In vivo synthesis of RNA by vesicular stomatitis virus and its mutants

J. Mol. Biol. (1974) 87, 31-53

In viva Synthesis of RNA by Vesicular Stomatitis Virus

and its Mutants A. FLAMAND AND D.H.

I,. BISHOP

The Institute of ilficrobiology Rutgers, The State University

New Brunswick N.J. 08903, U.S.A. (Received 28 January 1974, and in revised form 4 April 1974) The synthesis of viral complementary RNA by vesicular stomatitis virus has been examined. Both wild-type or temperature-sensitive mutant viruses belonging to Group I or Group IV (RNA-inhibited) or Group III (RNA-uninhibited) have been studied at permissive (31°C) or non-permissive (39.5 or 4072) temperatures for virus production. Transcription of RNA from the infecting virion genomes (primary transcription) as well as amplified (secondary) transcription has been demonstrated. Primary transcription at 31, 395 or 40°C has been observed for mutants from all three complementation groups as well as for the wild-type virus. Secondary transcription is inhibited at 39.5 and at 40°C for mutants of both Groups I and IV but not for the wild-type or (at 40°C) for a Group III mutant,. Secondary transcription is not inhibited when either the mutant or wild-type viruses are grown at 31°C. It is calculated that for the wild-type virus grown at 31 or at 38°C by five to six hour post-infection, there are about 500 transcriptive intermediates per cell irrespective of the multiplicity of infection. These intermediates are responsible for the accumulation of more than lo* viral complementary RNA genome equivalents per cell. At 38 or at 39*5”C during a wild-type virus infection more than lo* or lo5 virion particles are produced per cell. Evidence is presented to indicate that there is a disproportionate synthesis of the various vesicular stomatitis virus messenger RNA species.

1. Introduction Vesicular stomatitis virus is a member of the group of rhabdoviruses whose subjects include viruses capable of infecting mammals, insects, plants and fishes (Knudson, 1973). Among the animal rhabdoviruses, several have been shown to possess a viral RNA-dependent RNA transcriptase (Aaslestad et al., 1971; Baltimore et al., 1970; Chang et al., 1974). In vitro, VS virust transcriptase is capable of transcribing the viral RNA repetitively, sequentially and completely to give a series of product RNA molecules smaller than, but complementary to, the virion genome (Bishop, 1971; Bishop & Roy, 1971; Roy & Bishop, 1972). These product RNA molecules possess distinguishable 5’-nucleotide sequences starting with either adenoeine or guanosine nucleotides, which suggests that they are initiated at different sites on the templat,e molecule (Roy & Bishop, 1973 ; Chang et aE., 1974). t Abbreviations used: VS virus, vesicular temperature sensitive.

stomatitis

31

virus;

p.f.u.,

plague-forming

units;

ts,

32

A. FLAMAND

AND

D. H.

L. BISHOP

Messenger RNA isolated from VS virus-infected cells is also smaller than, and complementary to, the virion genome. It has been observed that VS virus messenger RNA is synthesized in cells in which protein synthesis is inhibited by cycloheximide or puromycin (Flamand & Bishop, 1973; Huang et al., 1970; Huang & Manders, 1972; Mudd $ Summers, 1970). These latter results indicate that either the virion polymerase or a pre-existing cellular enzyme is responsible for the initial intracellular synthesis of viral complementary messenger RNA (primary transcription). Although primary transcriptioncan be detected at 18°C it is optimal between 36 and 39*5”C, and for the best virus preparations between one-tenth to four-tenths of the absorbed virus particles (at multiplicities of from 5 to 20,000 virions per cell) can be shown to participate in primary transcription. The in vivo primary transcription process is both repetitive and complete taking only a few minutes to render a complete set of transcripts from the viral RNA (Flamand t Bishop, 1973). In relation to the overall synthesis of viral complementary RNA in VS virusinfected cells, we have sought answers to the following questions. (a) How long does primary transcription last? (b) In the subsequent synthesis of viral complementary RNA (secondary transcription), how many transcriptive intermediates are involved? (c) Are the virus messenger RNA species synthesized equally or disproportionately during primary and secondary transcription? (d) How many copies of viral complementary RNA are made per cell? (e) Are the amounts of viral complementary RNA which are synthesized during secondary transcription higher at high multiplicities of infection! There are five known complement&ion groups of temperature-sensitive mutants of VS virus (Flamand & Pringle, 1971). Synthesis of RNA at non-permissive temperatures is inhibited for mutants of Groups I and IV and for some mutants of Group II (Flamand & Lsfay, 1973; Lafay, 1969; Pringle & Duncan, 1971; PrintzA& et al., 1972 ; Unger & Reichmann, 1973). VVe have shown that particular mutants from all five complement&ion groups of VS virus, when grown at permissive or nonpermissive temperatures for virus production, initially perform essentially equivalent amounts of primary transcription (Flamand & Bishop, 1973). In this paper we have extended those studies by examining primary and secondary transcription for mutants of Groups I, III and IV at different temperatures of growth and in relation to the production of both temperature-sensitive and revertant wild-type progeny virions. Most studies on VS virus-directed RNA synthesis have relied on the use of [3H]nucleosides to label RNA species and, at early times of an infection, sctinomycin D to suppress host DNA-directed RNA synthesis. However, in the presence of this drug, residual cell RNA synthesis is about 5 to 15% that of untreated cells so that small amounts of viral RNA synthesized early in an infection are impossible to detect-even in the presence of actinomycin D. In quantitating the amounts of RNA synthesized, there is also a problem of the rate of uptake of [3H]nucleosides by infected cells, their dilution by unlabeled cellular nucleoside pools as a function of an infection time-course, and the difficulties of distinguishing residual cellular RNA synthesis from viral-like or viral complementary RNA species. We have developed a method for detecting viral complementary RNA synthesis in infected cells; this method avoids these problems and has allowed us to obtain answers to the questions and investigations enumerated above. The procedure involves infecting cells with very highly 3H-labeled virus (so that low multiplicities

VESICULAR

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33

of infection can be used), and extracting the infected cell nucleic acids after suitable incubation periods. The content of 3H label in the nucleic acids is ascertained in order to calculate the number of adsorbed virions. The [3H]ribonuclease resistance is then determined either directly to determine the number of adsorbed genomes involved in transcriptive intermediates, or after annealing to an excess of viral [3H]RNA extracted from unused virus to give the total content of viral complementary RNA. We have previously shown that after 20 minutes of incubation and before any annealing, the percentage of [3H]RNA that is ribonuclease resistant corresponds well to the number of transcriptive intermediates formed from the infecting virus particles, i.e. between 10 and 40% of the absorbed virus (Flamand & Bishop, 1973). Also this number corresponds reasonably well to the number of potential plaqueforming units in a virus inoculum. Therefore, it has been possible in the present, studies to determine, from the amount of viral complementary RNA synthesized, how many genome transcript equivalents are made per cell as well as the approximate number of progeny transcriptive intermediates.

2. Materials and Methods (a) Chemicals Cycloheximide

and puromycin

hydrochloride

were obtained from Sigma Chemical Co.,

St Louis, MO. (b) Virus strains and their

propagation

Vesicular stomatitis virus, Indiana serotype, was grown in baby hamster kidney cells (BHK21) in monolayer cultures using Eagle’s minimal essential medium (Eagle, 1959), fortified with non-essential amino acids (Gibco, Grand Island, N.Y.), glutamine and serum. Cells were maintained in 10% calf serum except that during infection with the virus, 5% fetal calf serum was used. Confluent monolayers of 3 x 10” cells in Blake bottles were infected with approximately 0.1 p.f.u./cell, then incubated in 50 ml of medium at 31’C for 48 h in the presence of 1 mCi each of [3H]uridine (21 Ci/mmol), [3H]adenosine (21 Ci/mmol) and [3H]guanosine (16 Ci/mmol) (although occasionally 1 mCi of [3H]cytidine (22 Ci/mmol) was used in lieu of one of the other nucleosides). The following VS virus Orsay mutants were used: Group I-5; Group I-80; Group 111-23; Group IV-62 and Group IV-194, in addition to the wild-type strain. (c) Virus purijcation In order to maintain infectivity ratios as high as possible, the purification procedure was kept short, Cell supernatant fluids (100 ml) were clarified by centrifugation at 10,000 g at 4°C for 30 min and precipitated by 7.5 g polyethylene glycol/lOO ml (Carbowax 6000, Union Carbide, Linden, N. J.) in the presence of 2.5 g NaCl/lOO ml and stirred at 4°C for 4 h. The polyethylene glycol precipitate was recovered by centrifugation (10,000 g at 4°C for 30 min), suspended in 3 ml of 0.14 m-NaCl, 0.003 M-KCl, 0.01 M-sodium phosphate buffer, 0.5 mm-EDTA, pH 7.4 (versene solution) and loaded on a g-ml gradient of 70% to 20% sucrose (w/v) in 0.15 M-N&Cl, 0.01 M-Tris.HCl buffer (pH 7.4) prepared in A Spinco SW41 centrifuge tube. The gradient was centrifuged at 4°C and 150,000 g for 20 min. No slower-sedimenting defective virion bands were observed at this stage of the purification procedure for any of the preparations used. The virus band was harvested by pipette, then one quarter of it was loaded on a lo-ml column of 4% agarose (Biorad Labs, Richmond, Cal.) freshly equilibrated, packed and washed at 4°C in Eagle’s medium. Fractions of 0.5 ml were collected and the samples containing an opalescent virus suspension were pooled. Portions of this [3H]virus in Eagle’s medium were used for infecting cells. The rest of the [3H]virus was used for preparing labeled viral RNA.

34

A. FLAMAND

AND

D. H. L. BISHOP

(d) Plaque amay~ Plaque assays were performed at 31 or at 39.5% using confluent monolayers of BHK cells and plaques were counted after 36 to 48 h of growth. Mutant virus stocks, which all contained less than O.Olo/o wild-type reverts&s, were verified by complement&ion tests es described previously (Flamand, 1969,197O). (e) Infection of cells by highly labeled [3H]virus The infection of confluent monolayers of 3 x lo6 BHK cells by, on average, 2 x lo* cte/min of [3H]virus at 4°C followed the procedure described previously except that to obtain adsorption of up to 15% of the virus inoculum, DEAE-dextran (10 pg/ml) was used (Flamand & Bishop, 1973). (f) Extmction

of nucleic acids, ribonucleme resicrtance determinations and RNA annealing procedures Unused [3H]virus was centrifuged at 4°C and 150,000 g for 2 h through a l-ml cushion of 30% (w/v) sucrose in 0.15 M-NaCl, 0.01 M TriseHCl buffer (pH 7.2) in a Spinco SW41 rotor. The virus pellet was suspended in O-4 M-N&~, 0.01 M-Tris.HCl buffer (pH 7.2), extracted for RNA by phenol/cresol and checked for homogeneity and lack of defective virion RNA as described previously (Flamand $ Bishop, 1973). The specific activities of the viral RNAs between different experiments varied from 5 x lo8 to 4 x lo9 cts/min per mg RNA. The extraction of uninfected or infected cell nucleic acids, in which better then 85% of the nucleic acids were recovered, involved using diethyl pyrocarbonate as a RNAase inhibitor, and sonic&ion to shear the cellular DNA followed by phenol/cresol extraction, as described previously (Flsmand & Bishop, 1973). All nucleic acid samples were finally suspended in 0.2 ml of 0.4 M-N&~, 0.01 M-Tris.HCl buffer (pH 7.2) and stored at - 20°C until required. Portions of RNA were diluted into 1 ml of 0.4 M-N&~, 0.01 M-TriseHCl buffer (pH 7.2) and equal volumes were used to determine the trichloroecetic acid-insoluble radioactivity either (1) directly or (2) after incubating at 37°C for 30 min with pancreatic RNAaae (20 pg RNAase per 0.5 ml containing no more than 100 pg RNA). Additional use of 20 pg RNAase T1 gave the same results. The annealing conditions followed the procedures described previously except that duplicate dilutions of infected cell nucleic acids were mixed with different amounts of viral [3H]RNA in a total volume of from 15 to 25 ~1 (depending on the experiment) containing 0.4 M-NaCl, 0.01 M-Tris.HCl buffer (pH 7.2) then sealed in a capillary and incubated at 60°C for 24 to 48 h (Flamand & Bishop, 1973). After annealing, the RNAase resistance WIXJdetermined. Usually only RNAase resistance values representing between 10 to 30% of the added [3H]RNA were used to calculate the amount of viral complementery RNA in an infected cell nucleic acids sample due to the possibility of disproportionate synthesis of the various VS virus messenger RNA species (see below). Only when similar amounts of RNA&se-resistant [3H]RNA were found for both quantities of added viral [sH]RNA, were the results used for calculating the total amount of viral complementary RNA in an infected cell nucleic acid sample. Parallel annealing8 with uninfected cell nucleic acids and viral [3H]RNA were used to determine the [3H]RNAaseresistant core of the viral RNA and this value (1 to 3% of the viral [3H]RNA) w&s subtracted from the annealing results of the infected cell samples.

3. Results (a) The primary

transcription of wild-type vesicular stomatitis in cells inhibited for protein synthesis

virus

In TVprevious communication we showed that the initial primary transcription of VS virus (i.e. transcription from the infecting virion genomes) is unaffected by pretreatment of cells by cycloheximide or puromycin (Flamand & Bishop, 1973). Those

VESICULAR

A-

Time(h) (a)

STOMATITIS

,I

35

VIRUS

Time(h) (b)

Fro. 1. The synthesis of VS viral complementary RNA in BHK21 cells inhibited for protein synthesis. A preparation of 3H-labeled, purified VS virus (2.6x IO4 ots/min/lOO pl of Eagle’s medium containing 1 pg DEAE-dextran to aid adsorption) was added to prewashed cold monolayers of BHKSI cells (3 x lo8 cells/small T flask of 26 cma) and allowed to adsorb for 30 min at 4°C. Infected monolayers were washed twice with cold Eagle’s medium (to remove excess virus), and then incubated with warm (38%) Eagle’s medium (containing 6% (v/v) fetal calf serum) in a circulating weter bath regulated to within ho.2 deg. C. In (a), the monolayers were either untreated (-O--O--), or treated 60 min before infection with 2 ml of 100 pg puromycin/ml Eagle’s medium containing 5% (v/v) fetal calf serum (a), or 50 pg cycloheximide/ml of the same medium ( x ), or 100 pg puromycin and 60 pg cycloheximide/ml of the same medium (A). After infection 4 ml of warm medium containing the same respeative concentrations of each drug were added to the monolayers. In (b), the cells were infeoted with 3.6 x 10’ cts/min of virus (2 x lo7 p.f.u.) but not pretreated with cyoloheximide. In this case the infected monolayers were incubated with 100 pg cycloheximide per ml of medium. After incubation at 38”C, each infected monolayer was washed twice with 6 ml of oold 0.16 JIN&l, 0.01 M-Tris*HCl buffer, pH 7.4, and then stored frozen at -20°C. The nucleic acids were extracted as described previously (Flamand & Bishop, 1973), and used in annealing experiments to determine the content of viral oomplementary RNA as described in Materials and Methods. In (a) the results obtained for all three drug treatments were absolutely identical so that only particular ones are shown for each time-point. In (b) the absorbed infecting virion label in the extracted nucleic acids recovered per monolayer for each time-point is shown. The average o, [3H]RNAase resistance before annealing ww 20& 7% and was within these limits for esch timepoint.

experiments did not measure the overall extent of transcription in terms of how long primary transcription persisted or how much viral complementary RNA was made. Such experiments have been conducted using cells pretreated and incubated with puromycin and/or cycloheximide or incubated from the time of infection with cycloheximide. The results are given in Figure 1. In each case using similarly treated BHKSI cell monolayers, the incorporation of l*C-labeled amino acids into acidinsoluble proteins was reduced by at least 98% and there was no increase in incorporation through the incubation time-course. When monolayers of BHK21 cells were pretreated with puromycin or cycloheximide or puromycin and cycloheximide (Fig. I(a)), then infected with [3H]virus in the presence of the drugs, essentially identical linear rates of viral complementary RNA synthesis were observed during five hours of incubation at 38°C. The accumulation

30

A. FLAMAND

AND

D.

H.

L.

BISHOP

of viral complementary RNA in the infected cells was measured by hybridizing the infected cell nucleic acids to an excess of viral E3H]RNA and determining from the [3H]ribonuclease resistance (post-annealing) the mass equivalents of viral complementary RNA. It should be noted that this measure does not differentiate between the individual types of VS virus messenger RNA (see later). We have previously shown that of the total adsorbed input virion [3H]RNA, the percentage that is recovered as double and multi-stranded RNA species (putative transcriptive intermediates) corresponds reasonably well to the percentage of ribonuclease-resistant 3H label in the infected cell nucleic acids (Flamand t Bishop, 1973). This observation therefore allows us to quantitate the synthesis of viral complementary RNA in terms of the minimum number of copies made per transcriptive intermediate (i.e. active genome) per hour. A preparation of highly labeled [3H]virus was made in which the specific activity of the viral RNA was 28 x log cts/min per mg RNA. It was calculated that 1 ct/min was equivalent to 5.6 x lo4 viral particles (on the basis of (1) Avogadro’s number, (2) the gram molecular weight of VS virus RNA is 382 x lo6 (Repik & Bishop, 1973) and (3) one virion particle contains one viral RNA). Portions of 3.5 x lo4 cts/min of virus (2 x lo9 particles, 2 x lo7 p.f.u.) were used to infect confluent, washed monolayers of 3~ lo6 BHK21 cells at 4°C. After removing unadsorbed virus, warmed medium containing 100 pg cycloheximide/ml was added and the monolayers incubated at 38°C for the indicated times. The infected cell nucleic acids were extracted and their content of [3H]RNA determined (Fig. l(b)). The mean adsorbed 3H label (and standard deviation) per monolayer was 1510f220 3H cts/min and the unannealed [3H]ribonuclease resistance was similar for all time points (20f4%, i.e. 302+60 3H cts/min). It was, therefore, calculated that there were in the neighborhood of 8.5 x 10” particles adsorbed per monolayer, i.e. an average of 28 particles per cell. Since 20&a% of the [3H]RNA in the infected cell nucleic acids was ribonuclease resistant, it was calculated that there were an average of six active virions per cell. Notice that the ratio of p.f.u. to particles in the inoculum (1: 100) corresponded reasonably well to the ratio of the intracellular ribonuclease-resistant 3H label to inoculum 3H label (i.e. 1: 120). The linear increase in total viral complementary RNA synthesis in the infected monolayers (Fig. l(b)) suggests that the transcriptive intermediates responsible for RNA synthesis were e.qually if not aimilurly active throughout the time-course of the experiment. The rate of synthesis was equivalent to 12,900 3H cts/min per hour (i.e. 12,900+302=43 mass equivalents of RNA/hour per active genome, or 43 x 6= 258 mass equivalents/hour per cell). Clearly these mass equivalents do not relate to which VS virus messenger RNA species are represented therein nor to their individual amounts (see later). In similar experiments with cells pretreated with cycloheximide and involving incubation of the infected cells in the presence of the drug at 31, 39.5 or 40°C the respective initial linear rate of viral complementary RNA synthesis was equivalent to 25, 36 and 20 mass equivalents per hour per active genome. These rates of RNA synthesis were linear for the 31 and 39.5% incubation temperatures, but decreased to ten mass equivalents per hour by five hours of incubation for the monolayers incubated at 40°C. In an experiment in which a 30-fold higher multiplicity of adsorbed virions was investigated, VS virus primary transcription in infected cells incubated at 39.5”C was linear for five hours in the presence of cycloheximide and

VESICULAR

STOMATITIS

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37

%I (-cycloheximide)

lnocdum pf.u pfu (-cycloheximide)

pfu I-cycloheximlde)

AbsorbedC3H1

*-‘.

vinons

_LIem.-.

-

Time(h)

Fro. 2. The production of infectious virus and intracellular viral complementary RNA in cells infected by wild-type VS virus at 31 or at 39.6”C in the presenoe or absence of cycloheximide. Monolayers of BHK21 cells were either pretreated for 60 min with 100 (~g cycloheximide/ml of medium or used directly for infection in the presence of DEAE-dextran with 3H-labeled, purified VS virus (3-2x lo* cts/min, 1-2x IOr p.f.u./monolayer). After infection, the cells were washed and incubated in appropriate media ot either 31 or 39.5% using a circulating water bath. After 30 min of inoubation the media were ohanged for fresh warm media to remove desorbed virus (Flamand & Bishop, 1973). Infected monolayers were withdrawn et the appropriate intervals and the total content of p.f.u. in the aupernatant fluids determined by essaying in BHK21 monolayers incubated at 31°C. The infected monolayers were extracted for nucleic acids and the content of [sH]RNA (2f0.4 x lo3 ots/min), its RNAaae resistance (23&8%), and oontent of viral complementary RNA determined. In this experiment, on the basis of the content of p.f,u. in the inooulum, it was osloulated that there were 4 p.f.u./oell, whereas on the basis of the specific aativity of the 3H-labeled absorbed virus and its RNAase resistance, it was calouleted that there were 7 intracellular active vu-ions/cell. Since determining the inoculum p.f.u. does not necessarily exactly mimic the absorption of virus by oonfluent monolayers, this differenoe may not be signifioent, especially when in other experiments the difference was negligible.

38

A. FLAMAND

AND

D. H.

L. BISHOP

equivalent to 33 mass equivalents per hour per active genome, with 18% of the adsorbed 3H label being recovered as ribonuclease-resistant RNA. In different experiments with wild-type virus we have observed rates of viral complementary RNA synthesis that vary by as much as 30% from the values given above. The reasons for these differences are not known. (b) The secondary transcriptions of wild-type vesicular stomatitis vir~ at different temperatures of incubation A preparation of highly labeled [3H]virus was used to infect confluent monolayers of 3 x lo6 BHK21 cells preincubated in Eagle’s medium in the presence or absence of 100 pg cycloheximide/ml. After virus adsorption at 4°C excess virus was removed and the infected cell monolayers were incubated at 31 or 39.5% for five hours. Fractions of the supernatant fluids were assayed for p.f.u. (Fig. 2) and the monolayers extracted for nucleic acids and their content of viral [3H]RNA and its ribonuclease resistance determined. Upon annealing the infected cell nucleic acids to an excess of added viral [3H]RNA, the total content of viral complementary RNA was determined (Fig. 2). Apart from the first hour’s point, the amounts of viral complementary RNA synthesized in the presence of cycloheximide at the two temperatures were essentially similar. In the absence of cycloheximide from one hour post-infection, amplified (secondary) transcription was apparent for both temperatures of incubation. However, the initial increase (from 1 to 3 h) in amount of viral complementary RNA in the absenceof cycloheximide was notably greater at 39*5”C, reflecting greater amounts of secondary transcription. By five hours of incubation, the levels of viral complementary RNA were almost the same for the two incubation temperatures. In contrast to this observation there was a 150-fold difference in released p.f.u. by the two sets of monolayers. By extrapolation, and in comparison to the p.f.u. present in supernatants from the cycloheximide-treated cells (probably representing desorbed virus), it appeared that the first progeny p.f.u. arose by two hours post-infection for the monolayers incubated at 31”C, but by one hour post-infection for the monolayers incubated at 39*5”C. In a separate experiment conducted at 38°C (Fig. 3), similar levels of viral complementary RNA synthesis and released p.f.u. have been obtained. When the amounts of viral complementary RNA were related to the number of intracellular active virions (see left-hand side ordinates of Fig. 2), it was calculated that by five hours post-infection there were of the order of 3 x lo3 mass equivalents of viral complementary RNA per active genome. Since in these experiments the number of intracellular active virions represented about seven of the virions per cell, this indicated that there were around 2 x lo4 mass equivalents of viral complementary RNA per cell. In a similar experiment in which the multiplicity of infection was 30-fold higher, similar accumulated amounts of viral complementary RNA have been obtained in the absence of cycloheximide. Notice also that in Figure 2 approximately 1 x lo4 p.f.u. were produced per cell. Assuming that the efficiency of virus adsorption in the p.f.u. assay corresponded to 10% of C3H]virus adsorbed from the inoculum (see Materials and Methods), then the particle production minimally represented 1 x IO6 virions per cell if every absorbed particle was productive. If only 20% of the adsorbed particles were productive this value would be five times greater.

VESICULAR

STOMATITIS

IO’ 4*--*

VIRUS .

NOW 3 2

/-x

/fs;::....

.

, .

IO5

--,z.--o---~ 3.-i/

IO4

/ .’

0

(2xlO’~p.fu.) 3

.x-

c-x-.-x---x

o-

2

lnoculum ,Y ptu. . /

/’ T Cycloheximide addition time(h)

x

f-t ,-.&.--x

.--

,.A-x

39

: Cycloheximide addiiion time (h)

-

E

d W Absorbed [‘HI

virions

103F *Active

lOZ(j

’

[3Hlvirions

’

2

’

’

’

4

’

6

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0

Y--x--y+--,--x

i ,

~.-~~--0--9-4

0

I

2

I

I

I

4

I

6

106

105

Time (h) (a)

(b)

Fro. 3. Effect of addition of oycloheximide at different times upon the produotion of virus and intrsoelluler accumulation of viral complementary RNA in BHK21 cells. Monolayers of BHK21 cells were infected in the presence of DEAE-dextran with 3.4~ lo4 cts/min (2 x 10’ p.f.u.) of 3H-lebeled virus and incubated at 38°C. For the monolayers not treeted with cycloheximide (continuous lines), the infected cell nucleic acids were extrtMted and the adsorbed sH label (1913.6 X 1Oa cts/min), its RNAase resistance (2014%) and the content of viral complementary RNA were determined (a). For each of these monolayers the medium w8s changed 1 h post-infection to remove desorbed virus and fresh warm medium added. At the time of harvest, the content of released p.f.u. in the supernetant fluids was determined ((b) continuous line). One series of infected monolayers was treated immediately post-infection with 100 pg cycloheximidelml (broken line, 0 h) end similerly incubated at 38°C. The relessed p.f.u. (b) or cellassociated sH label, its RNAase resistance and the intracellular virel complementary RNA were determined (8). For this series, 8s well as the next three series, the cell-associated sH label and its RNAase resistance were the same as in the untreated monolayers. For another series of untreated monolayers after 1 h of incubation at 38’C, the supernetant fluids were removed and repleced with medium containing cycloheximide (broken line, 1 h) end the subsequent release of p.f.u. determined (b). Monolayers were removed from this series et hourly intervals to determine the 3H label, its RNAase resistance and the intracelluler content of viral complementary RNA. Similar series of monolayers were treated 2 h or 3 h post-infection and anslyzed 8s described above. The hourly rates of viral complementary RNA synthesis were determined for each set of treeted monolayers. A mean velue ( ~s.D.) equivalent to 1.7hO.3 x lo4 sH cts/min viral oomplementary RNA per h was obtained for the zero-time cycloheximide-treated semple. For the l-h treated samples, the mean rate of RNA synthesis from 2 to 6 h post-infection was equivalent to l-2& 0.9 x lo5 3H ots/min of viral oomplementary RNA per h. For the 2-h treated samples, the mean rate of RNA synthesis from 3 to 6 h post-infection ~8s equivalent to S.Z&O.S x 10s sH cts/min viral complementary RNA per h, and for the 3-h drug-treeted samples a me8n rate equivalent to 11&l x lo6 sH cts/min viral oomplementery RNA was obtained between 4 and 6 h postinfection.

(c) Effect of ~~Zdingcycloheximide at different times of an infection time-course upon the synthesis of viral complementary RNA It has been shown in Figure 1 that upon addition of cycloheximide at the time of infection, or with cells preincubated in the presence of cycloheximide, there is a subsequent linear synthesis of viral complementary RNA through at least six hours of incubation. Also, it is clear that in the absence of the drug (Fig. 2), the rate of viral complementary RNA synthesis dramatically changes by one hour post-infection (secondary transcription). It can be concluded, therefore, that transcriptive intermediates are formed consequent on protein synthesis. Are the new transcriptive

40

A. FLAMAND

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D. H. L. BISHOP

intermediates derived from the infecting virion particles that are not initially active? As indicated in the legend to Figure 2 neither the amount of adsorbed [3H]virions nor the [sH]ribonuclease resistance significantly changed through an incubation timecourse. Consequently these results suggest that infecting virions that are initially inactive probably remain so during the life-cycle of an infection. The reason for this inactivity is not known. Do the new transcriptive intermediates represent refurbished infecting virion intermediates? The answer to this question cannot be ascertained directly at present. However, since the initial rate of viral complementary RNA synthesis (equivalent to I copy of about 12,000 nucleotidesJ90 s) increases dramatically (by over 60-fold, see later), it is difficult to imagine that all viral complementary RNA is synthesized from the infecting active virions. It is more likely that the increased rate of synthesis of viral complementary RNA reflects the presence of new progeny viral-type RNA involved in transcription. An experiment was conducted in which cycloheximide was added at various times during the infection time-course with wild-type VS virus and the subsequent accumulation of viral complementary RNA (or p.f.u.) measured. The results are shown in Figure 3. In terms of p.f.u. production, it can been seen that there were significantly more p.f.u. (though still less than 1 p.f.u./cell) in the supernatant fluids of the cells treated with cycloheximide at one hour post-infection than for the monolayers to which cycloheximide was added at the time of infection. This could have been caused by higher levels of virus desorption from the untreated cells or represent the initial release of progeny virus particles. At other times of cycloheximide addition, virus particles continued to be released for two to three hours (Fig. 3). We do not know if this slow curtailment of p.f.u. production represents depletion of intracellular virion protein or genome precursors of virus particles or impaired host functions required for virion production, or whether it just represents a lack of immediacy in cycloheximide inhibition of viral protein synthesis. However, from the point of view of incorporation of l*C-labeled amino acids into infected cell proteins, we have found that upon addition of the drug, labeled protein synthesis is inhibited 98% within five minutes. Therefore, the results suggest that sufficient pools of virus precursors are present in the cells to allow some continued synthesis of virus particles. The parallel accumulation of viral complementary RNA after the additions of cycloheximide is also shown in Figure 3. Except for the zero-time treated samples, the increase in viral complementary RNA after cycloheximide addition during the next hour was always greater than for the subsequent hourly increases for any of the series of experiments. Thus after addition of the drug at one hour post-infection there was an increase in viral complementary RNA equivalent to 2.6 x lo5 3H cts/min by two hours post-infection (see Fig. 3 legend). Addition at two hours gave an increase equivalent to 12 X 105 3H cts/min by three hours post-infection, and addition at three hours gave an increase equivalent to 20 x lo6 3H cts/min by four hours postinfection (see Fig. 3 legend). As in the case of the parallel p.f.u. production, the cause of these differences probably relates to assembly of new transcriptive intermediates from existing precursors. Treatment of infected cells with cycloheximide at three hours post-infection did not substantially change the overall accumulation of viral complementary RNA by comparison to the untreated control. The control gave an hourly increase in viral

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complementary RNA equivalent to 14 x lo5 3H cts/min between four and five hours post-infection and 12 x 105 3H cts/min between five and six hours post-infection. Contrast these results to the p.f.u. production by the same monolayers where, after drug treatment of the three-hour time point, there was a 30-fold difference in released p.f.u. by six hours post-infection. From the specific activity of the viral [3H]RNA and the presumed infecting virion tra,nscriptive intermediates, it was calculated that there were approximately six active virions per cell. Note that the innoculum p.f.u. were equivalent to seven per cell. These six active virions developed a rate of viral complementary RNA synthesis equivalent to 1.7 X 10* 3H cts/min per hour (i.e. 2.8 x lo3 3H cts/min per active virion). If we assume that t,he infecting virion transcriptive intermediates give the same rate of RNA synthesis as progeny transcriptive intermediates, then the number of progeny intermediates can be calculated from the linear rates of viral complementary RNA synthesis established in the cycloheximide-treated monolayers. The numbers obtained per cell for the one, two or three-hour drug-treated monolayers were 40, 300 and 400, respectively, and the probable number of transcriptive intermediates in the control monolayers between four and six hours post-infection was approximately 450 per cell. (d) Transcription

of mutant vesicular stomatitis virus i?z cel16incubated cct different temperatures

It has been shown previously that mutants belonging to all five complementary groups of VS virus are able to perform primary transcription at 31 and at 39,5°C. In order to determine the extent of such primary transcription as well as the existence of secondary transcription at permissive or non-permissive temperatures, highly labeled preparations of [3H]virus were prepared for five mutant viruses. The five viruses were two Orsay mutants of VS virus belonging to Group I (I-5, I-80), two belonging to Group IV (IV-62, IV-194) and one belonging to Group III (III-23). (i) Transcription of Group I mutants Analyses of the synthesis of viral complementary RNA during tsI-5 infections at 31 or at 39~5°C were performed in suitable infected cells in the presence or absence of cycloheximide (Fig. 4), as described for wild-type virus (Fig. 2). In addition, the production of p.f.u. at these temperatures was determined by plating supernatant fluids on monolayers of BHK21 cells and growing them at 39.5 or at 31°C to determine the cont.ent of wild-type (i.e. revertant) or temperature-sensitive virions. It was determined that in each case the 3H-labeled mutant virus preparations used in t’hese experiment,s all contained less that 0.01% wild-type revertants, as assayed by the relative ability to produce plaques in monolayers of BHK21 cells grown at 31 or at 39.5%. Similarly the supernatant fluids from the infected cells of all time points contained less than lo2 p.f.u. when assayed at 39.5”C. In t,he [3H]virus inoculum there were 15 p.f.u. per cell. From the specific activity of the viral RNA it was calculated that there were about 90 adsorbed virions per cell, of which ten exhibited transcriptase activity at either 31 or 39.5%. Therefore, it was calculated that there was less than 3 x lo3 (or 4.5 x 103) cells in the monolayer that could have been infected by wild-type revertant viruses in the inoculum [311]virus preparations. The lack of production of progeny virions able to produce plaques at 39.5”C is in agreement with this point,

42

A. FLAMAND

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(b)

IO’ M-5 39.5oc

2 d

3H (+cycloheximide) IO5

IOR h ee,py-b

IO’

.,~ e

i

‘no;ufk.m

g

q.f.u.(-c;clohelimide)

1’”

<

c L 107

106

I !-

i

10s

Time(h)

FIU. 4. Intracelhder RNA transcription and production of virus by tsI-6 et 31, 39.6 or 4O’C. Monolayers of BHK21 cells were used directly for infection by sH-labeled VS virus &I-6 or after pretreatment by 100 c(g oyoloheximide/ml (Fig. l(a)). After adsorption, the infected monolayers were washed and incubated in water baths regulated at 31, 39.6 or 40°C and the monolayers processed as described in the legend to Fig. 2 to determine the oontent of sH label, its RNAase resistanoe end the intracellular viral complementary RNA as well 88 the released p.f.u. For the experiment oonduoted at 40°C, a second betoh of 3H-labeled virus was used and the results of an equivalent monolayer incubated for 6 h at 31°C are given. All plaque assays were conducted et 31°C. When the supernatants were asseyed for wild-type virus by performing the plaque assay at 39.6”C, less then 1Oa p.f.u./time-point were observed. It was conoluded, therefore, that in the monolayer8 incubated at 31 or 39.6% only tempereture-sensitive virus was being produced,

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With regard to the production of temperature-sensitive I-5 virions at 31 or at 39.5”C (through leakiness), the following observations were made. First, that at 31°C in comparison to a wild-type infection at 31°C (Fig. 2), a similar number of p.f.u. were produced. Second, that at 39*5”C there was some production of temperature-sensitive p.f.u. through five hours of incubation (i.e. minus the cycloheximide control value, equivalent to 3 x lo6 p.f.u. net) and this was equivalent to 0.01 o/othat of the wild-type virus grown at 39.5% (Fig. 2; see Pringle, 1970) but represented 3% the value of p.f.u. produced by the tsI-5-infected cells incubated at 31°C. Whether this production of p.f.u. at 39.5% was due to leakiness or enhanced desorption (in the absence of cycloheximide) could not be determined. Third, that at both temperatures, progeny p.f.u. were detectable from two to three hours post-infection. The synthesis of viral complementary RNA in the tsI-5-infected monolayers incubated at the two temperatures were compared to those induced in wild-type infections. It was found that at 31°C in the presence or absence of cycloheximide essentially identical patterns of viral complementary RNA synthesis were observed to that shown in Figure 2 for wild-type virus grown at 31°C. At 39.5”C the results obtained in the presence or absence of cycloheximide were essentially equivalent to each other and also gave amounts similar to those obtained for the tsI-5 or wild-type infected cells grown at 31°C in the presence of cycloheximide. Since there was a significant release of temperature-sensitive progeny virus at 39.5”C!, it was decided to determine if 40°C would be more restrictive (if the virus released was due to leakiness) and what effect this temperature would have upon primary RNA transcription. The results of such an experiment using a fresh batch of 3H-labeled M-5 virus are also shown in Figure 4(c). A parallel control five-hour incubation at 31°C was carried out and the results are included in the graph and gave a similar value to the previous experiment in terms of p.f.u. production but one-half the amount of total complementary RNA. Whether this was due to the cells or virus batch is not known. The results for the incubation at 40°C of tsI-5-infected monolayers gave no evidence for more released virus than in the cycloheximide control as assayed as virus capable of giving plaques at 39.5 or at 31”C, i.e. no revertants or temperature-sensitive virions. Both in the presence or absence of cycloheximide there were similar levels of viral complementary RNA synthesized. However, these levels were one-half to onequarter those of the previous 39.5”C experiment depending on the time-point (see Fig. 4). In summary it was apparent that for M-5, intracellular primary transcription occurred at temperatures non-permissive for M-5 virus production, in amounts similar or less than that at permissive temperatures or obtained for wild-type virus infections. Whether the lesser M-5 transcription at 40°C (as observed for the wild-type virus; see Fig. 2) was due to impaired host or virus functions, the batch of virus or cells is not known. At both 39.5 and 40°C there was very little, if any, detectable secondary transcription when compared with the tsl-5-infected cells grown without cycloheximide at 31 “C.

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Almost identical results have been obtained for the viral complementary RNA synthesized in tsI-80-infected cells grown at either 31 or 396% (data not shown). (ii) Transcription of a Group III mutant Similar analyses of the syntheses of p.f.u. and viral complementary RNA during tsIII-23 infections at 31 or at 40°C were done in suitably infected cells in the presence or absence of cycloheximide (Fig. 5). The following comparisons to the infections by wild-type virus were made. (1) The p.f.u. produced at 31°C by MD-23 at five hours were twice as high as in the wild-type infection.

p t u (kcycloheximide)

Time(h)

FICA 6. Intracellular RNA transoription and production of virus by tsIII-23 at 31 or at 40°C. Monolayers of BHK21 cells were infected with or without oyoloheximide pretreatment by sH-labeled VS virus leIII-23 and inoubated again with or without the drug at 31 or at 40°C. Monolayers were extracted at various times end the nuoleio scids assayed for 3H label, its RNAase resistance and the oontent of viral complementary RNA as described in the legend to Fig. 2. The production of infectious virus w&9 essayed in the supernatsnt fluids by plating on BHK21 cells and growing at 31°C. No wild-type virus production (less than 10a/sample) was found when the supernatant fluids were plaque assayed at 39*6”C.

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(2) The amounts and rate of tsIII-23 viral complementary RNA synthesized at 31°C in the presence of cycloheximide were equivalent to those observed in wild-type infected cells grown at 31°C. (3) The tsIII-23 viral complementary RNA synthesized at 31°C in the absence of cycloheximide was three times higher than for the wild-type infection-even though for both viruses there was the same number of presumed infecting virion transcriptive intermediates per cell. From this it was calculated that at 31°C a total of 7 x lo3 mass equivalents of viral complementary RNA were synthesized per active virion and this corresponded to about 7 x IO4 mass equivalents per cell. (4) When the tsIII-23-infected cells were grown at 40°C no production of progeny virus (temperature-sensitive or revertant) was detected between two and five hours post-infection. Although at 4O’C the number of adsorbed virions per monolayers incubated at 31”C, the presumed infecting virion transcriptive intermediates were less (i.e. the unannealed [3H]RNAase resistance was 9% at 40°C and 17% at 31°C). Also the viral complementary RNA synthesized at 40°C from four or five hours post-infection in the presence of cycloheximide was one-third the corresponding value obtained at 31°C. In the absence of cycloheximide the viral complementary RNA synthesized at 40°C was one-half that sYynthesized at 31°C. In conclusion, for a mutant belonging to complementation Group III of VS virus, primary and secondary transcription of RNA could be demonstrated in infected cells grown at a temperature (40°C) totally non-permissive for virus production. (iii) Transcription of Group IV mutants The synthesis of viral complementary RNA and production of virions by two mutants from Group IV were examined. The results obtained for tsIV-194 are presented in Figure 6. As with the M-5 infection, monolayers that were incubated at 31 or at 39.5”C used one batch of tsIV-194 virus whereas monolayers incubated at 40°C used another batch of virus. At both 31 and 39.5°C progeny virion p.f.u. production (assayed at 31°C) was detected from two hours post-infection. The relative p.f.u. yield at 39.5”C by five hours post-infection was 10% that at 31°C (equivalent to 0.06% wild-type production at 395°C). At 40°C no progeny virion production was detected between two and five hours post-infection. The E3H]virus inoculum was determined to contain less than low4 revertant wild-type virions (i.e. p.f.u. assayed at 39+5”C in comparison to p.f.u. assayed at 31°C). None of the infected cell supernatant fluids from the three incubation time-courses contained detectable wild-type p.f.u. capable of giving plaques at 395°C (less than lo2 per monolayer). A comparison of the amounts of viral complementary RNA synthesized in the presence of cycloheximide for infected monolayers, incubated at 31 or at 39*5”C, indicated that equivalent amounts of RNA were synthesized at either temperature. Without cycloheximide at 39.5”C, the amounts of viral complementary RNA were similar to those in its presence (around 150 copies per active virion at 5 h) whereas at 31°C increased levels of viral complementary RNA were obtained through secondary transcription (about 3000 copies per active virion at 5 h). At either temperature the unannealed [3H]ribonuclease-resistant label represented 14% of the adsorbed

46

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p.f.u.(-cycloheximide)

Time(h)

Pm. 6. Intracellular RNA transcription and production of virus by tsIV-194 grown at 31, 39.6 or 40°C. Monolayers of BHK21 cells were infected with or without cycloheximide pretreatment by sH-labeled VS virus &IV-194 and incubated in the presence or absence of the drug at 31, 39.5 or 40°C. The infeoted cell nucleic acids were extracted and the content of sH label, its RNAase reaiatanoe and viral complementary RNA determined as described in Fig. 2. The produotion of infeotioua virus was assayed at 31°C. No wild-type virus production (less than lOs/sample) was deteoted in assaying the supernatant fluids in BHK21 oells grown at 39*6”C. The experiment at 4O’C was performed with a second.preparation of virus and the results for a control monolayer incubated for 6 h at 31% are given.

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virus In the experiment conducted at 4O”C, the unannealed [3H]ribonuclease-resistant label was 9% of the adsorbed virus and the amounts of viral complementary RNA synthesized in the presence or absence of cycloheximide were equivalent to those observed for the monolayers incubated at 39.5%. Similar results were obtained for the other Group IV mutant, tsIV-62 (data not shown). In conclusion, intracellular primary transcription has been demonstrated for two Group IV mutants of VS virus grown at non-permissive temperatures for virus production. At 39.5 and at 40°C there was very little, if any, detectable secondary transcription when compared to tsIV mutants grown at 31°C. (e) Evidence suggesting that there is disproportional synthesis of viral compleme&ry RNA in infected cells Both t,he messenger RNA isolated from VS virus-infected cells and the in vitrosynthesized viral complementary RNA transcripts are smaller than the viral RNA (Bishop & Roy, 1971; Huang et al., 1970; Mudd & Summers, 1970). In vitro it has been shown that the RNA transcription process is repetitive and sequential giving a disproportionate set of the various viral complementary RNA species (Roy & Bishop, 1972). These results suggest that in vivo there could be some regulation of individual VS virus messenger RNA species, which may be synthesized independently of each other. In order to examine this possibility, use was made of the observation that a defect,ive particle of VS virus (T particle:VSV-111) represents a unique onethird portion of the VS virus complete particle (VSV-1) (Repik t Bishop, 1973). TABLE

1

Presence of VSV-1 and VSV-111 viral complementary RNA in VS virus-infected cell nucleic acids Cell incubation time (4

Cycloheximide treetment

-

+ +

VSV-1 cRNA (3H cts/min

2.2 15 76 123 347 1.9 4.0 6.9 8.0 9.0

VSV-111 cRNA x 10e3 annealed)

0.8 2.8

Ratio VSV-1 cRNA VSV-111 cRNA

11.3 19 51 0.7

2.8 6.4 6.6 6..5 6.X 2.7

1.7 1.6 2.1

4.0 6.0 4.3

A preparation of 3H-labeled VS virus was used to infect confluent monolayers of BHK21 cells and the infected cell nucleic acids extracted at various times post-infection. Another preparation of sH-labeled VS virus w&s prepared which contained both complete virion B (VSV-1) particles as well w defective T (VW- 111) particles. Each was purified, extracted for RNA snd then snslyzed for homogeneity by polyacrylamide gel electrophoresis (Bishop & Roy, 1971). The 3H-Iabeied VSV-1 or VSV-111 RNA was used to determine the content of the respective complementary RNA in the infected cell nucleic acids. The ratio of the molecular weights of the two r3H]RNA speoies is 3.1: 1 (Repik & Bishop, 1973).

48

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A preparation of [3H]virus was used to infect confluent monolayers of 3 x lo6 BHK21 cells and, after adsorption and removal of excess virus, the monolayers were incubated at 38°C and the infected cell nucleic acids extracted as usual. A second preparation of [3H]nucleoside-labeled VS virus was prepared from a virus stock known to give a harvest containing both complete (VSV-I) and defective (VSV-111) virions (Repik & Bishop, 1973). The progeny-labeled virus was purified by sucrose-gradient centrifugation and the two particles were separated, repurified, and their RNA extracted as described previously (Repik & Bishop, 1973). By polyacrylamide gel electrophoresis it was determined that the VSV-111 RNA preparation contained less than 4% of its label in VSV-1 RNA, while the VSV-1 contained less than 5% of its label in VSV-111 RNA (Bishop & Roy, 1971). The two RNA preparations were used in annealing experiments to determine the amounts of VSV- 1 complementary RNA or VSV-111 complementary RNA in the infected cell nucleic acids obtained as described above. The results are presented in Table 1. The ratios of VSV-1 to VSV-Ill viral complementary RNA were determined for each time-point and gave values varying from 28 to 6.8 : 1. If all viral complementary VS virus messenger RNA species were synthesized (and accumulated) equally, one could expect a ratio equivalent to the molecular weights of the two virion RNA species (i.e. 3.1: 1). However, as seen from Table 1 this was not the case and these results suggest, therefore, that in vivo there may also be some disproportionate synthesis of the different viral complementary messenger RNA species.

4. Discussion A procedure has been developed for following the synthesis of only viral complementary RNA in VS virus-infected cells. This method of analysis is able to detect very low levels of transcription and has allowed us to monitor the process of viral RNA transcription both through its initial phase, involving the infecting virion genomes (primary transcription), and through a subsequent phase of amplified (secondary) transcription. As shown previously (Flamand & Bishop, 1973), there is a reasonable correspondence between the fraction of an inoculum’s adsorbed [3H]virus that exhibits transcriptase activity in vivo (as monitored by the percentage [3H]ribonuclease resistance) and the number of inoculum particles capable of developing productive infections. Plaque-forming unit determinations represent an initial cellular infection at a multiplicity of one virus per cell. In biochemical studies such as those involved here, higher multiplicities of infection have been used. Is there an inhibition of the productivity of one potentially active VS virus virion by another, in a multiply infected cell? From evidence presented previously (Flamand & Bishop, 1973), in cells that were infected with between 5 and 6000 adsorbed virions per cell, the percentages of viral adsorbed genomes exhibiting primary transcription were comparable (i.e. 18%). In these present studies we have observed that the primary transcription developed in cycloheximide-treated cells infected by 28 virions per cell (7 p.f.u./cell) or 840 virions per cell (210 p.f.u./cell) was 28-fold higher for the 30-fold higher multiplicity of infection and also involved similar percentages of the adsorbed virions (see text). However, the accumulated viral complementary RNA by five hours post-infection was essentially identical for both sets of monolayers. We conclude that the initial transcription processes are probably proportional to the multiplicity of infection

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within the limits studied but that later in infection (during the phase of secondary transcription) this proportionality does not hold, We are currently investigating the intriguing possibility that at a sufficiently high multiplicity of infection no secondary transcription may be observed ! We have found that up to 20% of the initially adsorbed virions (at 4°C) may be subsequently desorbed from BHK21-infected cells during the first 20 minutes of incubation (Flamand b Bishop, 1973). In unpublished results we have observed that the desorbed virus is as capable, on a particle (i.e. 3H label) to p.f.u. basis, of giving productive infections as the original inoculum. Further, that after 20 minutes of incubation there is very little (less than 3%) subsequent desorption of label or p.f.u. Consequently the content of label in the infected monolayers has been reasonably equivalent throughout an infection time-course (see Fig. l(c)). The variation that has been observed probably reflects differences in the individual adsorptions of virus by the different monolayers (relating to cell numbers, inoculum volume, physiological condition of the monolayers, etc.). We have previously shown that for the first hour of an infection, the label in the infecting virion genomes is conserved in VS virus RNA (Flamand & Bishop, 1973). Although the amount of label associated with the monolayers remains reasonably ronstant through up to six hours of an infection, we have not in the present investigation proved that this label is resident in VS viral RNA; however, we have indirect evidence to support this point, When a six-hour, 31”C, [3H]virus-infected cell nucleic acid sample was self-annealed, all the label was rendered ribonuclease resistant. Since viral-type RNA is in a minority in the infected cells (see below), this result suggests that the infecting viral 3H label was probably still resident in viral-type (as opposed to cellular or viral complementary RNA) sequences. In parenthesis, in unpublished experiments we have observed that with t3H]amino acid-labeled infecting virus, almost all of the absorbed viral proteins are conserved intact in the infected cell. (a) Primary transcription Primary transcription appears to be the main source of viral complementary RNA for the first 20 to 60 minutes of an infection by VS virus. Thereafter secondary transcription becomes apparent. We have obtained up to 40% of the adsorbed virion genomes exhibiting transcriptase activity in viva; most virus preparations however give about 20% activity. Since the drugs puromycin or cycloheximide are efficient inhibitors of protein synthesis, we have been able to analyze primary transcription in drug-treated monolayers not only as a function of the incubation temperature or multiplicity of infection, but also in terms of the number of mass equivalent copies produced per cell. The transcriptase activity developed in these conditions by the infecting virions is linear (at 31 to 39+“C) for six hours, but reduces with time when the infected cells are incubated at 40°C. At optimum temperatures the maximum activity corresponds to a rate of one genome mass equivalent copy per 90 seconds per active genome. In all probability the infecting virions bring into a cell several transcriptase enzyme molecules per virion (Bishop & Roy, 1972), some of which may be able to function consecutively or simultaneously and this may account for the repetitive nature of the transcription process (Bishop & Roy, 1971). Primary transcription has been observed at both permissive and non-permissive temperatures of virus production for five out of five VS virus temperature-sensitive 4

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mutants: two belong to Group I, two to Group IV and one to Group III. For all five mutants at 39*5”C, an equivalent amount of primary transcription has been observed to that obtained at 31°C with the same mutant, or for the wild-type virus. At 40°C there was less primary transcription for the tsI-5 mutant than at 39.5% but since the wild-type virus transcription was also inhibited at 4O”C, these results do not indicate that the tsI-5 enzyme is temperature sensitive. Our data on the occurrence of primary transcription at non-permissive temperatures for Group IV mutants of VS virus is comparable to similar observations by [3H]nucleoside labeling of infected cells obtained by Printz-An6 et al. (1972) and Unger & Reichmann (1973). However, in their studies little or no primary transcription at non-permissive temperatures was observed for Group I mutants. Unger & Reichmann used different mutants belonging to Group I than those used here, and although Printz-An6 and his co-workers used the same t&I-5 mutant we have employed they conducted their studies in HeLa cells. It is possible that different host cells may influence the expression of viral transcription and that different Group I mutants express themselves differently at non-permissive temperatures. In our studies we are able to detect much lower levels of transcription than those obtainable by alternative techniques-even to the extent of a few copies per genome. The transcription rates observed have been related to the number of adsorbed virions exhibiting enzyme activity and, for both the Group I and Group IV mutants we have studied, the number of active virions per infected cell found at permissive or non-permissive temperatures was similar. We have shown previously that the in vitro transcription process with w&&type virus is anomalous in that it is restricted and slower at 37°C (or 39.5”C) than at 31°C and involves unique initiation sequences (unpubhshed observations). Recently Hunt & Wagner (1974) have claimed from in vitro reconstitution experiments with separated virion template and enzyme fractions, that the virion enzyme fractions for Group I mutants are temperature sensitive. They have not yet examined the Group IV mutants. These results apparently do not agree with our observations of substantial in vivo primary transcription of Group I mutants at non-permissive temperatures. While in these studies we have not used all the mutants t,hat Hunt & Wagner used, one (tsI-5) has been used in both studies. The most probable explanation of the difference between the in vivo and in vitro results with Group I mutants is that in the in vitro reconstitution, the enzyme is partially denatured during the high-salt treatment and upon removal of the salt it renatures incorrectly. Our present results are compatible with the suggestion that the progeny enzymes responsible for secondary transcription are incorrectly folded during cell growth at high temperatures, but not at low temperatures. (b) Secondary transcription As indicated in the text, we have evidence to suggest that those viral genomes that are absorbed but are not initially active probably remain so throughout the phase of secondary transcription. Whether secondary transcription involves the active infecting virions or progeny viral-type species cannot be ascertained from these analyses although the latter appears more probable. No secondary transcription was observed for mutants of Group IV and Group I at temperatures non-permissive for viral production. The Group III mutant exhibited secondary transcription at 40°C as did the wild-type virus at 39.5 and at 40°C (data not shown).

VESICULAR

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.i 1

By equating the rates of RNA transcription in cycloheximide-treated cells, at different times of an infection time-course, to the rate established by the infecting active virions, we have calculated that of the order of 450 transcriptive intermediates are present in infected cells by five hours post-infection. The greatest increase in transcriptive intermediates occurs between one and three hours post-infection, In amount, the viral complementary RNA that accumulates per cell represents greater than lo4 mass equivalent copies of viral complementary RNA per cell.

(c) Virus production from infected cells It has been shown that virus production at 38 or at 39*5”C begins one to two hours post-infection and continues for at least another four to five hours. The number of plaque-forming units produced at 38 or at 39*5”C is of the order of lo* per cell by five or six hours post-infection, with the greatest rate of production occurring in the last hour analyzed (see Fig. 3; 1 x lo4 p.f.u./cell per h between 4 and 5 h postinfection). Since we do not know the efficiency of adsorption in the progeny p.f.u. assay, nor the percentage of those adsorbed virions that give productive infections, this value is a minimal estimate of the particle production and could be 50-fold higher (i.e. 5x lo5 particles/cell per h). It is evident from the results obtained with wild-type or mutant viruses grown at permissive temperatures that there is no direct correspondence between the synthesis of viral complementary RNA and virus production. Thus when the accumulation of wild-type viral complementary RNA at 39.5”C increased twofold between four and five hours post-infection (Fig. 3), progeny virus p.f.u. increased 20-fold. Note also that at 31°C when the accumulated viral complementary RNA was similar to that of monolayers incubated at 395°C (Fig. 3), the production of p.f.u. was more than lOO-fold less. Of interest is the observation that at 39.5”C with various temperature-sensitive mutants of VS virus (results with td-5 (Fig. 4) and tsIV-194 (Fig. 6) are shown) there is some production of temperature-sensitive mutants but little or no evidence of secondary transcription. The production of temperature-sensitive mutants at high temperatures is usually characterized as the leakiness of a particular mutant, and the degree of leakiness varies with the VS virus mutant genotype (Pringle, 1970). Although 40°C is a non-permissive temperature for the temperature sensitive mutants we have studied with regard to p.f.u. production (non-infectious particle production has not been studied), we have found that primary transcription becomes inhibited at 40°C for our wild-type virus in BHK21 cells although in the absence of cycloheximide, productive infections can be achieved giving progeny p.f.u. levels comparable to that obtained at 31°C (data not shown). The scatter of data points in some of the primary transcription assays at 40°C is also evidence of differences in t,he behavior of different monolayers at that incubation temperature. (d) RNA-inhibited

complementation groups of vesicular stomatitis virus

The results obtained with mutants for both Group I and Group IV of VS virus show that primary transcription occurs with these mutants at temperatures that are restrictive (e.g. 39.5”C) or non-permissive (e.g. 40°C) for virus production. Although not all Group I or IV mutants may behave in the same way as t.he ones we have studied, neither complementation group can be described as RNA negative. We would suggest a term, RNA inhibited, as being more appropriate.

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D. H. L. BISHOP

What are the temperature-sensitive defects of the two RNA-inhibited mutant groups we have studied? At the present time we cannot operationally distinguish the mutants of either Group I or Group IV. Either mutant could represent transcriptional or replicational defects. If the virion or progeny transcriptase (gene product A, say) is converted into a replicase function by gene product B, then it would be difficult to determine which function represented which gene in an in vivo situation where secondary transcription (mediated by progeny gene product A) relied on progeny viral-type RNA being formed by the gene product B. Clearly one method of unravelling this mystery will be to identify when viral-type RNA is synthesized, and which gene product is required for its synthesis. However, even this approach of looking for viral-type sequences is not necessarily going to yield a definitive answer to the question of which gene product represents which complementation group, since it is conceivable that replication only occurs with progeny gene product A and progeny gene product B and not with progeny gene product B plus the virion transcriptase. The virion transcriptase may be programmed for transcription of small messenger RNA species and not be able to be converted into a replicase. The inter-relations of the two gene products of complement&ion groups I and IV will be an interesting and challenging problem to unravel, let alone the additional complication of the mutants of Group II, some of which also appear to be RNA inhibited (Unger & Reichmann, 1973). (e) Viral-type RNA synthesis in injected cells We have presumed that the increase in viral complementary RNA synthesis in infected cells reflects the presence of new transcriptive intermediates involving progeny viral-type RNA species. On this assumption, both the increase in their number as well as the onset in released plaque-forming units indicate that progeny viral-type sequences are formed by one hour post-infection. The equivalence of these observations might be interpreted to mean that a progeny viral RNA strand can become a transcriptive intermediate or a new virus. Clearly, later in the infection where the number of transcriptive intermediates stabilizes, the majority of viral-type RNA species end up in progeny virions. It can be calculated from the data presented in Figure 3 that at two hours post-infection 0.5 p.f.u. per cell had been produced (possibly representing ten to 50 times more particles per cell) whereas between 40 and 300 transcriptive intermediates are present per cell (see text). In determining the viral complementary RNA accumulation in infected cells, we have used a hybridization procedure with an excess of labeled viral RNA as the probe. Hybridization has been performed as nearly as possible to saturation (Flamand t Bishop, 1973) using a procedure designed to compete out the hybridization by unlabeled viral-type sequences to the unlabeled viral complementary RNA sequences. Since in every assay two or more concentrations of viral [3H]RNA were used for the annealing procedure in which 10 to 30% of the label was rendered ribonucleaseresistant and the mass of ribonuclease-resistant RNA obtained was essentially similar for both viral [3H]RNA concentrations, it can be concluded that the amount of progeny viral-type unlabeled RNA in the infected cell nucleic acids was smaller than the unlabeled viral complementary sequences or the 3H-labeled viral RNA mass used in the assays. Moreover, for wild-type virus between four and five hours post-infection at 38 or at 39.5”C there are probably more released virus particles

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than intracellular mass equivalents of viral complementary RNA; this also suggests that there is a fast turnover of the intracellular pool of viral-type sequences as observed by Kiley & Wagner (1972). Experiments are in progress to quantitate the synthesis of viral-type RNA in infected cells as a function of the infection time-course. This investigation was supported by Public Health Service grant AI10692 from the National Institute of Allergy and Infectious Disease. One of us (A. F.) is on leave from the C.N.R.S., France. We thank Roger Van Deroef and DOME+ Cryan for excellent teohnical assistance. REFERENCES Aaslestad, H. G., Clark, H. F., Bishop, D. H. L. & Koprowski, H. (1971). J. did. 7, 726-735. Baltimore, D., Huang, A. S. & Stampfer, M. (1970). Proc. Nat. Acad. Sci., U.S.A. 66, 572-576. Bishop, D. H. L. (1971). J. ViroZ. 7, 486-490. Bishop, D. H. L. t Roy, P. (1971). J. Mol. Bid. 57, 513-527. Bishop, D. H. L. & Roy, P. (1972). J. Viral. 10, 234-293. Chang, S. H., Hefti, E., Obijeaki, J. F. & Bishop, D. H. L. (1974). J. Firol. 13, 652-661. Eagle, H. (1959). Science, 130, 432-437. Flamand, A. (1969). C. R. H. Ad. Sci. 268D, 2305-2308. Flamand, A. (1970). J. Gem. Viro2. 8, 187-195. Flamand, A. & Bishop, D. H. L. (1973). J. ViroE. 12, 1238-1252. Flamand, A. & Lafay, F. (1973). Ann. Inet. Pasteur, Pa&, 124, 261-269. Flamand, A. & Pringle, C. R. (1971). J. ffen. ViroE. 11, 81-85. Huang, A. S. BEManders, E. K. (1972). J. Viral. 9, 909-916. Huang, A. S., Baltimore, D. & Stampfer, M. (1970). ViTology, 42, 946-957. Hunt, D. M. t Wagner, R. R. (1974). J. ViroZ. 13, 28-35. K&y, M. P. & Wagner, R. R. (1972). J. Viral. 10, 244-255. Knudson, D. L. (1973). J. Gen. F&oZ. 20 (suppl.), 105-130. Lafay, F. (1969). C. R. H. Acad. Sci. 268D, 2385-2388. Mudd, J. A. & Summers, D. F. (1970). Virology, 42, 958-968. Pringle, C. R. (1970). J. Viral. 5, 659-567. Pringle, C. R. & Duncan, I. B. (1971). J. Viral. 8, 56-61. Printz-An& C., Combard, A. & Martinet, C. (1972). J. ViroZ. 10, 889-895. Repik, P. & Bishop, D. H. L. (1973). J. ViroZ. 12, 969-983. Roy, P. & Bishop, D. H. L. (1972). J. ViroZ. 9, 946-955. Roy, P. & Bishop, D. H. L. (1973). J. ViroZ. 11, 487-501. Unger, J. T. & Reichmann, M. E. (1973). J. Viral. 12, 670-578.