Synthesis and distribution of vesicular stomatitis virus-specific polypeptides in the absence of progeny production

VIROLOGY

81, 37-47 (1977)

Synthesis and Distribution of Vesicular Stomatitis Virus-Specific Polypeptides in the Absence of Progeny Production’ SHEILA Department

of Microbiology

P. LITTLE2

and Molecular

AND

ALICE

Genetics, Harvard Accepted April

S. HUANG3j4

Medical

School, Boston, Massachusetts

02115

4,1977

In cultures of Chinese hamster ovary cells infected by vesicular stomatitis virus (VW), viral polypeptides can be found in cellular, virion-associated, and soluble fractions. In order to determine whether or not the soluble polypeptides resulted from degradation of virions, progeny formation was inhibited and the distribution of polypeptides in these fractions was examined. Synthesis of infectious progeny was prevented in three ways: (1) addition of defective interfering (DI) particles; (2) utilization of a replication-minus mutant, tsG41; and (3) utilization of a glycoprotein mutant, ts045. In experiments involving either the coinfection of standard infectious particles and DI particles or the infection of cells at the nonpermissive condition for tsG41, overall virusspecific RNA synthesis was reduced by 80%, whereas the appearance of extracellular soluble viral polypeptides was not greatly inhibited. Cell-associated polypeptides were also present in normal amounts. By using ts045, it was shown that there was no virion maturation but, surprisingly, the appearance of soluble viral polypeptides was enhanced. Therefore, the distribution of viral proteins in the cell-associated and extracellular soluble fractions was not dependent on the production of infectious progeny. The major soluble viral polypeptide was a glycoprotein which migrated more rapidly than the virion-associated glycoprotein; these two glycoproteins had about the same amount of carbohydrate, even when the soluble glycoprotein was synthesized by ts045 under nonpermissive conditions. These results lead to the hypothesis that G protein interaction with the plasma membrane exists in three different states: (1) an initial very unstable association; (2) possible stabilization at the cell surface via interactions with another membrane protein called matrix protein; and (3) aggregation of G protein at the cell surface initiated by nucleocapsids interacting through the matrix proteins. The first two stages of this interaction probably result in the shedding of G protein from the cell surface. INTRODUCTION

coprotein (G); the inner envelope-associated protein is a matrix or membrane (M) protein; the RNA-binding (N) protein associates with the virion genome to form the nucleocapsid; and the largest (L) protein and a phosphorylated (NS) protein associate with the nucleocapsid (see Wagner et al., 1972). The synthesis of each of these proteins appears to be unregulated temporally, in that they are made at about the same relative proportions throughout infection (Wagner et al., 1970). Kang and Prevec (1971) examined cellassociated VSV proteins in conjunction with the appearance of both virion structural proteins and extracellular soluble

the multiplication of vesicular During stomatitis virus (VSV), five virus-specific proteins are synthesized (Kang and Prevec, 1971; Wagner et al., 1972). The outer envelope-associated protein is a glyI This investigation was supported by research grants VC-63B and VC-63C from the American Cancer Society, BMS74-09713 from the National Science Foundation, and AI10100 from the U.S. Public Health Service. 2 Predoctoral Fellow of the Ford Foundation. 3 Research Career Development Awardee of the U.S. Public Health Service. * Author to whom reprint requests should be addressed. Copyright 0 19’77 by Academic Press, Inc. All rights of reproduction in any form reserved.

37 ISSN

0042-6622

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antigens in the medium. We have confirmed the relative distribution of these polypeptides. Because an important biological role may exist, during viral pathogenesis, for extracellular soluble virusspecific polypeptides, it was necessary to determine the pathway by which newly synthesized viral polypeptides reached the soluble pool. Two possibilities exist: (1) that the extracellular soluble polypeptides arose from virion degradation or (2) that these polypeptides are shed directly from infected cells. Here we report the results of experiments designed to test these possibilities. Progeny formation was inhibited in three different ways, and the extracellular soluble polypeptides were examined. Under these conditions soluble virus-specific antigens were not greatly affected. Moreover, cell-associated polypeptides remained at the same amounts when progeny synthesis was prevented. In a particular case, by using a t.s mutant with a lesion in the glycoprotein, we were able to demonstrate that the lesion affected virion maturation but, surprisingly, had no inhibiting effect on the appearance of the soluble antigens. Thus, the presence of viral polypeptides in the cell-associated and soluble fractions was not dependent on the production of infectious progeny virions. This finding supports shedding from infected cells as a mechanism by which virus-specific polypeptides reach the extracellular environment. The interpretation of these findings in relation to viral morphogenesis and pathogenesis will be discussed. MATERIALS

AND

METHODS

Viruses and cells. The history of Chinese hamster ovary (CHO) cells and the Indiana serotype of vesicular stomatitis virus (VSV) used in these studies has been given (Stampfer et al., 1969). Mutants tsG41 and ts045 were kindly supplied by Dr. Craig Pringle (Pringle, 1970; Flamand and Pringle, 1971). Recently cloned and sucrose gradient-purified standard infectious particles and defective interfering (DI) particles were used throughout these studies (Stampfer et al., 1971; Huang et al., 1966). The determination of

HUANG

multiplicities of both types of particles has been described (Huang and Manders, 1972). Infection of cells and labeling with radioactive precursors. CHO cells in suspension at a concentration of 4 X 10” cells/ml were infected by the indicated virus particles at 34”. The medium consisted of Joklik’s modified Eagle’s minimal essential medium, supplemented with nonessential amino acids and 2% fetal calf serum and buffered at pH 7.4 with 25 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) and 10 mM N-tris(hydroxymethyljmethyl - 2 - aminoethanesulfonic acid (TES). This pH has been found to be optimal for the production of infectious VSV (Fiszman et al., 1974; Huang and Palma, 1974). During each experiment the infection was monitored by a pilot assay for cumulative virus-specific RNA synthesis as described previously (Huang et al., 1970). For radioactive labeling of polypeptides, the infected cells were centrifuged and resuspended in medium devoid of leucine and containing 2% dialyzed fetal calf serum. 13H]Leucine or [14Clleucine was added for the indicated periods at 34”. At the end of the labeling period infected cultures were processed into three different fractions. One fraction, the cellular cytoplasmic extract, was obtained by nonionic detergent solubilization of infected cells (Huang and Manders, 1972). Cytoplasmic proteins were precipitated by the addition of nine volumes of cold acetone to the cytoplasmic extract. The precipitate was collected by centrifugation at 15,000 g for 15 min at 4”. The second fraction was extracellular virions, defined as material which sedimented from the cell-free medium at 41,700g for 60 min at 4”. The third fraction consisted of “soluble” antigens (Kang and Prevec, 1970) which remained in the medium after both cells and virus particles had been removed. To harvest these antigens, proteins were precipitated from the medium by adding saturated ammonium sulfate to a final concentration of 60%. After 60 min at 4”, the precipitate was collected by centrifugation at 6660 g for 20 min at 4” and then washed once with 50%

DISTRIBUTION

OF VSV POLYPEPTIDES

saturated ammonium sulfate. The final pellet was dissolved in phosphate-buffered saline (PBS; Dulbecco and Vogt, 1954) and dialyzed for 18 hr against PBS according to the procedure of Kang and Prevec (1970). All three fractions were stored at -70”. Polyacrylamide-gel electrophoresis. The cytoplasmic and extracellular fractions were each suspended in a final volume of 2.0 ml. The soluble fraction was maintained in 2.0 ml of dialyzed PBS solution. Equivalent aliquots (12.5%) of each fraction were analyzed on 10% SDS-polyacrylamide gels, prepared by Laemmli’s modification of Maizel’s method, in Tris-glycine buffer (Laemmli, 1970; Stampfer, 1972). In Figs. 6 and 7 aliquots of each fraction were analyzed on Laemmli’s 10% SDS-polyacrylamide gels (Laemmli, 1970). The gels were cast either in cylindrical Perspex tubes (6 x 190 mm) or as slab gels of 1.5mm thickness. Electrophoresis was performed at 100 V for 5 hr for cylindrical gels and for 6 hr for slab gels. Cylindrical gels were fractionated into l-mm slices by elution with 0.1% SDS in a Maize1 Autogeldivider (Savant Instruments, Hicksville, N.Y.). The crushed gel pieces were suspended in Bray’s solution (Bray, 1960) and counted in a Beckman LS-233 scintillation counter. All results shown have been corrected for background and spillover radioactivity. Slab gels were dried in a Hoefer slab-gel dryer (Hoefer Scientific Instruments, San Francisco, Calif.) and exposed to X-ray film. Autoradiograms were scanned with an E-C transmission densitomer (E-C Apparatus Corp., St. Petersburg, Fla.). Materials. Radioactive precursors (L[4,B”H]leucine at 64 Ci/mmol, 14C-labeled L-amino acid mixture, L-[14C]-leucine at 302 mCi/mmol, D-[6(N)-“Hlglucosamine hydrochloride at 7.3 Ci/mmol, and Dl’4Clglucosamine hydrochloride at 237 mCi/mmoU were obtained from New England Nuclear, Boston, Mass. The organic buffers HEPES and TES were purchased from Mallinckrodt Chemical Works, St. Louis, MO. Actinomycin D was a kind gift from Merck, Sharp, and Dohme, Rahway, N. J. Marker 14C-amino acid-labeled virion proteins were a kind gift from Dr. Pamela

39

Siegel. All other chemicals and reagents were obtained as described previously (Huang and Manders, 1972). RESULTS

Reproducibility of the Fractionation cedures for Viral Proteins

Pro-

In order to be able to make quantitative comparisons among the cellular, virion, and soluble fractions of VSV-infected cells, the reproducibility of the fractionation procedure was first examined. Infected cells were labeled with radioactive leutine, and during each of the fractionation steps aliquots were taken for the determination of acid-precipitable radioactive material. In five separate experiments the total amount of labeled polypeptides obtained just prior to electrophoresis represented approximately 80% of the starting material from each of the.three fractions. Deviation in this percentage yield from one experiment to the next was as great as 20%. When different cultures of infected cells were handled on the same day, the deviation was reduced to 12% or less. Therefore, quantitative comparisons among the three different fractions appeared to be valid, especially when the differences were usually 50% or greater. Relative Amounts the Cellular, Fractions

of VSV Polypeptides in Virion, and Soluble

In order to demonstrate the distribution of viral proteins in each of the fractions, CHO cells were infected with wild-type standard VSV, and 13Hlleucine was added to these cells for a period of 3 hr during infection. The culture was fractionated into cellular, virion, and soluble fractions; each fraction was separated on 10% polyacrylamide gels together with a marker provided by 14C-amino acid-labeled virions. Figure 1A shows that host cell protein synthesis was inhibited and that all the stable cell-associated proteins labeled un der these conditions migrated almost identically with VSV structural proteins. The NS protein is not indicated in Fig. 1 or in subsequent figures because 10% acrylamide gels do not adequately separate this

LITTLE

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A.

AND

4

which migrated faster than the G protein from virions (Fig. 1C). The major soluble antigen, which we have designated Gs, has been previously identified by Kang and Prevec (1970) as a soluble glycoprotein with antigenic similarities to the G protein. There are two questions concerning the synthesis and distribution of VSV proteins: (1) What proportion of the proteins in each of the fractions is synthesized as a direct result of primary transcription by input virions? (2) Are soluble antigens present because of the degradations of virions or because they arise by direct shedding from infected cells?

16

Primary Translation Products Made in the Presence of Defective Interfering Particles

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FIG. 1. SDS-polyacrylamide-gel analysis of proteins synthesized by wild-type VSV. A sample of 4 x 10’ CHO cells was infected at 34” with wild-type standard VSV at a multiplicity of 40 PFU/cell. At 2.0 hr postinfection, infected cells were pelleted and resuspended in leucine-minus medium and returned to 34”. After 5 min, [4,5-3H]1eucine (100 &i) was added. At 5.0 hr postinfection, infected cells were harvested and fractionated into three parts. Oneeighth of each fraction plus W-labeled VSV marker proteins were analyzed by electrophoresis as described in Materials and Methods. (A) Cellular, (B) virion, and (C) soluble fractions; CO---- -0) [3H]leucine, (0-O) “C-labeled VSV marker.

from the N protein. A considerable amount, about 60%, of these stable virusspecific proteins synthesized during this period was found in the extracellular virion fraction (Fig. 1B). The soluble fraction which contained about 3% of the total, also, consisted of polypeptides which comigrated with VSV structural proteins except for one polypeptide, the major one,

One approach towards delineating the relationships between the proteins in each of the fractions is to inhibit the replication of the 40 S genome RNA by the addition of VSV-DI particles (Palma et al., 1974). Under conditions of maximal interference some DI-specific RNAs are replicated in the system but there is little or no amplification of mRNA beyond primary transcription (Huang and Manders, 1972). Therefore, few progeny virions besides DI particles are expected to be produced and almost all of the viral proteins that are synthesized should be the result of translation of primary transcripts. To demonstrate this, cells were infected by standard and DI particles, each at a multiplicity of 40. The amount of virus-specific RNA synthesized by cells that were coinfected was, as expected, only 20% of that synthesized by cells infected with standard virus alone (Fig. 2). When the distribution of proteins was examined from cells infected with both standard and DI particles, the amount in each of the three fractions was not reduced in the same proportion relative to the reduction in RNA synthesis (Fig. 3). The cellular fraction did not show a significant decline in the synthesis of proteins from cells infected with standard virus alone; this held true both for the total amount of proteins and for the relative proportion of

DISTRIBUTION

41

OF VSV POLYPEPTIDES

neither the cellular nor the soluble fraction reflect the 80% inhibition in the synthesis of RNA. Primary Translation Products Made by tsG41 at the Nonpermissive Temperature Another way to limit VSV-RNA synthe-

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ffOUR.5 FIG. 2. Cumulative virus-specific RNA synthesis in the presence and absence of superinfecting defective interfering (DI) particles. Two-milliliter pilot samples infected either with standard virus or with both standard and DI particles were assayed for incorporation of radioactive uridine by acid-precipitable radioactivity as described in Materials and Methods. This infection was carried out in parallel with that shown in Fig. 3. The level of incorporation of radioactive uridine by uninfected cells in the presence of actinomycin D has been found to represent reproducibly 5% of total incorporated radioactivity in other experiments and has not been subtracted from the assay points.

each polypeptide (Fig. 3A). Inhibition of host cell protein synthesis was not as effective in the coinfected cells as in cells infected with standard virus alone as indicated by the heterogeneous broad background. The virion fraction, as expected, indicated marked reduction in the synthesis of extracellular virions when cells were coinfected with DI particles (Fig. 3B). The higher ratio of G protein to the N protein from the mixedly infected cells confirms previous observations that only shorter, bullet-shaped DI particles were produced (Huang and Manders, 1972; Palma et al., 1974). Despite the reduction in virions, the soluble fraction still contained viral polypeptides from cells coinfected with DI particles (Fig. 3C). Here Gs and the other polypeptides were reduced by 40% under conditions of interference. It is clear that

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40

60

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SDS-polyacrylamide-gel electrophoresis of polypeptides synthesized by cells infected with standard particles alone or infected with both standard and defective interfering (DI) particles. Samples of 4 x 10’ CHO cells were infected with standard particles alone or with standard and DI particles, each at a multiplicity of 40. The two cultures were labeled with [4,5-3H]1eucine at 10 pCi/ml for 3.0 hr beginning at 2.5 hr after the initiation of infection. Labeled polypeptides from the cultures were distributed into three fractions analyzed separately by electrophoresis as described in Materials and Methods. The migration of marker virion proteins in each gel is indicated by the letters L, G, N, and M. (A) Cellular, (B) virion, and (C!) soluble fractions; (0- - - - -0) infected with standard virus, (0-O) infected with standard and DI particles.

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sis to only primary transcription is by the use of a ts mutant. Mutant tsG41 belongs to complementation group IV (Pringle, 1970) and has been shown to have a lesion affecting virion RNA synthesis (Perlman and Huang, 1974). When the replication of virion RNA is prevented from the beginning of infection by incubating at the nonpermissive temperatures, progeny virions are not produced and secondary transcription is prevented. Figure 4 shows the results of the pilot experiment in which RNA synthesis at the nonpermissive temperature was indeed only 20% of that at the permissive temperature when the infection by tsG41 was initiated and maintained at the respective temperatures. When the proteins synthesized at the permissive and nonpermissive temperatures were compared, it was apparent that the temperature of incubation had no effect on the synthesis of polypeptides found in the soluble fraction (Figs. 5E and F). A similar result was obtained for the cellular fraction except for the reduction of N protein at the nonpermissive

FIG. 4. Cummulative RNA synthesis of tiG41 at permissive and nonpermissive temperatures. Twomilliliter pilot samples of CHO cells were infected with tsG41 and either incubated at 31” (O- - - - -0) or 38” (O---O). RNA synthesis was assayed as described in Materials and Methods. This infection was performed in parallel with that shown in Fig. 5. The background level of incorporation of uridine into RNA of uninfected cells treated with actinomytin D was noted as in Fig. 2.

HUANG

temperature (Figs. 5A and B). In contrast, the proteins in the virion fraction were reduced to barely detectable levels at the nonpermissive temperature (Figs. 5C and D). Again, the inhibition of genome replication and the subsequent lack of mRNA amplification did not reduce the cellular and soluble fractions, but only affected the production of progeny virions. Parenthetically, the instability of N protein intracellularly probably reflects the lesion in tsG41, which has been shown to produce an unstable N protein at the nonpermissive temperature (Knipe et al., 1977). Continued Synthesis of Gs Protein by a Mutant with a Temperature-Sensitive Lesion in the G Protein Because the previous experiment indicated that the synthesis of soluble proteins was dependent neither on virion RNA replication nor on progeny formation, another method to test the uncoupling of progeny formation from the synthesis of Gs protein was utilized. This involved the mutant ts045 which has a heat-labile G protein (Deutsch and Berkaloff, 1971). G protein synthesized at the nonpermissive temperature does not appear to associate with plasma membranes and results in the subsequent inhibition of progeny formation (Lafay et al., 1975; Knipe et al., 1977). CHO cells were infected with ts045 at the permissive and nonpermissive temperatures and a comparison of the polypeptides in each of the three fractions was made. Figures 6A and B show the amounts of cell-associated proteins, with L and G polypeptides inhibited the most. The virion fractions indicate the expected inhibition of progeny virion formation at the nonpermissive temperature (Figs. 6C and D). G protein is notably absent from particles produced at the nonpermissive temperature. These particles were not infectious on CHO cells. A striking effect of the production of Gs protein by this mutant at the nonpermissive temperature is shown in Figs. 6E and F. Rather than the synthesis of this soluble antigen being inhibited, the amount of Gs was somewhat increased at the nonpermissive temperature (Fig. 6F). The other

DISTRIBUTION

OF VW

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43

POLYPEPTIDES

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FIG. 5. Densitometer tracings of autoradiograms of polypeptides synthesized by tsG41 at permissive and nonpermissive temperatures. Two samples of 4 x 10’ CHO cells were each infected with tsG41 at a multiplicity of 20 PFU/cell. One sample was incubated at 31” and the other at 38”. Each sample was labeled for 3.5 hr beginning at 1.5 hr after infection with 14C-labeled amino acids at 5 &Wml. The cultures were harvested and fractionated as described in Materials and Methods. The films were exposed for 3 days and scanned with an E-C transmission densitometer. (A,C,E) Cellular, virion, and soluble fractions, respectively, at 31”; (B,D,F) cellular, virion, and soluble fractions, respectively, at 38”; (G) VW marker proteins.

soluble proteins N and M were also present at the nonpermissive temperature. This result clearly shows that the presence of extracellular soluble proteins was independent of virion formation. Glycosylation of GS Protein Kang and Prevec (1970) initially described Gs protein and showed that it was readily labeled by glucosamine. To demonstrate further that the Gs protein detected under our conditions was also labeled by glucosamine, Gs protein synthesized by ts045 and tsG41 at the permissive and nonpermissive temperatures was compared to Gs protein made by wild-type virus with respect to its ability to incorporate glucosamine.

Figure 7E shows that Gs protein made at the nonpermissive temperature by tsO45 was glycosylated. Also, the cell-associated G protein synthesized at the nonpermissive temperature incorporated [14Clglucosamine. For comparison, glucosamine incorporation into Gs protein made by tsG41 is shown in Table 1, where the relative ratios of [3H]glucosamine to 14Clabeled amino acids in the virion-associated G protein and in the Gs protein are summarized. The relative ratios of carbohydrate to protein in the virion-associated G protein and in the soluble Gs protein were not markedly different. It is not known whether the striking difference in the ratio for G protein made by wild-type and by tsG41 was due to the difference in

44

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FIG. 6. Densitometer tracings of autoradiograms of labeled polypeptides synthesized by t.9045 at permissive and nonpermissive temperatures. A sample of 4 x 10’ CHO cells, infected with ts045 at a multiplicity of 20 PFU/ml, was equally divided and incubated at 31 and 38”. At 2.0 hr after infection, 14C-labeled amino acids (2 &i/ml) were added to each culture. At 5.0 hr after infection cells were harvested and fractionated as described in Materials and Methods. Equal aliquots of the cellular, virion, and soluble fractions were placed on a Laemmli-type 10% slab gel. Polypeptides were electrophoresed at 100 V for 6 hr. A film was exposed for 2 weeks and scanned as described in Fig. 5. (A,C,E) Cellular, virion, and soluble fractions, respectively, at 31”; (B,D,E) cellular, virion, and soluble fractions, respectively, at 38”; (G) VSV marker proteins.

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D/STANCE #/GRATED /cm/ 7. Densitometer tracings of n-glucosamine-labeled polypeptides of ts045 at permissive and nonpermissive temperatures. A sample of 4 x 10’ CHO cells, infected with ts045 at a multiplicity of 20 PFU/ml, was equally divided and incubated at 31” and 38”. Each sample was labeled for 3.0 hr beginning at 2.0 hr after infection with n-lW]glucosamine (5 &i/ml). Cells were harvested, fractionated, and analyzed as in Fig. 6. (A,C,E) Cellular, virion, and soluble fractions, respectively, at 31”; (B,D,E) cellular, virion, and soluble fractions, respectively, at 38”; (G) VSV marker proteins. FIG.

DISTRIBUTION TABLE

OF VSV POLYPEPTIDES

1

SUGAR TO AYINO ACID RATIOS OF VIRION-ASSOCIATED AND SOLUBLE GLYCOPROTEINS [3HlGlucosamine/W-labeled amino acids= Virus Temperature G Gs

wt+h tsG41’ tsG41c

34 31 38

1.76 5.28 -

1.64 6.89 8.19

Q The radioactivity found in the gel fractions containing the glycoprotein is expressed as the ratio of counts per minute of [“Hlglucosamine to the counts per minute of W-labeled amino acids. b A sample of 8 x 10’ CHO cells was infected with wild-type standard VSV at a multiplicity of 40 PFU/ cell. At 1.5 hr after infection, i4C-labeled amino acids (0.5 nCi/ml) and [3H]glucosamine (25 @X/ml) were added to the culture. At 5.5 hr after infection, cells were harvested, fractionated, and analyzed by electrophoresis as described in Materials and Methods. c A sample of 8 x 10’ CHO cells, infected with tsG41 standard particles at a multiplicity of 20 PFU/ cell, were equally divided and incubated at 31 and 38”. At 1.5 hr after infection, l”C-labeled amino acids (0.5 &i/ml) and [3Hlglucosamine (25 &i/ml) were added to each culture. At 5.5 hr after infection, cells were harvested and fractionated as described in Materials and Methods. Electrophoresis was performed on Laemmli’s 10% gels.

the viruses themselves or due to variations in cellular uptake and incorporation from day to day. DISCUSSION

These experiments were designed to examine the relative distribution of virusspecific polypeptides during the multiplication of VSV. Our results confirm those of Kang and Prevec (1970) in that the virusspecific proteins were distributed into the cell-associated, virion, and soluble fractions in the proportion 4:5:0.3. In addition, we showed that the amounts of proteins in the cell-associated and soluble fractions were largely unaffected when the production of infectious progeny was inhibited. These observations are discussed, especially in relation to cell-associated polypeptides or to soluble antigens. When infected cells were only permitted to synthesize primary transcripts from input genomes, the cells contained almost as

45

much virus-specific polypeptide as did productively infected cells. This surprising result serves to demonstrate the extent of primary translation of transcripts from negative-stand viruses. Support for a possible differentiation between the functional roles of primary and secondary translation products comes from the finding that DI nucleocapsids appear to have a lower efficiency for budding out with the resultant buildup of intracellular nucleocapsids (Palma et al., 1974). Such a control for differentiating the function between a primary and a secondary translation product is difficult to imagine, and these experiments do not suggest any possible mechanisms. The finding that soluble viral antigens are produced by infected cells without the production of infectious virions points to several interesting interpretations. Soluble antigens are clearly not degradation products of virions. A similar conclusion was made using the adenovirus system (Wilcox and Ginsberg, 1962). The mechanism by which soluble viral antigens reach the extracellular environment has not been analyzed in any detail, although the term “shedding” can be used to describe the loss of antigens from infected cells. Regarding the shedding of G protein, an initial very unstable interaction at the cell surface is suggested by the finding that ts045 produces more Gs protein at the nonpermissive temperature than at the permissive temperature. The lesion in tsO45 may be in the inability of G protein to recognize M proteins and may, thereby, result in a rapid loss from the cell surface. Experiments to test such a possibility using mutants with lesions in the M protein are in progress. Experiments with DI particles and tsG41 can be interpreted to be a measure, once G protein is at the cell surface and anchored by M proteins, of the finite chance that it has of being shed into the extracellular environment. The ratio of Gs protein to cell-associated G protein is approximately 1:6, indicating that one in six plasma membrane-associated G proteins can be shed. Throughout infection with wild-type VSV, this chance of being shed is highest for the newly synthesized

46

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G protein (Kang and Prevec, 1971). Therefore, G protein gradually stabilizes at the cell surface. This may be due to the aggregation of G protein in the process of viral maturation and budding-out. The results with tsG41 suggest that nucleocapsids are required for this aggregation. The increased mobility on acrylamide gels of Gs protein relative to G protein shows a 5000 to 7000-dalton difference. Because Gs protein appears to be fully glycosylated under the various conditions used in these studies, the difference in molecular weight is probably due to the loss of a hydrophobic portion imbedded in the membrane. Such a hydrophobic portion has been produced upon proteolytic cleavage of G protein from virions (Mudd, 1974; Schloemer and Wagner, 1975). The heterogeneity in Gs protein on acrylamide gels suggests that cleavage may not be the only mechanism by which this protein is shed from infected cells. The presence of these soluble antigens as well as plasma membrane-associated antigens is biologically significant. Soluble antigens not only serve to elicit an immune response in animals but also can combine with antibodies to form immune complexes and, thereby, lower the effective concentration of neutralizing antibodies. Moreover, membrane-associated antigens alter the cell surface and may subject the infected cell to immunologic surveillance. If surface antigens are reduced by the addition of DI particles or by shedding into the extracellular medium, then persistently infected cells may escape immune attack. Such a possibility has been observed for cells persistently infected with arenaviruses (Oldstone, personal communication). ACKNOWLEDGMENTS We thank Ms. Norma Hewlett for excellent technical support and Dr. Leevi Kaarilinen for stimulating discussions. REFERENCES BRAY, G. A. (1960). A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1, 279-285. DEUTSCH, V., and BERKALOFF, A. (1971). Analyse d’un mutant thermolabile du virus de la stomatite

HUANG vesiculaire (VSV). Ann. Inst. Pasteur Paris 121, 101-106 DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99, 167-182. FISZMAN, M., LEAUTE, J.-B., CHANY, C., and GIRARD, M. (1974). Mode of action of acid pH values on the development of vesicular stomatitis virus. J. Viral. 13, 801-808. FLAMAND, A., and PRINGLE, C. R. (1971). The homologies of spontaneous and induced temperaturesensitive mutants of vesicular stomatitis virus isolated in chick embryo and BHK 21 cells. J. Gen. Viral. 11, 81-85. HUANG, A. S., BALTIMORE, D., and STAMPFER, M. (1970). Ribonucleic acid synthesis of vesicular stomatitis virus. III. Multiple complementary messenger RNA molecules. Virology 42, 946-957. HUANG, A. S., GREENAWALT, J. W., and WAGNER, R. R. (1966). Defective T particles of vesicular stomatitis virus. I. Preparation, morphology and some biologic properties. Virology 30, 161-172. HUANG, A. S., and MANDERS, E. K. (1972). Ribonucleic acid synthesis of vesicular stomatitis virus. IV. Transcription by standard virus in the presence of defective interfering particles. J. Viral. 9, 909-916. HUANG, A. S., and PALMA, E. L. (1974). Vesicular stomatitis virus: Defectiveness and disease. In “Mechanisms of Virus Disease” (W. S. Robinson and E. F. Fox, eds.), pp. 87-99. Benjamin, Menlo Park, Calif. KANG, C. Y., and PREVEC, L. (1970). The proteins of the vesicular stomatitis virus II. Imunological comparisons of viral antigens. J. Viral. 6, 20-27. KANG, C. Y., and PREVEC, L. (1971). Proteins of vesicular stomatitis virus. III. Intracellular synthesis and extracellular appearance of virus-specific proteins. Virology 46, 678-690. KNIPE, D. M., LODISH, H. F., and BALTIMORE, D. (1977). Analysis of the defects of temperature-sensitive mutants of vesicular stomatitis virus. Intracellular degradation of specific viral proteins. J. Viral. 21, 1140-1148. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680X%2. LAFAY, F. (1974). Envelope proteins of vesicular stomatitis virus: Effect of temperature-sensitive mutants in complementation groups II and V. J. Viral. 14, 1220-1228. MIJDD, J. A. (1974). Glycoprotein fragment associated with vesicular stomatitis virus after proteolytic digestion. Virology 62, 573-577. PALMA, E. K., PERLMAN, S. M., and HUANG, A. S. (1974). Ribonucleic acid synthesis of vesicular stomatitis virus. VI. Correlation of defective particle RNA synthesis with standard RNA replication. J. Mol. Biol. 85, 127-136.

DISTRIBUTION

OF VSV POLYPEPTIDES

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