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During viral RNA synthesis by the viral RNA-dependent RNA polymerase (vRdRp) of vesicular stomatitis virus, the sequestered RNA genome must be released from the nucleocapsid in order to serve as the template. Unveiling the sequestered RNA by interactions of vRdRp proteins, the large subunit (L) and the phosphoprotein (P), with the nucleocapsid protein (N) must not disrupt the nucleocapsid assembly. We noticed that a flexible structural motif composed of an α-helix and a loop in the N protein may act as the access gate to the sequestered RNA. This suggests that local conformational changes in this structural motif may be induced by interactions with the polymerase to unveil the sequestered RNA, without disrupting the nucleocapsid assembly. Mutations of several residues in this structural motif-Glu169, Phe171, and Leu174-to Ala resulted in loss of viral RNA synthesis in a minigenome assay. After implementing these mutations in the viral genome, mutant viruses were recovered by reverse genetics and serial passages. Sequencing the genomes of the mutant viruses revealed that compensatory mutations in L, P, and N were required to restore the viral viability. Corresponding mutations were introduced in L, P, and N, and their complementarity to the N mutations was confirmed by the minigenome assay. Introduction of the corresponding mutations is also sufficient to rescue the mutant viruses. These results suggested that the interplay of the N structural motif with the L protein may play a role in accessing the nucleotide template without disrupting the overall structure of the nucleocapsid. IMPORTANCE During viral RNA synthesis of a negative-strand RNA virus, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the sequestered RNA in the nucleocapsid to use it as the template, but at the same time may not disrupt the nucleocapsid assembly. Our structural and mutagenesis studies showed that a flexible structural motif acts as a potential access gate to the sequestered RNA and plays an essential role in viral RNA synthesis. Interactions of this structural motif within the vRdRp may be required for unveiling the sequestered RNA. This mechanism of action allows the sequestered RNA to be released locally without disrupting the overall structure of the nucleocapsid. Since this flexible structural motif is present in the N proteins of many NSVs, release of the sequestered RNA genome by local conformational changes in the N protein may be a general mechanism in NSV viral RNA synthesis., (Copyright © 2018 American Society for Microbiology.)

Non-segmented negative strand RNA viruses belonging to the Mononegavirales order possess RNA-dependent RNA polymerase L proteins within viral particles. The L protein is a multifunctional enzyme catalyzing viral RNA synthesis and processing (i.e., mRNA capping, cap methylation, and polyadenylation). Using vesicular stomatitis virus (VSV) as a prototypic model virus, we have shown that the L protein catalyzes the unconventional mRNA capping reaction, which is strikingly different from the eukaryotic reaction. Furthermore, co-transcriptional pre-mRNA capping with the VSV L protein was found to be required for accurate stop?start transcription to synthesize full-length mRNAs in vitro and virus propagation in host cells. This article provides a review of historical and present studies leading to the elucidation of the molecular mechanism of VSV mRNA capping.

Sinefungin (SIN), a natural S-adenosyl-L-methionine analog produced by Streptomyces griseolus, is a potent inhibitor of methyltransferases. We evaluated the effect of SIN on replication of vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses. The 241-kDa large polymerase (L) protein of VSV methylates viral mRNA cap structures at the guanine-N-7 (G-N-7) and ribose-2'-O (2'-O) positions. By performing transcription reactions in vitro, we show that both methylations are inhibited by SIN and that methylation was more sensitive at the G-N-7 than at 2'-O position. We further show that SIN inhibited growth of VSV in cell culture, reducing viral yield by 50-fold and diminishing plaque size. We isolated eight mutants that were resistant to SIN as judged by their growth characteristics. The SIN-resistant (SINR) viruses contained mutations in the L gene, the promoter for L gene expression provided by the conserved sequence elements of the G-L gene junction and the M gene. Five mutations resulted in amino acid substitutions to conserved regions II/III and VI of the L protein. For each mutant, we examined viral gene expression in cells and cap methylation in vitro. SINR mutants upregulated RNA synthesis in the presence of SIN, which may be responsible for their resistance. We also found that some SINR viruses with L gene mutations were defective in cap methylation in vitro, yet their methylases were less sensitive to SIN inhibition than those of the wild-type parent. These studies show that the VSV methylases are inhibited by SIN, and they define new regions of L protein that affect cap methylation. These studies also provide experimental evidence that inhibition of cap methylases is a potential strategy for development of antiviral therapeutics against nonsegmented negative-strand RNA viruses.

For many viruses, primary infection has been shown to prevent superinfection by a homologous second virus. In this study, we investigated superinfection exclusion of bovine viral diarrhea virus (BVDV), a positive-sense RNA pestivirus. Cells acutely infected with BVDV were protected from superinfection by homologous BVDV but not with heterologous vesicular stomatitis virus. Superinfection exclusion was established within 30 to 60 min but was lost upon passaging of persistently infected cells. Superinfecting BVDV failed to deliver a translatable genome into acutely infected cells, indicating a block in viral entry. Deletion of structural protein E2 from primary infecting BVDV abolished this exclusion. Bypassing the entry block by RNA transfection revealed a second block at the level of replication but not translation. This exclusion did not require structural protein expression and was inversely correlated with the level of primary BVDV RNA replication. These findings suggest dual mechanisms of pestivirus superinfection exclusion, one at the level of viral entry that requires viral glycoprotein E2 and a second at the level of viral RNA replication.

Cytotoxic cells provide a crucial defense against DNA and RNA viral infections. Here we describe an in vitro model to study the fate of vesicular stomatitis virus (VSV) RNA in cells undergoing apoptosis. Using the [3H]uridine release assay, we show that human LAK cells induce the degradation of RNA in infected U937 cells in addition to inhibiting the production of infectious virions. LAK cell-mediated RNA degradation was blocked by the serine protease inhibitor, 3,4-dichloroisocoumarin. Purified human granzyme B but not inactivated granzyme B, granzyme A, or perforin rapidly induced degradation of RNA in VSV-infected U937 cells in a dose- and time-dependent manner without lysing the cells and suppressed viral production. Northern analysis of RNA extracted from infected cells with a VSV full-length cDNA probe confirmed that levels of viral transcripts were reduced by treatment with granzyme B. Nevertheless, the amount of host beta-actin mRNA was also reduced in infected cells, suggesting that treatment with granzyme B induced apoptosis. Consistent with this notion, infected cells exposed to granzyme B rapidly developed DNA strand breakage. Taken together, the data suggest that granzyme B in the absence of perforin reduced VSV production by activating a mechanism that degraded viral transcripts in infected U937 cells.

Brefeldin A is a macrolide antibiotic that interferes with membrane traffic and blocks the growth of several animal viruses including vesicular stomatitis virus (VSV). The inhibition of VSV by brefeldin A takes place at least at two different steps during the growth cycle: the glycosylation of VSV G protein and the replication of viral genomes. Our results indicate that interference with membrane traffic leads not only to inhibition of viral protein glycosylation, but also to the blockade of virus genome replication in several cytoplasmic RNA-containing viruses.

tsG16(l), a temperature-sensitive mutant of vesicular stomatitis virus, in vitro has at least three phenotypic differences from its parental wild-type (wt) virus due to mutation of the L gene. It was not known whether (i) the temperature-sensitivity of the transcriptase, (ii) the aberrant polyadenylation phenotype, and (iii) the extent of increased polyadenylation in response to S-adenosylhomocysteine (SAH) were associated with a single mutation. Spontaneous partial revertants were selected from tsG16(I) on the basis of the ability to form plaques at 34.7 degrees (35G16 revertants) or from 35G16 revertants on the basis of the ability to form plaques at 37 degrees (37G16 revertants). All six 35G16 revertants had fully (five) or partially (one) recovered the wt polyadenylation phenotype and the former five had also fully recovered the wt polyadenylation response to SAH. This suggested that a single mutation in tsG16(I) was probably associated with both of these phenotypes and also probably conferred the inability to grow at 34.7 degrees. None of the 35G16 revertants regained the wt phenotype for thermosensitivity of the transcriptase, although both of the 37G16 revertants did. This suggested that in vitro temperature-sensitivity of transcription by tsG16(I) might be due to a mutation different than the one affecting polyadenylation in the absence or presence of SAH.

Vesicular stomatitis virus (VSV) stocks isolated from two persistently infected mouse L-cell lines (designated VSV-PI stocks) express an altered phenotype of RNA synthesis. This phenotype is different from the RNA synthesis phenotype expressed by the viruses used to initiate the persistently infected lines, wild-type VSV and VSV ts-0-23 (a group III, ts-, RNA+ mutant). At 34 and 37 degrees C in L cells productively infected with VSV-PI stocks derived from the two cell lines, transcription of virus mRNA was significantly reduced, whereas replication of the 40S genomic RNA species was enhanced compared with wild-type VSV or ts-0-23. At 34 and 37 degrees C, both VSV-PI stocks replicated with equal or greater efficiency than wild-type VSV; 37 degrees C was the temperature at which the persistently infected cultures were maintained. At 40 degrees C, both VSV-PI stocks were temperature sensitive, and clonal VSV-PI isolates from both cell lines belong to complementation group I (RNA-). Standard ts- mutants (derived by mutagenesis of wild-type VSV) belonging to RNA- complementation groups I, II, and IV do not express the VSV-PI RNA synthesis phenotype at the permissive temperature, making this phenotype distinctive to persistent infection. Since the two VSV-PI populations from persistently infected cell lines initiated with different viruses both evolved this unique phenotype of RNA synthesis, the expression of this phenotype may play an important role in the maintenance of persistence.

Pulse-chase labeling and cell fractionation were used to examine the pathways taken by the three nucleocapsid polypeptide species of vesicular stomatitis virus into nucleocapsids and then into virions. An improved method of polyacrylamide gel electrophoresis resolved nucleocapsid polypeptides N and NS from cellular actin, facilitating accurate quantitation of the viral polypeptides. Contrary to previous belief, the rate of NS synthesis was found to be a constant fraction of total virus protein synthesis throughout infection, indicating a consistent mechanism of virus protein synthesis regulation. In the kinetic studies, each polypeptide species displayed the following characteristic behavior. (i) Structural polypeptide N was the only species that entered a metabolically active soluble pool before assembly into nucleocapsids. The size of this pool increased with time after infection, causing an increasing delay in the appearance of pulse-labeled N molecules in nucleocapsids. (ii) Throughout infection, the entire complement of L molecules entered nucleocapsids immediately after their synthesis, without diversion through a soluble pool. (iii) Although 75% of newly synthesized molecules of the transcriptase-associated protein NS entered a soluble pool, they never emerged from the compartment. At all times after infection, about 25% of the NS molecules bypassed the soluble pool and entered nucleocapsids directly after their synthesis, as if in concert with L. These results indicate that VSV nucleocapsid assembly in vivo is a stepwise process, comprising an initial condensation of N with the viral RNA, followed by attachment of L and NS, analogous to the stepwise assembly of Sendai virus nucleocapsids. (D. W. Kingsbury, C.-H. Hsu, and K. G. Murti. Virology 91:86-94, 1978). About half of the intracellular nucleocapsids were recovered in a form that sedimented at anomalously low centrifugal forces, reflecting an association with large cellular organelles. This attachment was mediated mainly by electrostatic forces, since these "bound" nucleocapsids were released by elevated salt concentrations. The kinetic behavior of nucleocapsid polypeptides was the same in both fractions, providing no evidence for a division of nucleocapsid functions between cellular compartments.

We have isolated and characterized the RNA of intracellular virus nucleocapsids recovered from a number of cell cultures persistently infected with rabies virus or vesicular stomatitis virus (VSV). VSV persistent infections in BHK21, L cells and Aedes albopictus (mosquito) cells generally showed the presence of large amounts of defective-interfering (DI) nucleocapsid RNA and much smaller amounts of standard (B) nucleocapsid RNA. Persistent infections of BHK21 cells by two rabies virus strains, challenge virus standard (CVS-11) or HEP-Flury, were followed for several months during which time the ratio of DI to B nucleocapsid RNA cycled dramatically. We also observed coordinated fluctuations in the absolute amount of incorporation of [3H]uridine into virus nucleocapsid RNA. Total incorporation was generally highest following a decrease in the relative amount of DI nucleocapsid RNA synthesis. At no time were DI nucleocapsids absent in any of the persistently infected cultures.

Poly(A)-containing vesicular stomatitis virus mRNA species synthesized in vesicular stomatitis virus-infected cells have been separated into four bands by electrophoresis on formamide-polyacrylamide gels. Two-dimensional fingerprints of ribonuclease T-1 and ribonuclease A digests of the RNA from each band show that they contain unique oligonucleotide sequences as well as 60 to 125 nucleotides of poly(A). The fingerprints were used to determine the nucleotide sequence complexities of RNA from three of the bands. Two contain nucleotide sequences which account completely for their molecular weights (0.70 times 10-6 and 0.55 times 10-6) determined by gel electrophoresis and sedimentation rate, and, therefore, these are radiochemically pure RNA species. The most rapidly migrating band must contain two ro three different RNA species since it has a molecular weight of 0.28 times 10-6, determined by physical methods, and a nucleotide sequence complexity two to three times that expected for a pure RNA species of this size. These data are in complete accord with translational studies (accompanying paper) which show that each of the two pure RNA species codes for a distinct viral protein, whereas the third codes for two viral proteins. From the molecular weight and sequence complexity determinations on mRNA from the bands, we conclude that most of the vesicular stomatitis virus genome is transcribed into discrete mRNA species.

Synthesis of vesicular stomatitis virus RNA and protein is almost completely inhibited in HeLa cells treated with relatively high doses of human fibroblast interferon. With lower concentrations of interferon virus replication is inhibited, but near normal amounts of virus RNA are found in cells infected at a m.o.i. of 10. All the virus RNA species are found in these cells with the exception of genomic size RNA. In contrast, synthesis of all the virus proteins is equally inhibited in proportion to the interferon concentration used to treat the cells. This inhibition is due to a decline in the rate of protein synthesis, which occurs in interferon-treated cells sooner after infection than in untreated cells. The decreased rate of protein synthesis is accompanied by a change in the polysome pattern of infected cells, characterized by polysome run-off and increase in 80S ribosomes. At the same time, a larger proportion of the virus poly(A)-containing RNA is not associated with polysomes in interferon-treated cells than in control cells. The non-polysomal virus RNA has a sedimentation rate identical with that of polysomal virus RNA. Possible causes for the decline in the rate of protein synthesis observed in interferon-treated cells are discussed.

Homologous and heterologous interference by Newcastle disease virus (NDV) was manifested in a marked inhibition of virus-specific RNA synthesis of both the interacting viruses. Inactivation of the interfering NDV by irradiation reduced its capacity to inhibit the synthesis of virus-specific RNA of the homologous virus, leaving its effect on the synthesis of RNA of the heterologous virus unchanged. In contrast, treatment of cell cultures with heparin leading to reduced interferon production eliminated the heterologous interference but did not affect the homologous interference.

The enhanced phosphorylation of specific protein(s) observed in extracts from interferon-treated cells (in the presence of ATP and double-stranded [ds] RNA) was also seen in intact mouse L929 cells upon treatment with dsRNA, polyriboinosinic.polyribocytidylic acid [poly(rI.rC)] or reovirus dsRNA, using 32Pi as radiolabel. Labeling of a 65,000-dalton protein(s) with 32P was greatly increased in interferon-treated cells in the presence of added dsRNA, suggesting that the expression in vivo of the kinase activity involved is regulated by dsRNA. This was used as a test system to investigate whether the activity of interferon-induced enzyme(s) is stimulated following virus infection, possibly owing to the accumulation of dsRNA. No obvious increase in 32P-labeling of 65,000-dalton protein(s) was observed upon infection of interferon-treated cells with mengovirus or vesicular stomatitis virus. A basal level of 32P-labeling of the 65,000-dalton protein(s) was detected in interferon-treated cells in the absence of added dsRNA, indicating a basal level of expression of the kinase activity involved. The possible implications of these results are discussed.

The synthesis of viral complementary RNA by vesicular stomatitis virus in permissive cells can be distinguished operationally into two phases, primary transcription, which can be observed in cycloheximide treated cells, and secondary transcription, which represents the further synthesis of viral complementary RNA obtained in untreated cells. Pretreatment of mouse L cells or chicken embryo fibroblasts with the homologous interferon and, for chicken embryo fibroblast cells, poly(rI):poly(rC), inhibits the production of infectious virus and reduces the synthesis of viral complementary RNA to levels comparable to that obtained during primary transcription. Treatment of mouse L cells with interferon plus cycloheximide also gives levels of vesicular stomatitis viral complementary RNA synthesis comparable to that observed with the cycloheximide treatment alone. Similar results have been obtained for interferontreated cells subsequently infected with influenza virus (strain WSN). The results are interpreted as indicating that inhibition of virus development by interferon does not act at the level of primary transcription but rather at an intermediate step between primary and secondary transcription, such as viral protein synthesis.

The ability of certain vesicular stomatitis virus (VSV; Indiana serotype) temperature-sensitive (ts) mutants to synthesize intracellular viral complementary RNA (vcRNA) at permissive or nonpermissive temperatures for productive infections has been investigated. Mutants belonging to complementation groups II, III, and V synthesize RNA at nonpermissive temperature in amounts essentially equivalent to that obtained at permissive temperatures. Mutant ts G I-114 possesses a thermolabile transcriptase and does not synthesize vcRNA at 40 degrees C; however, mutants ts O I-5, O I-53, O I-78, and O I-80 possess thermostabile transcriptases that are capable of some vcRNA synthesis at 40 degrees C. All five group I mutants are defective in their secondary transcription ability at 40 degrees C. Wild-type VSV New Jersey virus is able to complement the transcription defect of ts G I-114 at 40 degrees C. This complementation is inhibited by puromycin, suggesting that a viral gene product of VSV New Jersey (e.g., its transcriptase or a transcriptase component) is involved. Mokola virus is not able to complement the ts G I-114 defect, although Mokola does synthesize vcRNA in infected cells (in the presence or absence of cycloheximide).

Replicating vesicular stomatitis virus ribonucleoprotein (RNP) complexes were isolated in nonequilibrium Renografin density gradients. These nascent RNPs had the same buoyant density as virion nucleocapsids in both isopycnic Renografin and CsCl gradients. Both transcribing and replicating RNP complexes were shown to be stable in sucrose gradients, whereas only replicating RNP complexes were stable in Renografin gradients. Size analysis of the 5-min-pulse-labeled RNA species from the replicating RNPs using methylmercury gels revealed that the nascent strands were primarily less than full-length molecules. Longer times of radiolabeling demonstrated that the nascent RNA accumulated as 42S RNA, which was primarily of the same sense as the virion strand when it was radiolabeled at 5 h postinfection. The percentage of this radiolabeled RNA which was plus stranded was higher at 2.5 h postinfection, reflective of the shift in plus- to minus-stranded full-length 42S RNA synthesis which occurs in the cell. Addition of cycloheximide to the infected cells before the addition of the radiolabel prevented the formation of these RNP complexes. Both the change in the percentage of minus strands found in the RNP complexes at the different times postinfection and the sensitivity to cycloheximide indicate that the RNP complex which was isolated was indeed the replicative complex.

Vesicular stomatitis virus (VSV) isolated from two independently established lines of persistently infected mouse L cells expressed an altered phenotype of RNA synthesis at 37 degrees, the temperature at which the persistently infected cultures were maintained (T.K. Frey and J.S. Youngner, 1982, J. Virol. 44, 167-174). In comparison to the viruses used to initiate the two lines, wild-type (wt) VSV and ts-0-23 (ts-, RNA+ complementation group III), the VSV expressing this RNA phenotype synthesized much less mRNA but equal or greater amounts of 40 S genomic RNA (rt- phenotype). In the line initiated with wt-VSV, at 17 days after initiation, when 85% of the clones were ts-, 36% of the ts- clones were rt-. By 63 days the VSV-PI population was uniformly ts- and rt- and this phenotype prevailed for at least 2 years of persistence. In the line initiated with ts-0-23, the rt- phenotype was stable for at least 3 years of persistence. To study the relationship of the ts- and rt- phenotypes which were coselected during persistence, ts+ revertants of a ts- rt- VSV-PI clone were isolated. All of the ts+ revertants expressed a wt-VSV phenotype of RNA synthesis at 37 degrees (rt+), indicating that the two phenotypic markers may be pleiotropic manifestations of the same mutation. rt-VSV inhibited host cell RNA and protein synthesis more slowly than did wt-VSV. However, rt-VSV synthesized equivalent or greater amounts of all the virus proteins, compared to wt-VSV, despite the reduced amount of mRNA transcription. The attenuated shutoff of host cell macromolecular synthesis by rt- VSV and the concomitant efficient 40 S genome replication and virus protein synthesis may in part explain the selective advantage of the rt- mutation during persistence. The rt- phenotype was not unique to persistent infection; ts- rt- mutants also evolved during serial undiluted passages of wt-VSV in L cells and one ts- rt- mutant was identified in a group of spontaneous mutants isolated from a wt-VSV stock.

A permeable-cell system has been developed to study the replication of vesicular stomatitis virus. When vesicular stomatitis virus-infected BHK cells were permeabilized by lysolecithin treatment, they incorporated nucleoside triphosphates into RNA and amino acids into proteins at nearly normal rates. The viral mRNA's synthesized appeared normal in polarity, size distribution, and polyadenylation, and all five viral proteins were synthesized. Replication of the viral genome proceeded, and full-length RNA strands were synthesized in amounts and polarities resembling those found in intact cells. These full-length RNAs associated with viral N proteins to form RNase-resistant nucleocapsids of normal buoyant density. Permeable cells appear to represent ideal hosts for studying vesicular stomatitis virus replication since they closely mimic in vivo conditions while retaining much of the experimental flexibility of current in vitro systems.

Vesicular stomatitis virus RNA synthesis was investigated during the establishment of persistent infection in Drosophila melanogaster cells. The transcription rate declined as early as 5 h after infection and was strongly inhibited after 7 h, leading to a decrease in viral mRNA levels and in viral protein synthesis rates. Full-length plus-strand antigenomes and minus-strand genomes were detected after a 3-h lag time and accumulated until 15 h after infection. Short encapsidated plus-strand molecules were also generated corresponding to the 5' end of viral defective antigenomes. Assembly and release of virions were not restricted, but their infectivity was extremely reduced. In persistently infected cells, an equilibrium was reached where the level of intracellular genomes maintained was constant and maximal even after the rate of all viral syntheses had decreased. These results are discussed with regard to the establishment of persistent infection.

We have sequenced (via a product RNA) the 3' RNA terminus of a defective interfering particle that was generated from the standard virus isolated from a culture of BHK-21 cells persistently infected with vesicular stomatitis virus for over 5 years. By hybridization and RNA sequencing, seven mutations were identified in the 46 nucleotides at the terminus of this defective-interfering-particle RNA. It is likely that these mutations are a reflection of altered protein-nucleic acid interactions that the virus has evolved to maintain its persistently infected carrier state in vitro.

Pulse-labeling of vesicular stomatitis virus-infected HeLa and BHK cells with [3H]uridine throughout the infectious cycle demonstrated two peaks of uridine incorporation into virus-specific RNA molecules. By separating total RNA synthesis into replication and transcription products, we showed that replication occurs over a shorter period of time in one peak synthesis. The biphasic nature of uridine incorporation is in part due to a general membrane phenomenon of reduced metabolite transport during vesicular stomatis virus infection and in part due to the apparent uncoupling of replication and transcription. A change in the ratio of newly synthesized plus and minus strands of the genome length (42S) RNA was found as the infection proceeded. Early in the infection, plus-stranded 42S RNA comprised 40% of the total genome length RNA synthesis, whereas late in infection, only 15 to 20% of the 42S RNA synthesized was complementary to the virion minus strand. Our data suggest that the rate of synthesis of plus-stranded 42S RNA was constant throughout the infection. The rate of virus release was determined by monitoring the uptake of [3H]uridine into released virus particles. Virus maturation and release are closely associated with the assembly of 42S RNA-containing nucleocapsids.

The cytoplasm of vesicular stomatitis virus (VSV)-infected BHK cells has been separated into a fraction containing the membrane-bound polysomes and the remaining supernatant fraction. Total poly(A)-containing RNA was isolated from each fraction and purified. A 17S class of VSV mRNA was found associated almost exclusively with the membrane-bound polysomes, whereas 14,5S and 12S RNAs were found mostly in the postmembrane cytoplasmic supernatant. Poly(A)-containing VSV RNA synthesized in vitro by purified virus was resolved into the same size classes. The individual RNA fractions isolated from VSV-infected cells or synthesized in vitro were translated in cell-free extracts of wheat germ, and their polypeptide products were compared by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The corresponding in vivo and in vitro RNA fractions qualitatively direct the synthesis of the same viral polypeptides and therefore appear to contain the same mRNA species. By tryptic peptide analysis of their translation products, the in vivo VSV mRNA species have been identified. The 17S RNA, which is compartmentalized on membrane-bound polysomes, codes for a protein of molecular weight 63,000 (P-63) which is most probably a nonglycosylated form of the viral glycoprotein, G. Of the viral RNA species present in the remaining cytoplasmic supernatant, the 14.5S RNA codes almost exclusively for the N protein, whereas the 12S RNA codes predominantly for both the NS and M proteins of the virion.

Recent studies on the mechanism by which the virion-associated RNA polymerase of vesicular stomatitis virus transcribes RNA have revealed several new biological features of general interest. The mode of synthesis of the 5'-terminal cap structure of the mRNAs, the sequential transcription of the genes and the presence of a transcribed "leader" RNA segment are properties which are either not shown by other viruses, or have not yet been described. These features are probably inter-related with the primary transcription process, which itself may be a useful model for future studies on mRNA biosynthesis in eukaryotic systems.

Previous studies demonstrated that cytoplasmic extracts of cells infected with vesicular stomatitis virus contain plus-strand leader RNAs which sediment at 18S on sucrose gradients as a complex with viral N protein. The work presented in this paper demonstrated that these 18S complexes were stable on CsCl density gradients, banding at a buoyant density near that of genome nucleocapsids, and exhibited a morphology in an electron microscope similar to the disk structures found in virus genome nucleocapsids. Minus-strand leader RNAs were also found in 18S complexes on sucrose gradients. Quantitation of intracellular leader RNA suggested that, late in infection, approximately three-quarters of total intracellular leader RNA was encapsidated.

Several temperature-sensitive mutants of vesicular stomatitis virus in complementation group III produce, at nonpermissive temperature, noninfectious particles which contain the viral M (matrix) and G (glycoprotein) proteins but less than 10% of the normal proportion of N protein or RNA. Since group III mutants are thought to be defective in the structural gene for the virus M protein, these findings demonstrate that an interaction between M and the nucleocapsid is of importance in virus budding. Taken together with earlier results, they suggest that M is the key protein in bud formation.

The time course of defective interfering (DI) particle and B particle release from vesicular stomatitis virus-infected BHK-21 cells was studied at different multiplicities of defective and infective particles. Particle release was progressively delayed in cells infected with an increasing DI-to-B particle ratio. The delayed particle release during interference was found to be connected with a reduced but prolonged synthesis of viral proteins, a slower accumulation of viral proteins, and a delayed shutoff of cellular protein synthesis. The relative synthesis of M and G proteins was reduced during interference, whereas the relative synthesis of N and NS proteins was increased. On the level of genomic RNA replication, we found that DI RNA was replicated more slowly during interference than the standard genomic RNA was during acute infection. The ratio of DI particles to B particles which were released increased throughout the infectious cycle. At a given time in the infectious cycle, this ratio was independent of the multiplicity of infecting DI and B particles. On the basis of the kinetic studies, we argue that cells infected with higher amounts of DI particles compared with B particles synthesize a higher DI-to-B particle ratio and release these progeny particles later than cells infected with a low DI-to-B particle ratio.

We compared the ability of the leader RNAs of the New Jersey and Indiana serotypes of vesicular stomatitis virus to inhibit transcription in infected host cells. The level of cellular RNA synthesis in cells infected with either serotype was drastically reduced by 5 h after infection. Studies with UV-inactivated virus demonstrated that shutoff of cellular RNA synthesis directly correlated with the ability of the infecting virus to transcribe its plus-stranded leader RNA. Although both serotypes inhibited cellular RNA synthesis, the Indiana serotype reduced synthesis to lower levels. In addition, an examination of the kinetics of leader RNA synthesis in vivo indicated that up to four times more leader RNA was produced in cells infected with the Indiana serotype than in those infected with the New Jersey serotype. However, in vivo studies also suggested that the leader RNA of the New Jersey serotype was a more efficient RNA inhibitor than was the Indiana serotype leader RNA. Although up to 2,900 copies of the leader RNA per cell could be detected in infected cells, only 550 copies of the Indiana and 100 copies of the New Jersey leader RNAs per cell were present in infected cells that were demonstrating 50% of the maximal inhibition of RNA synthesis. In an in vitro system, leader RNAs of both serotypes inhibited DNA-dependent transcription of the adenovirus late promoter and adenovirus-associated RNA genes, but the New Jersey serotype leader was also a better inhibitor in this reconstituted system. Data from the dose response of inhibition by each leader suggest that polymerase III transcription was more sensitive to inhibition by viral leaders than was polymerase II transcription. Polyadenylated viral mRNAs and the NS and N gene starts transcribed by both serotypes did not significantly inhibit transcription at levels at which the corresponding leader RNAs were inhibitory. Overall, our results strongly suggest a role for the plus-stranded leader RNAs of the New Jersey and Indiana serotypes of vesicular stomatitis virus in inhibiting cellular transcription in vivo. We discuss differences in the nucleotide sequences of the two leader RNAs in relation to their differences in biological activity and to potential regulatory sequences.

The temperature-sensitive mutant ts114 of vesicular stomatitis virus is temperature-sensitive in both primary and secondary transcription, but not in the replication of 40S RNA. The synthesis of 40S RNA is specifically inhibited when protein synthesis is shut off. The addition of cycloheximide to cells infected by ts114, rapidly inhibits RNA replication at the permissive and nonpermissive temperatures. However, the addition of cycloheximide at the nonpermissive temperature also results in almost complete recovery of transcription to the level found at the permissive temperature. Other inhibitors of protein synthesis applied at the nonpermissive temperature result in the same recoverability of the apparent temperature-sensitive lesion. Ribonucleic acid products synthesized by ts114 at the nonpermissive temperature in the presence of cycloheximide are identical to virus-specific messenger RNA by the criteria of size and complementarity to virion RNA. When mutant-infected cells are shifted to the nonpermissive temperature, incubated for a period of time, and then treated with cycloheximide, the ratio of transcriptive to replicative activity varies depending on the time at which radioactive precursor is added to cells. This suggests an interdependence between replication and transcription during virus-specific RNA synthesis.

The wild-type strain of vesicular stomatitis virus (VSV) contains in its complete virion (VSV-1, B particles) a minus strand RNA. The principle defective particle of the wild-type strain (VSV-111, T particles) contains a shorter minus strand, homologous to part of the VSV-1 genome. Neither virion contains any detectable complementary (plus) strand RNA. In contrast, a preparation of a heat-resistant (HR) strain of VSV containing defective virions was found to contain both plus (21%) and minus strand RNA, present in several distinct size classes. It was found that the RNA in the HR virion preparation was at least 94% single-stranded and principally (96%) in ribonucleoprotein complexes. On extraction the plus and minus strand RNA species partially annealed to give a population of double- and multistranded RNA species. A small amount of RNA polymerase activity was associated with the HR defective virus preparation.

The synthesis of viral RNA by wild-type vesicular stomatitis virus (L(1)VSV) and a small, plaque-size mutant (S(2)VSV) was studied in vitro and in chicken embryo (CE) and mouse L-cell cultures. Virus-specific RNA synthesized in CE or L cells infected with either L(1) or S(2)VSV at low multiplicity was of the same size classes, 12 to 15S, 28S, and 38S. The major differences were in the proportion of RNA produced of each size class. L(1)VSV always synthesized larger proportions of 38S RNA, and S(2)VSV produced larger proportions of 12 to 15S RNA. Both S(2) and L(1)VSV exhibited RNA transcriptase activity in vitro and in cell culture. The products of the in vitro reaction were the same, 12 to 15S for both. The products of the virion-associated transcriptase in CE or L-cell cultures in the presence of cycloheximide were also the same for both viruses but differed from the in vitro products in that 28S and 12 to 15S RNA were made. The effects of addition of cycloheximide at various times after infection demonstrated that new protein synthesis is required early (0-2 h) for both S(2) and L(1)VSV to initiate and maintain the normal rate of viral RNA synthesis. However, the overall rate of RNA synthesis in L(1)VSV infections became independent of protein synthesis after 2 h whereas the rate in S(2)VSV infections did not. With either virus, synthesis of 38S RNA did not occur in the absence of protein synthesis. Moreover, continuous 38S RNA production required continuous protein synthesis. Production of 38S RNA ceased within 30 min after addition of cycloheximide to S(2) (-) or L(1)VSV-infected CE or L cells that had already begun to synthesize the 38S form. The cycloheximide-induced cessation of 38S RNA synthesis was accompanied by a marked increase in production of 12 to 15S and 28S RNA in L(1)VSV-infected cells, but no increase in synthesis of small RNA species occurred in S(2)VSV-infected cells.

Different temperature-sensitive mutants of vesicular stomatitis virus have been characterized in terms of their ability to induce synthesis of viral ribonucleic acid (RNA) in BHK-21 cells at 39 C (the restrictive temperature for these mutants). Mutants belonging to complementation groups I and IV (and probably II) did not induce actinomycin-resistant RNA synthesis in infected cells incubated at 39 C. All three mutants comprising complementation group III induced viral RNA synthesis at 39 C. The temperature sensitivity of the defective viral functions has also been studied by temperature-shift experiments. The functions associated with the mutants of groups I, II, and IV were required early, whereas the function associated with the group III mutants was not required until a late stage of the viral cycle. The heat sensitivity of extracellular virion was not correlated with complementation group.

T-particle-free stocks of temperature-sensitive mutants representing the four Glasgow complementation groups of the Indiana serotype of vesicular stomatitis virus were used to study RNA synthesis at the permissive and nonpermissive temperatures of 31 and 39 C, respectively. Mutants selected from the four Glasgow complementation groups were characterized on the basis of particle and ribonucleoprotein formation. Intracellular RNAs were further characterized by polyacrylamide gel electrophoresis. ts G22 (group II) and ts G41 (group IV), previously characterized as RNA negative at the nonpermissive temperature, synthesized low levels of RNA which could not be attributed to contaminating levels of revertants. Furthermore, the levels of synthesis could not be reduced by the addition of cycloheximide. These data suggest that ts G22 (group II) and ts G41 (group IV) contain a thermally stable, virion-encapsidated transcriptase, but fail to amplify RNA synthesis due to a thermally labile function presumably necessary for the synthesis of viral RNA. ts G31, a group III mutant, synthesized intracellular RNA at amplified levels at the nonpermissive temperature. Intracellular ribonucleoprotein complexes were isolated in copious amounts; however, no particles corresponding in size to finished virions were observed. These data suggest a thermally labile maturation factor or envelope associated structural protein to be defective in ts G31 (group III). ts G11 (group 1) showed no detectable RNA synthesis at the nonpermissive temperature. These data suggest ts G11 (group I) contains a thermally labile component involved in early transcription. This group may contain a number of mutants defective in different components of the transcription apparatus, which may not complement in vivo because of the physical improbability of subunit exchange between virion particles of the incoming inoculum.

Some temperature-sensitive mutants of vesicular stomatitis virus were tested for their ability to block the initiation of deoxyribonucleic acid (DNA) synthesis and division in serum-stimulated hamster embryo fibroblasts at the nonpermissive temperature. Although the parental strain blocked these processes, one particular mutant allowed essentially normal DNA synthesis and division. By autoradiography, it was shown that individual cells infected with this mutant could synthesize viral ribonucleic acid and at the same time initiate DNA synthesis and divide. Cells infected with such conditional defective mutants appear to be suitable for studies on the effects of persistent viral infections on molecular and cellular functions in proliferating cell populations.

Exposure of vesicular stomatitis virus-infected Chinese hamster ovary cells to cycloheximide results in the complete transcription of virion ribonucleic acid (RNA) into only 28S and 13 to 15S viral-specific RNA species. These RNA are identical to viral messenger RNA by the following criteria: size, single-strandedness, complementarity to virion RNA, and formation of messenger ribonucleoproteins. This transcription represents the intracellular enzymatic activity of the virion-associated polymerase and is shown to be dependent on input multiplicity. Intracellular transcription differs from in vitro polymerase activity in having a temperature optimum of 34 to 37 C and in synthesizing 28S as well as 13 to 15S messenger RNA species. Addition of interfering quantities of defective T particles to these cycloheximide-treated cells, either an hour before or at the same time as standard B particles of vesicular stomatitis virus, does not alter the rate of transcription nor does it change the sucrose gradient pattern of the viral RNA species. From these results it is concluded that the RNA of defective T particles does not serve a transcriptive function and probably interferes through the replicative mechanism for virion RNA synthesis.