTS


ABSTRACT ............................................... iv

CHAPTERS

1 INTRODUCTION.................................. 1

2 MATERIALS & METHODS........................... 8

DNA Manipulation and
Deletion Mutant Construction............. 8
Bacterial Transformation and Growth.......... 9
RNA Sequencing............................... 11
RNA Transcription.............................. 12
Mammalian Cells and RNA Transfection
Reactions................................ 13
Synthesis of 32P-Labeled Viral RNA In Vivo... 14
RNA Isolation................................. 14
Radiolabelled Probe.......................... 15
Gel Fractionating, Northern Blotting
and Hybridization of Probe............... 15
Quantitation.................................. 16
3D P and 2C Mutants.......................... 17

3 RESULTS...................................... 19

Introduction.................................. 19
Synthesis of Infectious RNA Transcripts...... 20
Replication of T7D-polio RNA in
Transfected Cells......................... 25
Deletion in the Capsid Coding Sequence....... 30
Large In-Frame Deletion........................ 33
Deletion in the 2A Coding Region............. 36
Deletion in the 2B Coding Region............. 36
Deletion in the 3C Coding Region............. 39
3DPol Point Mutation.......................... 44
2C Insertion Mutation.......................... 50
Minus Strand Replication....................... 53
















4 DISCUSSION..................................... 57

Introduction ................................. 57
Deletion Mutants ............................. 57
2C and 3DP01 Mutants........................... 62
Temperature Sensitivity of RNA2............... 62
Interference................................. 64
Cis and Trans Activity....................... 65
Minus Strand Transcripts...................... 66
Conclusion................................... .. 67

REFERENCES................................ ............... 69

BIOGRAPHICAL SKETCH....................... ............ 72


iii















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


A GENETIC ANALYSIS OF POLIOVIRUS
RNA REPLICATION IN VIVO

By

Philip Schuyler Collis


May 1991


Chairman: James B. Flanegan
Major Department: Immunology and Medical Microbiology


In this study, both full-length and subgenomic poliovirus

RNAs were transcribed in vitro and were transfected into

HeLa cells. RNA replication was analyzed using a Northern

(RNA) blot procedure. RNAs with either deletion, insertion,

or point mutations were characterized for their ability to

replicate in the presence or absence of helper RNA. An RNA

with an in-frame deletion in the capsid (Pl) coding region

(RNA 2) replicated very efficiently over a 24 h period. In

contrast, an RNA with an out-of-frame deletion in P1 did not

replicate either alone or with helper RNA. Subgenomic RNAs

with larger in-frame deletions were tested for their ability

to replicate alone or in the presence of helper RNA. A

mutant RNA with an in-frame deletion in the P1-2A coding

sequence was self-replicating










but at a very reduced level. Cotransfection of this mutant

with helper RNA significantly increased its replication

efficiency. Thus, it is possible to complement this 2A

mutant in trans using the cotransfection procedure. Other

subgenomic RNAs with larger in-frame deletions were not

self-replicating and could not be complemented. In

particular, an RNA with an in-frame deletion in 2B did not

replicate either alone or with helper RNA. Similar results

were obtained with an in-frame deletion mutant in 3CPro. The

results indicated that 2B and 3C have cis active functions.

Two temperature sensitive mutants were also characterized.

Tsl0 is an RNA-negative mutant which contains a point

mutation in the polymerase (3DPOl). Ts2C-31 is an RNA-

negative mutant which contains an insertion mutation in 2C.

Both mutants were complemented by helper RNA at the

restrictive temperature. Therefore, both 3DP01 and 2C have

activities that can be supplied in trans. In preliminary

experiments, minus-strand RNAs were evaluated for their

ability to replicate when cotransfected with plus-strand

helper. Neither full-length transcripts nor subgenomic

transcripts of the 3' end of the minus-strand showed any

evidence of replication. Thus, it may not be possible to

provide the replication proteins in trans to copy minus-

strand templates.















CHAPTER 1

INTRODUCTION

Poliovirus is a small RNA virus which is a member of

the picornaviradae family. This family includes a group of

small, non-enveloped viruses which include a number of

important human and animal pathogens. Other pathogens in

this group include the human rhinoviruses, hepatitis A

virus, the human enteroviruses, and foot and mouth disease

virus. The typical picornavirus genome is 7000-9000

nucleotides long and consists of a single strand of positive

sense RNA. The genome is translated into a polyprotein

which is subsequently processed by virally coded proteases.

Poliovirus contains a genome approximately 7500 nucleotides

long. It has a small viral protein, VPg, covalently linked

to the 5' end of the virion RNA and to all newly synthesized

viral RNA (Flanegan and Baltimore, 1977; Lee et al., 1977;

Pettersson et al., 1978). Virion RNA contains a large 5'

terminal noncoding region (745 nucleotides) and a large open

reading frame that encodes the viral polyprotein which

includes three domains (P1, P2, and P3). The open reading

frame is divided into these regions based on protein

function. The P1 region encodes the capsid proteins. The

P2 region encodes a viral protease (2A) and two other









proteins. While the 2A product has also been implicated in

host cell shutdown, the 2B and 2C product functions remain

unclear. The P3 region encodes the replication proteins and

includes 3A (function unclear,) 3B (VPg), 3C (the major

protease) and 3D (the RNA polymerase). The 3' end of virion

RNA contains a noncoding sequence (71 nucleotides) and a

heterogeneous poly (A) sequence 75-100 nucleotides long that

is required for infectivity of both virion RNA (Spector et

al., 1975) and RNA transcribed in vitro from plasmid DNA

(Sarnow, 1989).

Poliovirus replicates in the cytoplasm of infected

cells. The process begins when the virus binds to a

specific cellular receptor. Following binding to the

cellular receptor the virus is internalized and the RNA

uncoated. The plus strand RNA then acts as a messenger and

is translated into its polyprotein by the host cell

machinery. The polyprotein is then processed by two viral

protease (2APrO and 3CPro) to generate the virus specific

proteins necessary to begin replication. Plus strands are

then copied to generate minus strand templates which in turn

are used to regenerate plus strands. Each minus strand is

seen to have multiple plus strands initiating from it, and

the entire complex is membrane associated. Replication is

asymmetric and produces at least ten times as many plus

strands as minus strands. Following their synthesis, the

plus strands are subsequently packaged into virions and

released by cell lysis, completing the cycle.









Understanding the molecular mechanisms involved in the

replication of poliovirus and other plus stranded RNA

viruses remains of fundamental importance. Although many

aspects of poliovirus replication have been studied in

detail, including replication in vitro, little is known

about the RNA sequences and structures that are required for

replication, and only limited information is available

regarding the specific roles of the viral proteins during

replication.

Historically, information about which poliovirus

sequences were required for replication came from the study

of defective interfering particles (DI). Studies have shown

that poliovirus generates DI particles during replication.

Detailed analysis of cDNAs from several DI genomes revealed

that the DIs were deletions in the capsid coding region and

further that they were all in-frame deletions (Kuge et al.,

1986). This was contrary to the findings with Sindbis virus

where it was found that only the 5' and 3' terminal

sequences were necessary to generate a functional replicon

(Levis et al., 1986). This raised the question as to why

neither smaller nor noncapsid deletions were seen with

poliovirus DIs. Was there a packaging problem or were there

cis requirements or both?

Genetic studies of poliovirus have been conducted

previously but were limited to the analysis of mutants

showing conditional phenotypes. This precluded the ability

to assess the effects of mutants which did not allow for the









recovery of virus (lethal) or which had phenotypes

indistinguishable from wild type. More recently, the

isolation of an infectious cDNA clone (Racaniello and

Baltimore, 1981) provided a means to generate, isolate and

characterize specific mutants in poliovirus. Various

conditional mutants (ts, host range, plaque size) were

generated using this clone.

In addition to the information about the capsid coding

region provided by the cited DI particle studies, analysis

of various conditional mutants yielded information about the

sequences coding for nonstructural proteins. The Baltimore

lab has isolated a number of insertional mutants showing

that the 2A (tyr/gly protease), 2B, 3A, and 3D (viral RNA

polymerase) products or sequences were required for viral

RNA replication (Bernstein et al., 1986). It was shown

further that both the 2A and 3A mutations could be

complemented but that the 2B and 3D mutations could not.

More recently, the 2C region was also studied (Li and

Baltimore, 1988). While most mutants were RNA negative, two

viable ts mutants were found. One of these mutants, 2C-31,

has been investigated as part of this study.

The Wimmer group has constructed a number of mutations

in the 3B product (VPg) confirming that it is essential for

poliovirus replication (Kuhn et al., 1988). The 3B product

has been suggested to have primer activity, nuclease

activity for hairpin resolution and possibly a role in






5


encapsidation. Unfortunately, no conditional mutants have

been found in 3B to assist in understanding it's function.

The 3C product is the protease responsible for most of

the poliovirus polyprotein processing and cuts at gln/gly

sites. Viable mutants showing small plaque phenotypes have

been isolated (Dewalt and Semler, 1987). At least one of

these mutants showed nearly wild type levels of replication

even though only a low level of proteinase was observed.

In addition to the noncomplementable 3D mutation

reported by Bernstein et al.(1986), Agut et al.(1989)

reports that the 3D mutant ts035 is complementable. This

suggested that the 3D product, the viral RNA polymerase,

could be supplied in trans.

It has now been demonstrated that infectious viral RNA

transcripts can be synthesized in vitro (Kaplan et al.,

1985; Nomoto, 1989; Sarnow, 1989) and that the specific

infectivity of the synthesized RNA is within an order of

magnitude of the infectivity of poliovirion RNA (1.5 x 106

PFU/Ag). RNA transcripts with extended poly(A) sequences

have infectivities equivalent to that found in virion RNA

(Sarnow, 1989). By preparing mutant RNA transcripts with

in-frame deletions in the P1 region of the genome, Kaplan

and Racaniello (1988) were able to confirm that the capsid

sequences were not required for RNA replication or

translation in cultured cells. In addition, it was observed

that on cotransfection, transcripts lacking their 3'

sequences were inhibitory to wild type vRNA replication.









Curiously, this was not seen when using the capsid mutant

transcripts which contained the correct 3' sequence. It

should be noted, however, that this analysis was predicated

on evaluation by plaque assay and cytopathic effect and thus

required the presence of packaged virus.

In this study, I have evaluated the replication of

poliovirus RNAs by transfecting or co-transfecting mutant

and helper RNAs into cultured cells. The evaluation of

replication by Northern (RNA) blot analysis of RNA produced

during a single replication cycle has eliminated the need to

employ conditional mutants and allowed the investigation of

mutants that were previously unable to be analyzed. RNAs

with both in-frame and out-of-frame deletions, as well as

insertional and point mutants occurring in various regions

of the genome, were characterized.

Our results indicated that neither the capsid proteins

nor the amino terminus of the 2A product was required for

RNA replication. While the deletion mutant which extended

into the 2A region was able to self-replicate at a reduced

level, it could be complemented to wild type levels by

providing 2A in trans. In addition, ts mutants in both the

2C and the 3D coding regions were also shown to be

complementable.

Neither a large in-frame deletion (Pl-P2-P3) nor an

out-of-frame deletion restricted to the capsid region was

able to be complemented with helper RNA, suggesting that

poliovirus does indeed have cis requirements during plus to









minus strand synthesis. This was further evidenced by the

fact that deletion mutants extending into the 2B coding

region or occurring in the 3C coding region could neither

self replicate nor be complemented, providing evidence that

at least these two products have cis acting activities.

This study establishes that poliovirus RNA replication

can be studied in cultured cells transfected with viral RNAs

synthesized in vitro. It provides a powerful new approach

for studying the unique replication strategies that are

utilized by poliovirus and reports a number of interesting

findings.















CHAPTER 2

MATERIALS AND METHODS



DNA Manipulation and Deletion Mutant Construction

pT7D-polio DNA was generously supplied by Dr. Peter

Sarnow (University of Colorado, Denver, CO) and was used as

the parental clone for all mutants constructed (Sarnow,

1989). Digestion of plasmid DNA with restriction enzymes,

filling recessed 3' ends or polishing 3' extensions using

Klenow polymerase, and ligation reactions were all according

to the specification sheets provided by the manufacturer.

NruI, BstEII, SalI and DNA polymerase I large fragment

(Klenow) were purchased from Bethesda Research Laboratories,

Gaithersburg, MD; AvaI, SnaBI, and BanII were purchased from

New England Biolabs, Beverly, MA; AsuII and the synthetic

SalI linker (pGGTCGACC) were obtained from the Promega

Corporation, Madison, WI; bacteriophage T4 DNA ligase was

purchased from International Biotechnologies, Inc., New

Haven, CT.

RNA 1 was constructed by digesting pT7D-polio DNA with

both Aval and Nrul, filling the recessed 3' end of the Aval

site with Klenow polymerase, and blunt end ligation of the

resulting fragment with T4 DNA ligase (see figure 3-1). RNA









2 was constructed by digesting pT7D-polio DNA with NruI and

SnaBI and blunt end ligation of the resulting fragment with

T4 DNA ligase. RNA 3 was constructed by digesting pT7D-

polio DNA with AsuII and ligation of the resulting fragment

with T4 DNA ligase. RNA 4 was constructed by digesting

pT7D-polio DNA with BanII, removing the 3' extensions with

Klenow polymerase, and lighting a synthetic oligonucleotide

(8 mer) containing a unique SalI site onto the blunt ends.

The resulting construct was digested with SalI and the

resulting fragment was ligated with T4 DNA ligase. RNA 5

was constructed by digesting pT7D-polio with NruI and

BstEII, filling the recessed 3' ends of the BstEII site with

Klenow polymerase, and blunt end ligation of the resulting

fragment with T4 DNA ligase. RNA 6 was constructed by

digesting RNA 2 with BglII and NarI, filling the recessed 3'

ends with Klenow polymerase, and blunt end ligation of the

resulting fragment with T4 DNA ligase. The deletion clones

are diagrammed in figure 3-1.

Bacterial Transformation and Growth

Competent HB101 cells (BRL) were transformed with

plasmid DNA according to the specification sheet provided by

the manufacturer for small scale transformation. Briefly,

20 pA ligation reactions containing 200 ng DNA were diluted

1:5 with H20 and 1 .l was added to 20 pA HB101 cells in a

microfuge tube. The mixture was incubated on ice 30 min,

heat shocked 40 s at 42*C, and placed on ice for 5 min.

Eighty microliters of SOC media (2% bactotryptone, 0.5%









extract, 10 mm NaCi, 2.5 mM KC1, 10 mM MgC12, 10 mM MgSO4,

20 mM Glucose) was added and the tube was incubated at 37C

for 1 h while being shaken at 225 rpm. The entire 100 Jl

was then spread on an LB plate containing 100 Ag/ml

ampicillin and incubated overnight at 37C.

Resulting colonies were inoculated into 5 ml L Broth

(Gibco/BRL L Broth Base) starter cultures for miniprep

analysis. Minipreps were conducted as follows (Zhou et al.,

1990): An overnight culture (1.5 ml) was pelleted 10 s in a

microfuge tube. The supernatant was decanted and the pellet

was resuspended in 50 pl cleared supernatant. Three hundred

microliters TENS (TE buffer with 0.1N NaOH and 0.5% SDS) was

added and the mixture vortexed for 5 s. One hundred fifty

microliters 3.0 M sodium acetate pH 5.2 was added and the

mixture was vortexed for 5 s. The solution was then

centrifuged for 2 min in a microcentrifuge. The supernatant

was then combined with 0.9 ml ethanol which had been

precooled to -200C. The precipitate was collected by

centrifugation for 2 min in a microcentrifuge and rinsed one

time with cold 70% ethanol. The resulting pellet was dried

and resuspended in 30 pA H20.

Large preparations of plasmid DNA for the putative

mutant constructs were isolated as recommended by the

manufacturer for p-GEM plasmids (Promega Corporation).

Briefly, a 250 ml culture was grown overnight in L Broth

containing 50 Ag/ml ampicillin. The culture was centrifuged

5000 x g and the cells were resuspended in 6 ml cold 25 mM









Tris HC1, pH 7.5, 10 mM EDTA, 15% sucrose, 2 mg/ml lysozyme

and incubated at 0 for 20 min. Twelve milliliters of 0.2 M

NaOH, 1.0% SDS was added and the mixture was incubated 10

min at 0. Seven and one-half milliliters of 3 M sodium

acetate, pH 4.6, was then added and the mixture incubated an

additional 10 min at 0C. After clearing for 15 min at

10,000 x g, 50 pl of 1 mg/ml DNAse free RNAse A (Sigma

Chemical Co., St. Louis, MO) was added to the supernatant

and incubated 20 min at 37C. The RNAse A was heated to

1000C for 15 min prior to its use. The supernatant was

extracted twice with 1 volume of chloroform/phenol (1:1,

saturated with TE buffer) followed by extraction with 1

volume of chloroform/isoamyl alcohol (24:1). The resulting

solution was precipitated by adding 2 volumes of ethanol and

incubating at -200C for 60 min. The solution was

centrifuged for 20 min at 10,000 x g and the pellet

dissolved in 1.6 ml H20. Four hundred microliters of 4 M

NaCl was added and mixed, 2.0 ml 13% PEG was added, and the

mixture was incubated at 4*C for 60 min. The mix was then

centrifuged at 10,000 x g for 10 min, the pellet was washed

once with 70% ethanol, and then dried and resuspended in

H20.

RNA Sequencing

All mutant constructs were analyzed to confirm that

they contained the correct junction sequence. RNA

transcripts (see following section) were sequenced using the









Gemseq sequencing kit (Promega Corporation) according to the

manufacturer's directions. Briefly, 1 Ag RNA transcript was

annealed with 5 ng of the appropriate primer. All primers

were synthetic DNA oligonucleotides (21 mers) that were

minus-strand sequences 50 bases 5' of the junction sequence.

RNA transcripts were synthesized using AMV reverse

transcriptase in the presence of di-deoxynucleotides and

[35S]dATP (Amersham Corporation, Arlington Heights, IL).

The resulting transcripts were fractionated on 6%

polyacrylamide gels with 7 M urea using 0.5 x TBE buffer.

The gels were dried and radiographed directly.

RNA Transcription

All plus strand RNA transcripts were generated as

follows: 5 Mg of plasmid DNA cut with MluI restriction

endonuclease was incubated in the presence of 40 mM Tris, pH

7.9, 6 mM MgCl2, 2 mM spermidine, 10 mM DTT, 40 units RNasin

(Promega Corporation), 1 mM ATP, 1 mM UTP, 1 mM GTP, 1 mM

CTP, 10uCi [3H]UTP (Amersham, 40Ci/m mol), 2000u

bacteriophage T7 RNA polymerase. The reaction volume was

100 Al and the mixture was incubated for 4 h at 37C. A 5

Ml aliquot was precipitated in trichloroacetic acid and

counted to determine the efficiency of each reaction. The

reaction mixture was then used directly without further

processing.

Minus strand transcripts were generated as described

above. For full-length minus-strand RNA, p0F2612 DNA (M.S.









Oberste, Ph.D. dissertation, University of Florida) was

linearized with SacI prior to transcription with T7 RNA

polymerase. Two subgenomic RNA transcripts representing the

3' terminal sequences of poliovirus minus-strand RNA were

generously supplied by Dr. Daniel Brown, University of

Florida, Gainesville, FL. These two transcripts were made

from PCR amplified DNA which had used the T7D-polio plasmid

DNA as the template. Both a 127 nucleotide transcript and a

789 nucleotide transcript were made using the standard T7

RNA polymerase reaction conditions.

Mammalian Cells and RNA Transfection Reactions

All transfection reactions utilized HeLa spinner cells

placed in monolayer 24 h prior to use. In general, 3 x 106

cells were plated onto 6 cm dishes resulting in 60-70%

confluence. The HeLa cells were grown in Joklik's modified

MEM (Flow Laboratories, Inc., McLean, VA) and once in

monolayer were incubated in Eagles minimal essential medium

(EMEM-Flow Laboratories). Both media contained 10% fetal

calf serum.

For transfection, the medium was removed and the cells

were rinsed once for 1 min with 1 ml phosphate buffered

saline supplemented with magnesium and calcium (PBS+).

Cells were then transfected for 60 min at 37*C with 0.5 ml

of PBS+ containing 500 gg/ml DEAE DEXTRAN (Pharmacia,

Piscattaway, NJ) and 1-5 pg RNA transcript in the

transcription buffer. Following the 60 min incubation, the










DEAE dextran mixture was removed, the cells were rinsed once

for 1 min with 1 ml PBS+, and 5.0 ml EMEM with 10% FCS was

added. The cells were then incubated 0-24 h prior to

harvesting.

Synthesis of 32P-labeled Viral RNA In Vivo

Following transfection, the cells were rinsed three

times for 5 min with 2 ml phosphate-free EMEM. The cells

were then exposed to 5 gg/ml actinomycin D for 15 min in 2

ml phosphate-free EMEM with 10% dialyzed FCS. Following

this incubation, 1 mCi [32P]H3PO4 (Amersham) was added and

the cells were incubated as usual.

RNA Isolation

Whole cell RNA was harvested using a guanidinium

isothiocynate (GTC) procedure (Maniatis et al., 1982).

Cells were scraped from the dishes with a sterile rubber

policeman and pelleted by spinning at 1000 x g in a clinical

centrifuge. The supernatant was removed, the tube was dried

by blotting, and the pellet was resuspended in 0.25 ml lysis

buffer (4 M GTC, 25 mM sodium citrate pH 7.0, 0.5% Sarcosyl,

0.1 M B-mercaptoethanol). The pellet was resuspended by

repeated micropipetting and placed into a sterile microfuge

tube. Thirty microliters 2 M sodium citrate pH 4.0 was

added and the solution vortexed for 5 s. Three hundred

microliters phenol saturated with H20 was added and the

mixture was vortexed for 5 s. Following the addition of 60

pA of chloroform/isoamyl alcohol (24:1), the mixture was









vortexed 3 times for 5 s and placed on ice for 10 min. The

solution was then centrifuged for 10 min at 4*C and the

supernatant was transferred to another sterile microfuge

tube. Three hundred microliters isopropanol was added, the

mixture was vortexed for 5 s, and the RNA was precipitated

overnight at -20C.

Radiolabelled Probe

RNA transcripts were made as described previously with

the following exceptions. Fifty microcuries [32p]UTP was

added in place of [3H]UTP and the cold UTP was omitted. For

these experiments, the probe was a poliovirus minus strand

RNA transcript which was complementary to nucleotides 5240-

6775 (poF1265--Oberste, Ph.D. dissertation, University of

Florida).
Gel Fractionation, Northern Blotting
and Hybridization of Probe

These procedures were performed according to the

recommendation of the nitrocellulose manufacturer

(Schleicher and Schuell, Keene, NH). Briefly, the RNA was

fractionated on a 2.2 M formaldehyde, 1% agarose gel

containing 0.5 x MOPS buffer (20 mM 4-

morpholinopropanesulfonic acid, 5 mM sodium acetate, 1 mM

EDTA). A fraction (15 Ag) of the whole cell RNA preparation

was added to each lane and electrophoresed for 4 h at 150 V

on a 15 cm long gel. Following electrophoresis, gels

containing radiolabeled RNA were dried directly. For

Northern blot analysis, the gels were rinsed twice for 5 min









with H20 and then capillary blotted onto 0.45 J

nitrocellulose paper overnight using 20 x SSC (3.0 M NaCl,

0.3 M sodium citrate pH 7.0). Following this step, the

nitrocellulose was dried 5 min under a heat lamp and baked

for 30 min at 80C in vacuo. The nitrocellulose was then

prehybridized for 20 min at 420 in 20 ml of 50% formamide, 5

x Denhardts solution (0.2% ficoll, 0.2%

polyvinylpyrrolidone, 0.2% bovine serum albumin), 0.3%

sodium dodecyl sulfate, 5 x SSPE (0.9 M NaCl, 0.05 M NaPO4

pH 7.7, 5 mm EDTA) and 100 ug fragmented DNA. The

nitrocellulose was hybridized overnight at 42C in 20 ml

fresh prehybridization solution to which 5 x 106 cpm probe

and 2.0 mls 50% dextran sulfate was added. Following

hybridization, the nitrocellulose was rinsed twice for 10

min at 22C in 100 ml 2 x SSPE with 0.1% SDS, once for 10

min at 22C in 100 ml 0.2 x SSPE with 0.1% SDS and once for

60 min in 100 ml 0.2 x SSPE with 0.1% SDS. Radiolabeled

probe was then detected by autoradiography.

Quantitation

All radiograph bands were quantitated by scanning on a

Molecular Dynamics Model 300A computing densitometer

(Molecular Dynamics, Sunnydale, CA). In general, each

experiment was repeated a minimum of two times, and each

probed blot was used to generate radiographs of varying

exposure times. Based on these radiographs, the lower limit

of detection is approximately 3% of the level of replication

observed for the positive control in each experiment. For









example, in a given experiment the control RNA band would be

visible following a 2h exposure, whereas, a 72h exposure of

the same blot would yield no evidence of a band for the

experimental RNA.

3DPOI and 2C Mutants

Poliovirus mutant 2C-31 plasmid DNA was generously

supplied by Dr. Jing Po Li and Dr. David Baltimore

(Massachusetts Institute of Technology, Boston, MA). This

DNA was used to transform HB101 E. Coli as described to

prepare large scale plasmid DNA preparation. Plasmid DNA

was transfected into mammalian cells as described except

that BSC 40 cells were used. After picking a plaque from

overlayered cells, vRNA was prepared as described (Young et

al., 1986). Briefly, virions were banded on cesium chloride

density gradients and the RNA extracted with phenol and

subsequently ethanol precipitated. A portion of both the

inoculum and the resulting virus were titered to confirm the

ts phenotype and the virus titer (Table 3-1).

Mutant tslO virus was generously provided by Dr.

Martinez Hewlett (University of Arizona, Tempe, AZ). Virion

RNA was prepared as described above except that the initial

plaque was generated by infection as opposed to transfecting

DNA.

Both tslO and ts2C-31 were analyzed by transfecting

vRNA into mammalian tissue culture as described previously.

In addition, similar experiments were conducted in which

BSC40 cells were infected with virus. In these cases, the







18


multiplicity of infection was approximately 1.0 and the

harvest time was 6 h post-infection.















CHAPTER 3

RESULTS


Introduction

I report here the development of an assay system which

allowed me to evaluate poliovirus RNA replication by

transfecting or co-transfecting mutant and helper RNAs into

mammalian tissue culture cells. Replication was evaluated

by isolating whole cell RNA, fractionating the RNA by

formaldehyde-agarose gel electrophoresis, blotting on to

nitrocellulose and probing with 32P-label poliovirus minus

strand RNA.

Since both the mutant and helper RNA were made as

transcripts from cDNA clones, there was no need to isolate

conditional mutants to prepare stocks of mutant RNA. In

this system, even "lethal" mutants could be evaluated, a

significant and unique improvement over previous methods of

analysis. Using this assay I evaluated a number of

poliovirus deletion mutants, as well as some minus strand

poliovirus RNA transcripts for their ability to replicate in

transfected cells in the presence and absence of helper RNA.

RNA transcripts of the parental clone, pT7D-polio,

contained a complete copy of the poliovirus genomic









sequence, two extra 5' terminal GMP residues and twelve 3'

terminal AMP residues followed by CGCG (Sarnow, 1989).

Using pT7D-polio we constructed a number of deletion clones

(Figure 3-1). These included both out-of-frame and in-frame

deletions in the capsid coding (Pl) region (RNA 1 deletes

nucleotides 1175-2978, and RNA 2 deletes nucleotides 1175-

2986, respectively), a large in-frame deletion encompassing

virtually the entire coding sequence of the viral genome

(RNA 3, deletes nucleotides 867-6011), an in-frame deletion

encompassing the P1-2A coding sequence (RNA 4, deletes

nucleotides 910-3519), and an in-frame deletion encompassing

the P1-2A-2B coding sequence (RNA 5, nucleotides 1175-3025).

Using RNA 2 as the parent clone, an in-frame deletion in the

3C coding region was constructed (RNA 6, deletes nucleotides

1175-2976 and nucleotides 5606-5824). In addition to these

deletion clones we have also evaluated the RNAs of a point

mutant in the 3D polymerase (tslO) and an insertional

mutation in protein 2C (ts 2C-31).

Synthesis of Infectious RNA Transcripts

Following synthesis, RNA transcripts were transfected

into cell cultures to determine their infectivity. T7D-

polio RNA transcripts resulted in the development of a

normal cytopathic effect (cpe) when transfected into HeLa

cells in culture (Figure 3-2). Conversely, none of the

transcripts of the deletion clones (RNA 1, RNA 2 and RNA 3)

caused a cpe when transfected into HeLa cells. The unstained

































Figure 3-1. Bacteriophage T7 RNA polymerase transcripts from
the DNA of pT7D-polio, and the various subclones, which had
been linearized with Mlul. A poliovirus protein map and
restriction site map are shown for reference.

























Kb 0 1
1 I 1 1


2 3 4
I I I I


5 6 7
I I I I I


pT7D-polio DNA Ban II
Asu II Nru I

T7 promoter 86 11 75
910
Protein
Map I o vPo


Sna B1 Ava I


Ban II
I Bst E2


Nar I
Bg9 II Asu II


II I I I


2956 2978


i 3925
3519


5605 6011
5824

2c 1A cPr* I--p1


VP3 I VP1 ApI2B I


pT7D-polio RNA
GG (A)n"T7D polio"


1175 CF 2978
(Nru I to Ava I)


IF
1175 (Nru I to Sna B) 2956


pT7D-polio RNA Al 175-2978
(A


pT7D-polio RNA A1 175-2956
(A


IF pT7D-polio RNA A867-6011
.....) (A n
867 (Asu II to Asu II) 6011



IF pT7D-polio RNA A910-3519
910 (Ban II to Ban II) 3519




IF pT7D-polio RNA Al 175-3925
1175 (Nru I to Bst E2) 3925




pT7D-polio RNA A 1175-2956,
IF IF 5606-5824
GG -- 'A.'.... ........ .................,- A L


1175 (Nru I to Sna BI) 2b56


)n RNA 1




')n RNA 2


RNA 3





RNA 4





RNA 5


RNA R


5606 5824
(Bgl II to Nar I)


.3'NC


E,


Lir"


L]L

































Figure 3-2. Cytopathic effect observed on Hela cell
monolayers at 72 hours post-transfection. Cell cultures
were transfected with RNA as indicated in Materials and
Methods and stained with crystal violet.





















T7D RNA


MOCK








RNA 1







RNA 2








RNA 3


'K.
K


4..

Jr


T7D RNA
and RNA 1







T7D RNA
and RNA 2


T7D RNA
and RNA 3


J









area seen on the plate transfected with RNA 3 alone was due

to the monolayer being incomplete prior to transfection. In

co-transfection experiments, however, both nonreplicating

(RNA 1 and RNA 3) and self-replicating (RNA 2) RNAs

interfered with the development of cpe in cells transfected

with T7D-polio RNA.

Replication of T7D-polio RNA in Transfected Cells

The replication of T7D-polio RNA was examined by using

a Northern (RNA) blot analysis and by measuring the

synthesis of 32P-labeled poliovirus RNA in transfected

cells. The transfection of T7D-polio RNA into HeLa cells in

the presence of actinomycin D and [32P]H3PO4 resulted in the

synthesis of 32P-labeled RNA that comigrated with

poliovirion RNA and that was not present in mock transfected

cells (Figure 3-3, lane 2 vs. lane 1). The bands seen in

the mock transfected lane are cellular nucleic acids whose

synthesis was not totally suppressed by the actinomycin-D

treatment. As expected, the 32P-labeled RNA was

polyadenylated and could be selected on an oligo(dT) column

(Figure 3-3, lane 3). This result indicated the poly (A)

tail on the 32P-labeled RNA synthesized in vivo was

elongated, since the input T7D-polio transcript RNA with a

poly(A)12 sequence did not bind to an oligo(dT) column (data

not shown). A Northern blot analysis of whole cell RNA

isolated from cells transfected with T7D-RNA confirmed that

poliovirus specific RNA of the expected size was present

(Figure 3-4, lane 5). In addition, an analysis of RNA






























Figure 3-3. Formaldehyde-agarose gel analysis of 32P-labeled
RNA synthesized in T7D-polio RNA transfected cells. Whole
cell RNA was isolated from HeLa cells which were either mock
transfected or transfected with T7D-polio RNA. RNA was
labelled by adding [32P]H3PO4 to the cell culture following
treatment with actinomycin-D (see text for details). A
portion of the whole cell RNA was further purified by
chromatography on oligo(dT) cellulose. Following gel
fractionation, the gel was dried and analyzed by
autoradiography.













8

Sz
T7D RNA -




T7D RNA































Figure 3-4. Northern blot analysis of the replication of
T7D-polio RNA and RNA 2 in transfected cells. RNAs were
transfected as indicated in Materials and Methods. Whole
cell RNA was isolated at 2 h or 8 h post-transfection,
fractionated on a formaldehyde-agarose gel, blotted onto
nitrocellulose and probed with a 32P-labelled poliovirus
minus strand probe.





















z z

z z







RNA 2 -









isolated at 2 h and 8 h post transfection showed that the

RNA detected at 8 h was newly synthesized RNA since the

input RNA could not be detected at 2 h (Figure 3-4, lane 4

vs. lane 5). The viral RNA present at 8 h post-

transfection could also be selected on an oligo (dT) column

which again indicated that this was newly synthesized viral

RNA with an elongated poly(A) sequence (data not shown).

Deletions in the Capsid Coding Sequence

Previous studies with poliovirus DI particles have

shown that in-frame deletions in the coding sequence for the

capsid proteins (i.e., the P1 region of the genome) do not

inhibit viral RNA replication (Cole and Baltimore, 1973a;

Hagino-Yamagishi and Nomoto, 1989; Kaplan and Racaniello,

1988). I constructed and evaluated both an out-of-frame

deletion (RNA 1) and an in-frame deletion (RNA 2) in the P1

region as described in Materials and Methods and as detailed

in Figure 3-1. Evaluation using a Northern blot analysis

showed that RNA 1 did not replicate either alone or when co-

transfected with T7D-polio RNA (Figure 3-5, lanes 4 and 5).

Conversely, RNA 2 replicated to relatively high levels in

transfected cells, both alone and in the presence of T7D-

polio RNA (3-4, lanes 2 and 3). RNA 2 was a very efficient

replicon and replicated to a higher level than did full-

length T7D-polio RNA (Figure 3-5, lane 2 vs. lane 6). One

final observation was that upon co-transfection with either

RNA 1 or RNA 2, T7D-polio RNA replicated at a reduced

efficiency relative to the level of replication observed
































Figure 3-5. Northern blot analysis of the replication of RNA
1 and RNA 2 in transfected cells. RNAs were transfected or
co-transfected as indicated and whole cell RNA was isolated
at 8 h post-transfections and analyzed as in Figure 3-4.
RNA 1 and RNA 2 contained out-of-frame and in-frame
deletions in the capsid coding region respectively.























z z
cr

z z
2 Z






T7D RNA ..
RNA 1 orRNA 2-









when T7D-polio RNA was transfected alone (Figure 3-5, lane 3

vs. lane 6 and lane 5 vs. lane 6). RNA 2 also replicated

less efficiently when co-transfected with T7D-polio RNA

(Figure 3-5, lanes 2 and 3). These results were consistent

with the reduction in the cpe observed when T7D-polio RNA

was co-transfected with the subgenomic RNA transcripts

(Figure 3-2).

Large In-Frame Deletion

Since RNA 1, with an out-of-frame deletion in the

capsid coding region, was not complemented by co-

transfection with T7D-polio RNA, further investigation was

restricted to RNAs containing in-frame deletions and point

mutations. RNA 3 contained a large in-frame deletion

spanning the P1, P2, and P3 coding regions, and was used to

determine if the 5' and 3' terminal sequences in poliovirus

RNA were sufficient to support replication in the presence

of helper RNA. Unexpectedly, bands of RNA 3 were detected

at 8 h post-transfection in cells transfected with RNA 3

alone and in cells co-transfected with RNA 3 and T7D polio

RNA. Since it was highly unlikely that RNA 3 was able to

replicate alone, it was hypothesized that the RNA being

detected was input RNA. This was confirmed in a second

experiment where it was shown that the same amount of RNA 3

was detected at 2 h and 8 h post-transfection in the

presence or absence of T7D-polio RNA (Figure 3-6). Thus,

RNA 3 appeared to be significantly more stable than T7D-

polio RNA and RNA 2, but it was not able to replicate in the
































Figure 3-6. Northern blot analysis of the RNA recovered from
cells transfected with the P1-P2-P3 in frame deletion mutant
(RNA 3). Cells were transfected or co-transfected as
indicated and whole cell RNA was isolated at either 2 h or
8 h post-transfection and analyzed as in Figure 3-4.

































T7D RNA -



28S rRNA -




RNA 3 -


j









presence of helper RNA. It was also noted that RNA 3

significantly interfered with the replication of T7D-polio

RNA (Figure 3-6, lane 3). The reason for this interference

is not yet understood but was consistent with the

interference observed in other co-transfection experiments.

Deletion in the 2A Coding Region

Because RNA 3 was not able to replicate in the presence

of helper RNA, subgenomic RNAs with smaller in-frame

deletions were tested for their ability to replicate in co-

transfection experiments with helper RNA. The basic

approach was to systematically expand the in-frame deletion

in the P1 region to include the P2 coding sequence. RNA 4

was constructed by deleting nucleotides 910-3519

(Figure 3-1) which included a significant amount of the 2A

coding sequence. RNA 4 was found to be self-replicating,

but at a reduced level relative to T7D-polio RNA and RNA 2

(Figure 3-7, lane 2 vs lanes 3 and 4). It was clear that

RNA 4 was replicating since no input RNA was detected at 2 h

(Figure 3-7, lanes 1 and 2). Co-transfection with both RNA

2 and T7D-polio RNA increased the amount of replication

observed with RNA 4 (Figure 3-7, lanes 5 and 6). This

result was reproducible and indicated that the 2A gene

product could be complemented in trans.

Deletion in the 2B Coding Region

The preceding analysis was continued by expanding the

P1-2A deletion to include sequences in the 2B coding region.































Figure 3-7. Northern blot analysis of the replication of the
P1-2A deletion mutant (RNA 4) in the presence or absence of
helper RNA. The RNAs were transfected or co-transfected as
indicated. Whole cell RNA was isolated at 8 h post-
transfection unless otherwise indicated. The RNA was
analyzed as in Figure 3-4.




























T7D RNA -
RNA2 -
RNA4 -


N 0
< <
z z
cr cr


z


z


z
c8
o0
CM
cc 2


li fa ik '1
^^ ^H ^" ^B ^
l^^^B kk.--j ^^^B
JkftA ^^H^B -m ^^^^H
^v ^^ -- ---









RNA 5 constructed to delete nucleotides 1175-3925 in-frame

(Figure 3-1). RNA 5 was not able to replicate in

transfected cells either alone (Figure 3-8, lanes 2 and 3)

or when co-transfected with either RNA 2 or T7D-polio RNA

(Figure 3-8, lanes 6 and 7). This suggested that either the

deleted sequences, the protein product, or both may be

required in cis for poliovirus replication to proceed. Some

interference with the replication of T7D polio RNA and RNA 2

was again noted (Figure 3-8, lane 4 vs. lane 6 and lane 5

vs. lane 7).

Deletion in the 3C Coding Region

Since the RNA 5 could not be complemented by co-

transfection with helper RNA, a continued expansion of the

deletion in the Pl-P2 coding sequence was not possible. The

analysis was continued by making an in-frame deletion in the

3C coding region using RNA 2 as the parental construct.

Thus, RNA 6 contained an in-frame deletion of nucleotides

5605-5824 and nucleotides 1175-2986. Analysis of RNA 6

indicated that it was not self-replicating (Figure 3-9,

lanes 2 and 3), and that it did not replicate upon co-

transfection with T7D-RNA (Figure 3-9, lane 5). Once again,

this suggested that either the 3C sequence or product or

both are required in cis for poliovirus RNA replication to

proceed.
































Figure 3-8. Northern blot analysis of the P1-2A-2B deletion
mutant's (RNA 5) ability to replicate in the presence or
absence of helper RNA. The RNAs were transfected or co-
transfected as indicated. Whole cell RNA was isolated at 8
h post-transfection unless otherwise indicated. The RNA was
analyzed as in Figure 3-4.







41













I',
a: <











RNA2
z 5 s z










... -- RNA5
































Figure 3-9. Northern blot analysis of the 3Cpro deletion
mutant's (RNA 6) ability to replicate in transfected cells.
The RNA's were transfected or co-transfected as indicated.
Whole cell RNA was isolated at 8 h post-transfection unless
otherwise indicated. The RNA was analyzed as in Figure 3-4.


















T7D RNA -
RNA6 -


F'


S









3DPOt Point Mutation

To evaluate the sequence coding the poliovirus RNA

polymerase (3DPOt), a point mutation in 3D was used. Tsl0 is

a temperature sensitive RNA negative mutant (Hewlett et al.,

1982). The titer of this mutant drops about two logs in

going from the permissive temperature (32C) to the

restrictive temperature (39.50C) (Table 3-1). Previous

work in our lab indicated that purified tslO polymerase was

sensitive to heat-inactivation when assayed in vitro

(unpublished result, B.J. Marasco and J.B. Flanegan). On

sequencing, the 3DPOt coding sequence was found to contain a

single nucleotide change at position 7167 (Figure 3-10).

This would result in a single amino acid change from Met to

Thr at position 394 in the tslO0 polymerase. This mutation

was constructed in a wild type background using a poliovirus

cDNA clone and was shown to again confer the ts phenotype

(personal communication; K.Kirkegaard, University of

Colorado, Boulder, CO).

To determine if it was possible to complement the

mutation in tslO0, cells were transfected either with tslO0

vRNA alone, or co-transfected with tslO0 vRNA and RNA 2.

Transfections were done both at 320C and 39.5C. Since RNA

2 was self-replicating and smaller than tslO0 vRNA, it was

possible to follow the replication of each RNA using a

Northern blot analysis. As expected, tslO0 RNA was found to

replicate a a much lower level at 39.50C than at 32C

(Figure 3-11, lane 6 verses lane 2). The small amount of
















TABLE 3-1. TITER OF ts 10 and ts 2C-31 VIRAL STOCKS




TEMPERATURE ts 10 (pfu/ml) ts 2c-31 (pfu/ml)



32.0* C 1.0 x 109 3.1 x 109
39.50 C 7.5 x 106 2.1 x 107































Figure 3-10. Sequence analysis of polymerase coding
sequences in viral RNA from wild type poliovirus (Mahoney)
and tslO mutants. Shown above is the observed nucleotide
change at position 7167 and the resulting amino acid change.
This was the only difference found in the 3DP01 coding
region. RNAs were sequenced using the primer-extension
dideoxy method as described in materials and methods.
























WT T810




'-






(MET) U
A,9 C (THR)


U C G A U C G A

































Figure 3-11. Northern blot analysis of the replication of
tslO in transfected cells at 32C and 39.5*C. RNAs were
transfected or co-transfected as indicated. Whole cell RNA
was isolated at 8 h post-transfection and was analyzed as in
Figure 3-4.







49















32 C 39 C




z z


< -- <




co Z' o gZ .
i-- ccI. -









RNA replication observed in cells transfected with tslO

alone at the restrictive temperature was similar to the

level of RNA synthesis observed in infected cells at the

restrictive temperature (Figure 3-11, lane 6 verses Figure

3-12, lane 3). In the presence of helper RNA (RNA 2),

however, ts 10 replicated at essentially the same level seen

at the permissive temperature (Figure 3-11, lane 8 verses

lane 2). Thus, the results indicated that tslO was

complemented by the helper RNA at the restrictive

temperature. In addition, the results indicated that the

replication of tslO was also enhanced at 32*C in the

presence of RNA 2. A surprising result was that the

replication of RNA 2 was inhibited in the co-transfection

experiment at 39.5*C although RNA 2 was able to replicate

when transfected alone at 39.50C (Figure 3-11, lane 7 verses

lane 8). At 32*C, the replication of RNA 2 was totally

inhibited (Figure 3-11, lane 3). Thus, the in-frame

deletion in the capsid coding region has a "cold-sensitive"

phenotype. At the present time it is not known if this

phenotype results from an effect on the RNA or the protein

or both.

2C Insertion Mutation

Mutant 2C-31 was constructed by making a twelve base

pair insertion at position 4886 in the poliovirus genome (Li

and Baltimore, 1988). This insertion conferred a ts

phenotype on the virus such that the titer of the mutant was

150-fold higher at the permissive (32*C) temperature































Figure 3-12. Northern blot analysis of RNA replication in
virus infected cells at the permissive (32*C) and non-
permissive (39.5C) temperatures for tslO and ts2C-31.
Monolayers were infected using a moi of approximately 1.0.
Whole cell RNA was isolated at 6h post-transfection and was
analyzed as in Figure 3-4.











Ts 10


MOCK 32 C


39 C


2C-31


32 C 39 C


-4









compared to the titer at the restrictive temperature

(39.50C) (Table 3-1).

An analysis of RNA replication in transfected cells

confirmed the ts phenotype of 2C-31 (Figure 3-13, lanes 3

and 7). This was similar to the results obtained in virus

infected cells at the permissive and restrictive

temperatures (Figure 3-12, lanes 4 and 5). RNA replication

in the presence of helper virus (RNA 2) at 39.5C was

similar to the level seen with 2C-31 alone at the permissive

temperature (Figure 3-13 lane 8 vs. lane 3). Thus, it was

clear that the 2C-31 mutant could be complemented in trans.

Minus Strand Replication

In the previous analysis, my efforts were directed at

following poliovirus RNA replication starting with plus

strand transcripts. Since poliovirus replication includes

both plus to minus strand synthesis as well as minus to plus

strand synthesis, one can also ask questions regarding the

ability of poliovirus minus strand transcripts to be copied

in transfected cells. Since minus strands are unable to

make any poliovirus-specific protein products, minus strand

RNA would require helper RNA to provide these proteins in

trans. To determine whether minus strand transcripts can be

copied in vivo, I assayed for the synthesis of plus-strand

RNA products in cells co-transfected with a self-replicating

helper RNA.

Full-length poliovirus minus-strand RNA transcripts of

p0OF2612 were co-transfected with RNA 2 transcripts as helper
































Figure 3-13. Northern blot analysis of the replication of
ts2C-31. RNAs were transfected or co-transfected as
indicated. Whole cell RNA was isolated at 9 h post-
transfection for cells transfected at 32 C and 7 h post-
transfection for cells transfected at 39.5'C. The RNA was
analyzed as in Figure 3-4.



















320 C


390C


cm


SCci
8
2 a









RNA. In a Northern blot analysis of the RNA extracted from

the transfected cells, no full-length plus-strand RNA was

detected (data not shown). The pOF2612 minus-strand RNA

transcripts, however, contained extra vector sequences at

their 3' ends. In a first attempt to determine if this was

blocking the replication of these RNAs, we proceeded to

analyze sub-genomic minus-strand RNA transcripts which had

the correct 3' terminal sequence.

A minus strand transcript 127 nucleotides long which

contained the correct 3' end was received from Dr. Daniel

Brown, University of Florida (see Materials and Methods). A

Northern blot analysis indicated that co-transfection of

this RNA with either T7D-polio RNA or RNA 2 did not result

in the synthesis of plus-strand RNA of the appropriate site.

Since 127 nucleotides is a relatively short sequence, a

larger transcript was tried next. A minus strand RNA

transcript 789 nucleotides long which contained the correct

3' end was also co-transfected with either T7D-polio RNA or

RNA 2. Again, there was no evidence that complementary

plus-strand RNA was synthesized in transfected cells. These

data suggest that the poliovirus RNA replication proteins

cannot be provided in trans for the replication of minus-

strand RNA. Alternatively, the replication of minus-strand

RNA in transfected cells may require full-length transcripts

that have the exact 3' terminal sequence. Thus, a

significant amount of additional experimental work will be

required to answer this important question.















CHAPTER 4

DISCUSSION


Introduction


The replication of poliovirus RNA in transfected cells

was examined in this study. Mutant RNAs containing specific

deletions, insertions, and point mutations were evaluated

for their ability to replicate either alone or in the

presence of helper RNA. This general approach had the

advantage of providing a means to investigate the role of

specific sequences that are important for RNA replication

without isolating conditional lethal mutants. By co-

transfecting a mutant RNA with a self-replicating helper

RNA, cis and trans active genetic elements could be

identified.

Deletion Mutants

Both the full-length viral RNA (T7D-polio) and RNA 2

which contained an in-frame deletion in the capsid encoding

region were shown to be efficient replicons. In accord with

previous work (Kaplan and Racaniello, 1988), RNA 2 was

observed to replicate more efficiently than T7D- polio RNA

in a standard 8 h experiment. In addition, a time course

experiment with RNA 2 showed that this RNA continued to









replicate for at least 24 h post transfection. This made

the RNA 2 construct a valuable tool for increasing the

sensitivity in experiments where a second-site mutation was

assessed for its effect on RNA replication.

In co-transfection experiments, RNA 2 and T7D-polio RNA

were found to interfere to some degree with each other's

replication. From the results of previous studies with DI

RNAs (Cole and Baltimore, 1973a; Cole and Baltimore, 1973b)

and subgenomic replicons (Kaplan and Racaniello, 1988), we

expected RNA 2 to interfere with the replication of the full

length RNA. It was clear, however, that the full length RNA

also had a small but reproducible effect on the replication

of the subgenomic replicon. RNA 1 which contained an out-

of-frame deletion in the P1 region and RNA 3 which contained

a very large in-frame deletion of the P1-P2-P3 regions did

not replicate either alone or when co-transfected with T7D-

polio RNA. In co-transfection experiments, RNA 1 and RNA 3

significantly interfered with both the cpe of T7D-RNA on

cultured cells and the replication of T7D-RNA. Similar

results were obtained with other nonreplicating subgenomic

RNAs examined by us. It should be noted that during the

course of this study other laboratories have also reported

interference by both out-of-frame mutants (Hagino-Yamagishi

and Nomoto, 1989) as well as nonreplicating subgenomic

constructs (Kaplan and Racaniello, 1988).

This observation of interference indicated that both

RNAs were being taken up by the same cells during the co-









transfection procedure, a result consistent with the results

of many DNA co-transfection experiments described in the

literature. Thus, neither the replication of RNA 1 nor RNA

3 were supported by supplying the replication proteins in

trans. This further indicated that one or more of the viral

replication proteins was cis acting, and was consistent with

the previous report that 2B and 3D mutants could not be

complemented by other poliovirus mutants (Bernstein et al.,

1986).

Analysis of two of the deletion mutants further

supported the contention that certain poliovirus proteins or

sequences are required in cis for replication. Neither RNA

5 which is a deletion extending into the 2B coding region,

nor RNA 6 which is a small deletion in the 3C region, were

capable of replication either alone or on cotransfection

with helper virus. These two results coupled with the

result for RNA 3 suggest that poliovirus replication does

have cis requirements and that both the 2B and 3C products

may be involved. It should be noted that Dewalt and Semler

(1989) have reported a 3C point mutant which is

complementable. This would suggest that it may be the 3C

sequence which is being required in cis for our mutant.

The mounting evidence that one or more of the virus-

encoded replication proteins are cis acting supports the

model for RNA replication previously proposed by Bernstein

et al. (1986). This model explains the cis requirement for

certain proteins by suggesting that the replication of a









molecule of plus-strand RNA requires a complex of viral

proteins which are translated from that same RNA molecule.

This model predicts that minus-strand synthesis is directly

linked to protein synthesis. The mechanism would have the

obvious advantage of preventing the replication of mutant

plus-strands that contain defective cis active genetic

elements that are required for RNA replication. Considering

the high error frequency that has been determined for

poliovirus RNA polymerase, both in vitro (Ward et al., 1988)

and in vivo (C.D. Ward, Ph.D. dissertation, University of

Florida), this could be a significant factor in insuring

that a large number of viable progeny are produced during

each round of replication.

The finding that RNA 4 (a P1-2A in-frame deletion) was

self-replicating was very interesting. Although most

subgenomic replicons replicate more efficiently than full-

length viral RNA, RNA 4 was shown to be a less efficient

replicon (compare RNA 4 with T7D-polio RNA in Figure 7). In

the presence of either helper RNA, however, there was a

significant increase in the replication of RNA 4. This was

especially significant in view of the interference that was

repeatedly observed between different RNAs in our previous

experiments. Thus, these results indicated that it was

possible to complement a 2A deletion mutant in trans by co-

transfecting with either T7D RNA or RNA 2. This was

consistent with the previous results of conventional genetic

studies where it was shown that a small plaque variant of 2A









could be complemented by other site specific virus mutants

(Bernstein et al., 1986). I feel that this is a very

important result since it demonstrates for the first time

that it is possible to use the co-transfection approach to

characterize cis and trans acting genetic elements in the

viral genome. In addition, it may be possible to construct

even smaller replicons by removing additional sequences that

code for other trans acting gene products. The above

results raise many interesting questions about this 2A

mutant. The deletion in RNA 4 would result in removing 45

amino acids from the N-terminus of the 2A protein and in the

formation of a fusion protein containing 55 amino acids from

the N-terminal end of VP4. I would not expect this fusion

protein to retain the normal activity associated with 2A for

several reasons. It has been proposed that 2A is homologous

to the trypsin-like family of small serine proteases (Bazan

and Fletterick, 1988). This group of proteases are all

found to contain an active triad site of conserved His, Asp,

and Cys residues. The deletion in RNA 4 removes the His

residue. In a study by H. Toyoda et al. (Toyoda et al.,

1986), it was shown that a simple four amino acid insertion

near the N-terminal end of 2A inactivated the protease in an

in vitro assay. Thus, it is reasonable to suggest that the

modified 2A coded for by RNA 4 is inactive. This in turn

suggests that the inhibition of host protein synthesis and

the 2A-mediated cleavage of the polyprotein to form 3C' and

3D' are not absolute requirements for viral RNA replication.









2C and 3DP0t Mutants

Analysis of pre-existing insertion and point mutants

has also yielded interesting results and confirmed our

system's ability to detect complementation. In the case of

tsl0, a point mutation in the 3DPt0 sequence, I was able to

confirm that RNA replication was indeed ts. In addition,

the reduced replication seen at the restrictive temperature

could be complemented back to levels seen at the permissive

temperature by cotransfection with a helper virus. Previous

reports in the literature suggested both that 3DP'1 could be

complemented (Agut et al., 1989) and that it could not

(Bernstein et al., 1986). The occurrence of both

complementable and noncomplementable mutants in the same

gene suggests that individual proteins may have multiple

domains and include both cis and trans functions.

The second mutant, 2C-31, was initially isolated as a

ts mutant (Li and Baltimore, 1988). It exhibited a small

plaque phenotype and further analysis showed it to be RNA

negative at the nonpermissive temperature. On

investigation, both plaque assays and replication assays

confirmed the ts phenotype. Co-transfection with helper RNA

further revealed that the 2C-31 defect could be complemented

to permissive replication levels.

Temperature sensitivity of RNA2

During our co-transfection experiments with the two ts

mutants, it appeared that RNA2 was also being effected by

the temperature shifts. The observation that RNA2 was









unable to replicate at 32*C was surprising. It appears

that RNA2 is translated at 32C since tsl0 replication is

enhanced during co-transfection at 320C, however, there is

absolutely no evidence that RNA2 replicates. Possible

explanations include aberrant folding of the RNA2

polyprotein or transcript at 32*C vs. 39.5C. Another

possibility is that all the transcripts from T7D-polio and

it's subclones are effected by the two extra G's at the 5'

terminus or the Ai2CGCG at the 3' end. Experiments which

evaluate the replication efficiency of T7D- polio

transcripts at 32*C should distinguish between these

possibilities. If T7D-polio reacts the same as RNA2, the

cold sensitivity is probably a result of the unique

characteristics cited. If not, aberrant folding is the more

likely explanation.

It was noted, additionally, that at 39.5*C RNA2

replication was almost completely inhibited during co-

transfection even though it self-replicated and functioned

effectively as a helper RNA. Once again, this could be the

result of the unique 5'and 3' sequences of this transcript

versus the wild type ends. It is known that the poly(A)

sequence is necessary for replication, and the (A)12

transcript may not compete well with the wild type (A)7-10oo

at 39.5C. It is also possible that a change in the

secondary structure due to the deletion makes this template

less functional than the full length vRNA. Co-transfection

experiments comparing T7D-polio and RNA2 at 39.50C should









answer these questions. If the problem is secondary

structure, T7D-polio would be expected to replicate normally

and RNA2 to be suppressed. If the problem is the short

poly(A) sequence, both RNA2 and T7D-polio should be effected

similarly, since they both have the same 3' end.

Interference

As stated previously, the results of co-transfection

experiments indicated that both replicating and non-

replicating poliovirus transcripts interfered with the

replication of the helper transcripts. While the reason for

this is not understood, possible explanations include simple

competition between the two nucleic acids during

transfection and/or replication (i.e. the number of input

molecules of one transcript versus the other), as well more

complex explanations, such as a differential affinity for,

or use of some replication co-factor during the replication

process. Experiments evaluating different ratios of the co-

transfected nucleic acids should distinguish between these

choices. If the amount of replication observed following

co-transfection mirrors the input levels, a simple

competition is the most simplistic and reasonable

explanation. If the replication levels do not follow the

input pattern, then differential affinity for, or use of a

replication factor is probably a more likely explanation.

While the current study was not directed at this

question, our results suggest that the second explanation

may be more likely. All our experiments used equimolar









amounts of the co-transfected transcripts, but the

inhibition observed was not always in the 50% range. When

T7D-polio transcripts were used as the helper RNA, non-

replicating transcripts (RNAs 1,3,5 and 6) were shown to

inhibit T7D-polio replication by approximately 75% (63-81%).

On the other hand, self-replicating co-transfectants only

inhibited T7D-polio by 32% (RNAs 2 and 4). One possible

explanation is that the non-replicating transcripts may be

removing some replication component by binding it

irreversibly to a nonfunctional template.

Cis and trans activity

During the course of this study, it was determined that

various poliovirus proteins had either cis or trans activity

relative to the replication process. This observation is

consistent with the concept of a replication complex as

proposed by Bernstein, et al. (1986). Their hypothesis

proposes that poliovirue RNA replicates via a complex of

proteins which use the plus strand molecule from which they

were translated, as a template for minus strand synthesis.

Proteins which are irreversibly bound to the complex would

be cis active and those which are not would be trans active.

The hypothesis also suggests that some proteins might bind

in a reversible fashion and thus show both cis and trans

activity during the course of replication. I would extend

this point to suggest that individual proteins might have

multiple functions, which include both cis and trans

activities, and further that protein activities that require









movement (i.e. multiple active sites or movement along a

template) might be more likely to be trans acting.

Our results are consistent with this model. We have

observed that both an out-of-frame capsid deletion (RNA 1)

and a large in-frame deletion (RNA 3) are unable to be

complemented, suggesting the presence of cis active

elements. More specifically, we have seen that the 2B

product is apparently required in cis. We have also

observed that both the 2A and 2C products are able to be

provided in trans, and finally, in conjunction with existing

data, we have shown that both 3C and 3D seem to have both

cis and trans active regions. It should be noted, however,

that while our results are consistent with Bernstein's

hypothesis, there may be other possible explanations. Until

protein analysis of these mutants is conducted, aberrant

polyprotein processing will remain a valid concern regarding

cis activity, a basic premise of the hypothesis.

Minus Strand Transcripts

The fact that there are multiple initiation events on

minus strands templates during plus strand synthesis

suggests that viral replication proteins can be supplied in

trans during plus strand synthesis. To test this idea, I

cotransfected a number of minus strand transcripts into cell

culture with plus stranded RNA helper transcripts. In the

first experiment, a full-length minus strand RNA was

cotransfected with a self-replicating subgenomic RNA as

helper (RNA 2). On analysis there was no evidence that full









length plus stranded RNA was synthesized. It should be

noted, however, that these minus strand transcripts

contained some additional vector sequence at their 3' ends

which could conceivably block plus strand synthesis. In

hopes of overcoming this potential problem, subgenomic

transcripts were generated which contained an exact 3'

poliovirus end. On analysis, neither the 127 base

transcript nor the 789 base transcript showed evidence of

replication.

Taken together, these results suggest a couple of

possible explanations. The first would be that one needs to

have both a correct 3' end and more than 789 nucleotides of

sequence to allow for minus strand RNA to serve as a

template for plus strand synthesis. This hypothesis could

be tested definitively by the construction of a clone

capable of generating a full length minus strand poliovirus

RNA that has an exact 3' end. If this construct was capable

of replication, one could then determine cis and trans

requirements more specifically by constructing various

subclones and point mutants similar to those used for the

plus to minus strand analysis. Should the full length

construct prove unable to replicate, it would suggest that

the minus strand template may be part of a growing

replication complex which remains associated throughout the

replication process.
Conclusion

This study establishes that poliovirus RNA replication

can be studied in cultured cells transfected with viral RNAs









synthesized in vitro. It provides a powerful new approach

for studying the unique replication strategies that are

utilized by poliovirus and reports a number of interesting

findings. It was shown that neither the capsid proteins or

their coding sequence, nor the amino terminus of the 2A

protein or it's coding sequence, was necessary for

replication. Assuming the 2A product is no longer active,

this further implies that the 3C', 3D' products may also be

unnecessary for viral replication. The fact that neither a

large in-frame deletion nor a small out-of-frame deletion

were complementable indicates that poliovirus does indeed

have cis requirements during minus strand synthesis.

Additional analysis showed that the 2B and 3C products seem

to have cis activities.

It was further determined that the 2A, 2C and 3D

products could be complementable in trans. Since at least

one other 3D mutant has been shown to be noncomplementable,

these results further support the idea that the various

polio proteins may have multiple domains and include both

cis and trans activities within a single protein.















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BIOGRAPHICAL SKETCH


I was born on June 5, 1949 in Utica, New York, to

Ramsay and Eva Collis, the first of two sons. I was raised

in Utica, where I attended St. Francis de Sales Elementary

School and Notre Dame High School. Following high school, I

was married to Lynne A. Nowak. We have one son, David, who

currently attends graduate school at Georgetown University.

We lived in Rochester, New York, from 1968 to 1971 while I

attended the University of Rochester. We then moved to

Storrs, Connecticut, where I did graduate work at the

University of Connecticut. In 1974 I accepted a research

position at the Institute for Cancer Research in

Philadelphia, Pennsylvania, and we moved to Cheltenham,

Pennsylvania, a suburb of Philadelphia. In 1980, I accepted

an administrative position at the University of Florida and

we have lived in Gainesville ever since.















I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.



times B. Flanegai ,hair
Professor of Imminlogy and
Medical Microbiology


I certify that I have read this study and that in my
opinion it conforms to acceptable standards o scholarly
presentation and is fully adequate, in scope d quality, as
a dissertation for the degree of doctor of P losophy.



4W liam W. Hauswirth
Professor of Immunology and
Medical Microbiology


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.



Edward K. Wakeland
Professor of Pathology and
Laboratory Medicine


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.



Sue A. Moyer
Professor of Immunology and
Medical Microbiology








I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.


obert 4. Yerl
Professor of Botany

This dissertation was submitted to the Graduate Faculty
of the College of Medicine to the Graduate School and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
May, 1991 -A ., .A__/_
Dean, College of medicine


Dean, Gi(Aduat6 School












































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