Macromolecular synthesis in cells infected by frog virus 3 X. Inhibition of cellular protein synthesis by heat-inactivated virus

VIROLOGY 98, 319-327 (1979)

Macromolecular X. Inhibition

Synthesis

in Cells Infected

by Frog Virus 3

of Cellular Protein Synthesis by Heat-Inactivated

RAJENDRA

RAGHOW AND ALLAN

Virus

GRANOFF’

Division of Virology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38101 Accepted July 6, 1979 Heat-inactivated frog virus 3 (AFV3) inhibits host cell protein synthesis without interfering with protein synthesis directed by active FV3. An examination of various parameters of cellular protein synthesis after exposure of cells to AFV3 revealed the following. (i) The rate of inhibition was multiplicity dependent. (ii) There was a rapid disaggregation of both free and membrane-bound polysomes. (iii) Rates of protein chain elongation remained unaltered. (iv) There was neither a detectable breakdown nor modification of cellular mRNAs; the mRNAs isolated from AFV&treated cells could be translated in vitro and the pattern of the translational products appeared identical to that of mRNAs from untreated cells. These data are consistent with the conclusion that inhibition of host cell protein synthesis by AFV3 is the result of a selective effect on a step(s) essential for the initiation of translation of cellular mRNAs. MATERIALS

INTRODUCTION

Frog virus 3 (FV3) inhibits DNA, RNA, and protein synthesis in cells from a variety of species at temperatures permissive and nonpermissive for FV3 replication (Goorha and Granoff, 1979;for review). Biosynthetic activity of the viral genome is not required for this suppression; cellular macromolecules are inhibited efficiently with FV3 inactivated by heat (AFV3), ultraviolet light, or y-radiation (ibid, 1979). Inhibition of cellular macromolecules by AFV3 is selective. When cell macromolecular synthesis is suppressed by AFV3, subsequent infection with active FV3 results in normal virus replication (Goorha and Granoff, 1974). To determine the basis for selective inhibition of host cell macromolecules by AFV3 we have focused our studies on inhibition of cell protein synthesis. The experiments reported here are the first in a series designed to determine the component(s) of the cellular translational apparatus that is sensitive to inactivation by AFV3. The data presented indicate that AFV3 selectively alters some step(s) in the initiation of cellular mRNA translation. * To whom reprint requests should be addressed. 319

AND METHODS

Cells and virus. Procedures for growth of FHM and BHK cells, virus propagation and purification, and for plaque assay have been reported (Naegele and Granoff, 1971; Tan and McAuslan, 1971). Inactivation of purified FV3 was by heating at 56’ for 20 min, a treatment that resulted in greater than P.lo”-fold reduction in PFU titer. Measurement

of total protein

synthesis.

Cell monolayers grown in 35mm-diameter petri dishes were infected at appropriate input multiplicity with AFV3 or mock infected with PBS. After 1 hr adsorption at room temperature (23”), cells were rinsed with prewarmed (37”) MEM-10 and incubated in MEM-10 at 37”. At various intervals, duplicate cultures were labeled with [35S]methionine (1 @i/ml in methionine-free MEM-10) for 15 min. After the labeling period, the monolayers were washed with PBS (4”), scraped off the dishes, and the cells were pelleted by centrifugation (800 g, 5 min) and solubilized in 0.1 M NaOH containing 1.0% SDS (37”, 10 min). Protein was precipitated in 10% trichloroacetic acid (TCA), dissolved in 0.5 ml of NCS, and mixed with 7 ml of Omnifluor scintillant. 004%6822/79/140319-09$02.00/0 Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.

320

RAGHOW

AND GRANOFF

Radioactivity was determined in a Beckman scintillation spectrometer.

activity. The second cell pellet was resuspended in RSB containing 0.5% Noniodet-P40 Preparation and analysis of polysomes. (NP-40), stirred vigorously, and nuclei were The methodology for extraction and analysis removed by centrifugation (1000 g, 5 min). of polyribosomes was essentially that de- Cytoplasmic extracts were layered on 3.5 ml scribed by Noll and Burger (1974). All opera- of 15%sucrose-RSB, centrifuged (50,000rpm, tions were performed at O-4”. Cell mono- 90 min, SW 50.1 rotor) to pellet polyribolayers (lOO-mm-diameter petri dishes) were somes and monosomes and the TCAwashed with PBS, scraped off, and pelleted. precipitable radioactivity in the supernatant Cell pellets were allowed to swell in a 0.5 ml fluid (soluble counts) was determined. The of reticulocyte standard buffer (RSB) and separation of the two curves (total versus homogenized with 20 strokes of a tight-fitting soluble radioactivity) at the intercept with 2-ml Dounce homogenizer. The cell homoge- the time axis denotes half of the average nate was centrifuged (5000 g, 5 min) to give ribosome transit time (Fan and Penman, a supernatant “cytoplasm&? fraction con- 1970). taining free polysomes and a “nuclear” pellet. Extraction and analysis of poly(A) celluThe nuclei were extracted with RSB con- lar RNA. Cell monolayers were prelabeled taining 1% Triton X-100 for 10 min and with either [3H]- or [14C]uridine for 18 hr. centrifuged again (5000 g, 5 min); this super- The [3H]uridine-labeled cells were inoculated natant contained membrane-bound poly- with AFV3 and the [14C]uridine labeled somes. For total polysomes (membrane ones were mock infected with PBS. After bound and free), PBS-washed cell pellets virus adsorption, incubation at 37” in MEM-10 were extracted with RSB containing 1% was continued for another 3 hr, at which time Triton X-100 and the 5000 g supernatant the cell monolayers were washed with PBS was obtained. Polysome extracts were lay- and the cells were scraped off and collected ered on top of 12 ml, lo-50% (w/w> linear by centrifugation. The cell pellets were comsucrose-RSB gradient and centrifuged bined and cytoplasmic RNA was coextracted (40,000 rpm, 90 min, SW 41 rotor). Some with water-saturated phenol-chloroform polysome preparations were analyzed on (Willis and Granoff, 1976); RNA from the sucrose gradients in high salt buffer (0.5 M aqueous phase was precipitated with ethanol. NaCl in RSB) to distinguish between mono- Extraction of RNA and oligo(dT)-cellulose somes with or without bound mRNA (Zybler chromatography was according to Glazier and Penman, 1969). An absorbance scan at et al. (1977). The poly(A) RNA was taken 254 nm was obtained by monitoring the up in TENS buffer (0.005 M Tris-HCl, gradient fractions on an ISCO uv scanner. 0.001 M EDTA, 0.1 M NaCl, 0.5% SDS, To quantitate polysome disaggregations in pH 7.4) and centrifuged on a linear 15-30% AFV3-treated cells we compared either the sucrose-TENS gradient (27,000 rpm, 16 hr, relative areas covering the absorbance scan SW 27.1 rotor). An absorbance scan of riboof polysomes (larger than monosomes) or somal RNA centrifuged in an identical determined the radioactivity profile of poly- gradient was used to denote the positions somes from cells labeled with [3H]uridine of known markers. overnight. In vitro protein synthesis and electroMeasurement

of ribosome transit

times.

The ribosome transit times were measured by the technique of Fan and Penman (1970), adapted to cell monolayers. Cells in 35-mmdiameter petri dishes were labeled with 1 $?i/ml [35S]methionine in methionine-less MEM-10 and at 2-min intervals after the addition of the label, cells from two petri dishes were scraped off in PBS (O-4”) and pelleted. One of the cell pellets was processed to determine total TCA-precipitable radio-

phoretic analysis of proteins synthesized in vitro. An mRNA-dependent protein

synthesizing system from rabbit reticulocytes was prepared as described by Pelham and Jackson (1976). Each 25 ~1 of cell-free translational extract contained 20 ,ul of nuclease-treated lysate and the following assay components: 120 PM potassium acetate, 0.8 mM magnesium acetate, 12 mM creatine phosphate, creatine kinase 46 pg/ml, 4.6 mM dithiothreitol, 90 mM of each ofun-

INHIBITION

OF CELLULAR

labeled amino acids, 0.86 m&f ATP, and 0.17 mll/l GTP. Polyy(A) mRNAs taken up in distilled water were incubated at 30” in the reaction mixture for 90 min and then aliquots containing IOO,OOO-200,~ cpm were solubilized in an SDS buffer (Wilson et al., 1977). The buffer composition of the solubilized extracts was adjusted to the lysis buffer of O’Farrell(l975) and proteins were separated by two-dimensional gel electrophoresis (Raghow et al., 1978). Chemicals. [35S]Methionine (sp act. -1000 Ci/mmol), Amersham U. K.; [3H]uridine (24 Ci/mmol) and [14C]uridine (‘24Ci/mmol), B/D Immunodiagnostics; NP-40, Shell Chemical Company England; oligo(dT)-cellulose, T3, Collaborative Research; Ampholines, LKB Research Labs. RESULTS

The influence of input multiplicity of AFV3 on suppression of cellular protein synthesis in BHK cells is illustrated in Fig. 1. Trichloroacetic acid (TCA)-precipitable incorporation of [35S]methionine was reduced by 80% or more in cells treated with 10 PFIJ equivalents/cell of AFV3 for 4 hr. However, if an input multiplicity of 100 or more PFU equivalents/cell of AFV3 was used, maximal ambition was reached sooner; i.e., the rate of inhibition was multiplicity dependent. A semilog plot of inhibition of protein synthesis vs viral multiplicity indicated that the kinetics of inhibition by AFV3 was exponential. Comparable results were obtained with FHM cells. Drillien et al. (1977) also reported that the kinetics of cell killing by active FV3 was exponential. Although the preceding experiment demonstrated that AFV3 caused a drastic inhibition in the overall rate of protein synthesis it gave no clue as to the site(s) of protein synthesis at which the action of AFV3 was directed. The following experiments examined various parameters of protein synthesis in cells treated with AFV3 and include the size of poly~bosomes, rates of polypeptide chain elongation, and ~nctional competency of cellular mRNAs. In all subsequent experiments cells were infected with

PROTEIN SYNTHESIS

321

BY FV3

0’

--0

I 2 3 HOURS AFTER AooITION OF MV3

4

FIG. 1. Inhibition of cellular protein synthesis by AFW. BHK cell monolayers were either mock infected with PBS or treated with AFV3 at the indicated input multiplici~es of PFU equivalents. After virus adso~tion, replicate cultures were pure-~beIed with [3sS]methio~ne (15 min) at hourly intervals and TCApreeipitable incorporation from two cultures was determined for each time interval.

10 PFU equivalents/cell of AFV3, as this was sufficient to inhibit cellular protein synthesis maximally without causing severe cytopathic effects within the period of the experiments. Since similar results were obtained with both BHK and FHM cells, we show only representative data from experiments with BHK cells.

Since the average size of pol~bosomes is proportional to the rates of polypeptide chain initiation and inversely proportional to the rate of chain elongation (Lodish, 19’76), measurements of polyribosome size can provide information about relative changes in the average rates of these steps in AFV3infected cells. To determine if there was a selective effect on a particular class of polyribosomes (i.e., membrane bound versus free) we compared the size of the total, membr~e-bound, and free polyribosomes in cells at various times after AFV3-treatment. Figure 2 shows that in AFV3-treated cells there was a rapid and pronounced decline in the large pol~bosomes with a concurrent increase in the small polyribosomes

322

RAGHOW AND GRANOFF MEMBRANE BOUND

FREE

IHRAFV3

2 HR AFV3

0.2 3.1 3

3 HR AFV?

nToM

TC

hJ mm

TC

02 01

DTTOM

0

FIG. 2. Effect of AFV3 on polyribosome profiles in BHK cells, Cell monolayers were either mock infected or treated with AFV3 at an input multiplicity of 10 PFU equivalents/cell. At the indicated times after adsorption, cytoplasmic extracts were prepared, and total, membrane-bound, or free polyribosomes were analyzed on lo-50% sucrose gradients.

and 80 S monosomes. Disaggregation of both free and membrane-bound polyribosomes was virtually complete within 2 hr. The step in protein synthesis affected by AFV3 appeared to be initiation, i.e., formation of mRNA ribosome complexes; the monosomal

material in AFV3-treated cells was free of mRNA as indicated by disaggregation of the 80 S monosomes into ribosomal subunits when analyzed in RSB-sucrose gradient containing 0.5 A4 NaCl (data not shown), conditions under which monosomes with

INHIBITION

OF CELLULAR

mRNA do not disaggregate (Zybler and Penman, 1969). Rates of Protein Chain Elongation in AF’V$ Treated Cells Remain Unaltered ~though the rapid disag~gatio~ of polyribosomes after treatment with AFV3 indicated that the ~tiation of cellular protein synthesis, or specifically mRNA attachment, was markedly retarded, we could not rule out an independent effect of AFV3 on the rate of protein chain elongation on previously bound messages. To determine whether chain elongation was also affected we measured the ribosome transit time in the following experiment. It is important to realize that the ribosome transit time (the length of the time required for a ribosome to complete translation and release a finished polypeptide after it has become attached to mRNA) is independent of the rate of ribosome attachment (initiation). The data that describes the ribosome transit times in uninfected and AFV&infected BHK cells are shown in Fig. 3 and Table 1. Plots of total and released protein counts as a function of time yielded essentially parallel lines that extrapolated to a separation of about 1 min on the time axis (Fig. 3). Therefore, an average ribosome transit time of approximately 2 min in both untreated and AFV&treated cells at 2 hr postinfection (when protein synthesis was inhibited by ~50%) suggested that once polypeptide synthesis was initiated, the rates of protein chain elongation

PROTEIN SY~HESIS

323

BY FV3 TABLE 1

THE EFFECT OF AFV3 ON RIBOSOME TRANSIT TIME”

Transit time Experiment 1 2 3 4

Mock-infected 1.98 2.10 2.00 1.34

AFV3-treated 2.10 1.90 1.88 1.90

0 Control (mock infected with PBS) and AFV3treated (10 PFU equivalents/cell of AFV3,2 hr p.i.) cells were labeled with [35S]methionine. Cell extracts were processed to determine incorporation into total (nascent and released) and released polypeptides and the ribosome transit times were calculated as detailed under Materials and Methods. Each transit time represents the mean value of two separate deter~nations.

by the ribosomes in AFV3-treated cells were not detectably altered (Table 1). Since average ribosome transit times were determined, a comparison with untreated cells is valid only if the proteins synthesized in AFV&treated cells have the same size distribution of those of control cells. As the size dist~bution of proteins synthesized in control and AFV~t~ated cells were not app~c~bly different (data not shown) the transit times could be directly compared. These results also ruled out the possibility of premature ribosome termination, resulting in the synthesis of subsize proteins, as the cause of protein synthesis inhibition.

AFV3 Does not Degrude C~~~~~arMessenger RNAs The decrease in the size of pol~bosomes, combined with marked inhibition of protein synthesis in AFV3-treated cells, suggested that either the cellular mRNAs were degraded or that the mRNAs were prevented from attaching to the ribosomes (i.e., initiation was blocked). The former possibility FIG. 3. Ribosome transit times are unaltered in was more than theoretical since the FV3 AFVB-treated BHK cells. Control or AFV3-treated virions have endogenous viral nucleases (10 PFU eq~v~ent~eell, 2 hr after virus adsorption, capable of degrading both single- and doubleX0% in~bition of protein synthesist cultures were labeled with ~]met~o~ne (10 &Xtrl~. At the denoted stranded RNAs (Palese and Koch, 19’72). Although heat inactivation destroys these times, cells were processed to determine radioactivity in the total (nascent and released, 0) and released enzymatic activities in v&o (IX Willis, (0) acid precipitable material. personal communication) reactivation of these

324

RAGHOWANDGRANOFF TABLE 2

RECOVERYOFCELLULARRNASAFTER AFV3 TREATMENT" Experiment No.

3H cpm x 1Om6 Nonpolyadenylated RNA

3H cpm x 10m4 Polyadenylated RNA

I

Control AFV3

1.97 2.01

1.92 1.98

II

Control AFV3

3.64 3.59

3.80 3.82

a Cells labeled for 12 hr with [3H]uridine (50 @.X100-mm dish), were either mock infected (with PBS) or infected with AFV3 (10 PFU equivalent/cell) and 3 hr postadsorption cytoplasmic RNA was extracted (Willis and Granoff, 1976). After fractionation on oligo(dT)-cellulose columns to separate polyadenylated and nonpolyadenylated RNAs (Glazier et al., 1977) radioactivity in the ethanol-precipitable RNA was determined.

nucleases intracellularly could not be excluded. In cells labeled for 12 hr with [3H]uridine the poly(A) RNAs comprise about l-1.5% of the total cytoplasmic RNA; the relative proportion of polyadenylated and nonpolyadenylated RNAs remains unchanged in cells treated with AFV3 (Table 2). To further assess the possible breakdown of mRNAs, we prelabeled cells with [14C] (mock-infected)- or [3H]uridine (AFV3 treated), isolated the poly(A)-containing RNAs, and analyzed their size distribution by sucrose gradient sedimentation. The

FIG. 4. Sucrose gradient velocity centrifugation of poly(A) RNA from AFV3-treated BHK cells. Cell monolayers were prelabeled either with [3H]- or [“Cluridine for 18 hr. [3H]Uridine-labeled cells (0) were treated with AFV3 and [14C]uridine-labeled (0) cells were mock-infected with PBS. RNA was extracted and polyy(A) material was selected and centrifuged as described under Materials and Methods. Ribosomal RNA from chicken embryo lung cells was centrifuged in a similar gradient as a molecular weight marker.

poly(A) RNAs sedimented heterogenously between 12 to 28 S as judged by the relative positions of marker ribosomal and transfer RNAs (Fig. 4). Significantly, mRNAs isolated from AFV&treated cells were similar in size and distribution to the poly(A) mRNA from uninfected cells indicating no appreciable breakdown of cellular mRNAs in cells. Translation of mRNAs from AFVS-Treated or Untreated Cells in Vitro Yields Identical Polypeptides Although the experiments just described allowed us to conclude that the breakdown of host polyribosomes and the decline in the rate of protein synthesis could not be explained by random cleavage of the mRNA, they did not rule out the possibility of a small modification in the cellular mRNAs that could render them nonfunctional. To establish whether the cellular mRNAs in AFV3infected cells were functional, we programmed an mRNA-dependent translational extract from rabbit reticulocytes with poly(A) RNA isolated from untreated cells or from cells treated with AFV3 for 3 hr. The polypeptide products of translation in vitro were then resolved by bidimensional gel electrophoresis (O’Farrell, 19’75;Raghow et al., 1978). Polypeptides made in response to mRNAs from untreated or AFV&treated cells exhibited an almost identical pattern (Fig. 5). It is clear, therefore, that the majority of poly(A) mRNAs in the AFV&treated cells can function in a heterologous protein synthesizing system.

INHIBITION

OF CELLULAR

We have already shown that there was no appreciable change in the recovery of poly(A) RNAs from AFV3-treated cells as compared to untreated cells (Table 2). It should be emphasized that we obtained optimal incorporation of [35S]methionine in vitro with similar final concentrations of poly(A) RNA (80 pg/ml) irrespective of its source (mock-infected or AFV3-infected cells) suggesting similar translational efficiencies of these RNAs (data not shown). Similar relative intensities of corresponding polypeptide spots in the two autoradiograms (Fig. 5) further reinforce our conclusion that the rates of translation in vitro of mRNAs from mock-infected or AFV&treated cells are similar.

PROTEIN SYNTHESIS

pH 8.0-

325

BY FV3

I EF+

pH 4.0

DISCUSSION

Although the phenomenon of viral inhibition of cellular protein synthesis was discovered almost two decades ago, the underlying mechanisms of action remain unclear (Bablanian 1975). The principle aim of our experiments was to determine which of the major steps of protein synthesis (i.e., initiation, elongation, mRNA breakdown, etc.) was altered in cells treated with AFV3. The rapid disaggregation of polyribosomes, accompanied by unaltered rates of polypeptide chain elongation, and the lack of detectable breakdown of mRNA, as revealed by their translational competency in mRNAdependent reticulocyte extract, is consistent with the conclusion that the initiation of polypeptide synthesis is the step in protein synthesis affected by AFV3. Since viral proteins are synthesized normally when AFVS-treated cells are superinfected with active FV3 (Goorha and Granoff, 1974), the effect on initiation of translation is discriminatory. In addition to inhibiting cellular mRNA translation, AFV3 also inhibits translation of vesicular stomatitis virus (VSV) mRNA (Tannenbaum et aZ., 1978) and, as with cellular mRNAs, VSV mRNAs are not degraded. The results with VSV emphasize the specificity of the inhibitory activity of FV3. Although our data allow us to conclude that AFV3 inhibits host protein synthesis at the initiation step, the mechanism that

FIG. 5. Comparison of polypeptides synthesized in vitro by mRNA from control or AFV3-treated cells. Nuclease-treated rabbit reticulocyte extracts (Pelham and Jackson, 19’76) were programmed with poly(A) mRNA isolated from either mock-infected (control) or AFWtreated BHK cells. [35S]Methionine-labeledpolypeptides were subjected to two-dimensional O’Farrell gel analysis and fluorography as outlined under Materials and Methods.

discriminates between host and viral mRNA translation remains to be elucidated. Conceivably, a lesion in the initiation of translation could represent a modification in the structure of the ribosome or of one of the several initiation factors involved in assembly of the eucaryotic translation initiation complex. Both kinds of modifications have been shown to occur; phosphorylation of ribosomal proteins in vaccinia virus-infected HeLa cells (Kaerlin and Horak, 1976) and unidentified modification of an initiation factor(s) in poliovirus-infected HeLa cells (Helentjaris and Ehrenfeld, 1978; Rose et al., 1978). Precisely which initiation factor(s) is the primary target of viral action is controversial; while inactivation of eIF-4B was demonstrated in poliovirus-infected cells (Padilla et al., 1978; Rose et al., 1978), Kempfer et aZ., (1978) identified eIF-2 as the target in encephalomyocarditis virus-infected HeLa cells. Since initiation of protein syn-

326

RAGHOWANDGRANOFF

thesis is a multi-step process involving several initiation factor(s) (Lodish, 1976; Weissbach and Ochoa, 1976) it is possible that any one of them can be the target of a virus and lead to a similar manifestation, i.e., selective initiation of translation of different mRNAs. To identify the specific target of FV3 action will require an in vitro translational system reconstituted from the proteinsynthesizing components (e.g. ribosomes, various protein factor(s), and mRNAs, etc.) of control and AFV3-infected cells. For such studies, FV3 offers an added advantage over other virus systems; a viral protein(s) is the mediator of the mechanism of switch-off, it can be solubilized from purified FV3 (Aubertin et al., 1973) and it suppresses protein synthesis in a cell-free proteinsynthesizing system (Aubertin et al., 19’78) as well as in intact cells (Aubertin et al., 1973). It should be feasible, therefore, to isolate and characterize the viral switch-off protein(s) and to determine the mechanism of its action from its molecular interaction with components of the cellular translational apparatus. ACKOWLEDGMENTS Ms. Dorothy Neale and Ms. Susan Carr provided expert technical assistance. This study was supported by Research Project Grant CA-07055 and CORE Grant CA-21765 from the National Cancer Institute, and by ALSAC. REFERENCES AUBERTIN, A. M., CORDIER,O., and KIRN, A. (1978). The effect of FV3 structural proteins on cellular protein synthesis. In “Abstracts, 4th International Congress for Virology.” The Hague. AUBERTIN, A. M., HIRTH, C., TRAVO, C., NONNENMACHER,H., and KIRN, A. (19’73).Preparation and properties of an inhibitory extract from frog virus 3 particles. J. Virol. 11, 694-701. BABLANIAN, R. (1975). Structural and functional alterations in cultured cells infected with cytocidal viruses. Progr. Med. Virol. 19, 40-83. DRILLIEN, R., SPEHNER,D., and KIRN, A. (1977). Cell killing by frog virus 3: Evidence for cell killing by single viral particles or single viral subunits. Biochem. Biophys. Res. Commun. 79, 105-111. FAN, H., and PENMAN, S. (1970). Regulation of protein synthesis in mammalian cells. II. Inhibition of protein synthesis at the level of initiation during mitosis. J. Mol. Biol.

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INHIBITION

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in polypeptides of negative strand RNA viruses. Virology 99, 214-225. ROSE, J. K., TRACHSEL, H., LEONG, K., and BALTIMORE, D. (1978). Inhibition of translation by poliovirus: inactivation of a specific initiation factor. Proc. Nat. Ad. Sci. USA 75, 2732-2736. TAN, K. B., and MCAUSLAN, B. R. (1971). Proteins of polyhedral cytoplasmic deoxyvirus. I. The structural polypeptides of FV3. Virology 45, 200-207. TANNENBAUM, J., GOORHA, R., and GRANOFF, A. (1979). The inhibition of vesicular stomatitis virus protein synthesis by frog virus 3. Virology 95,227231. WEISSBACH,H., and OCHOA,S. (1976). Soluble factors

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