Vesicular Stomatitis Virus Genome RNA Research Articles

Unconventional Mechanism of mRNA Capping by the RNA-Dependent RNA Polymerase of Vesicular Stomatitis Virus

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.

Replication and Propagation of Attenuated Vesicular Stomatitis Virus Vectors In Vivo: Vector Spread Correlates with Induction of Immune Responses and Persistence of Genomic RNA

Live-attenuated vesicular stomatitis virus (VSV) vectors expressing foreign antigens induce potent immune responses and protect against viral diseases in animal models. Highly attenuated (VSV-CT1) or single-cycle VSV (VSVDeltaG) vectors induce immune responses lower than those generated by attenuated wild-type VSV vectors when given intranasally. We show here that reduced spread of the more highly attenuated or single-cycle vectors to other organs, including lymph nodes, correlates with the reduction in the immune responses. A reverse transcription, real-time PCR assay for VSV genomic RNA (gRNA) sequences showed long-term persistence of gRNA from replicating vectors in lymph nodes, long after viral clearance. Such persistence may be important for induction of potent immune responses by VSV vectors.

Two RNA polymerase complexes from vesicular stomatitis virus-infected cells that carry out transcription and replication of genome RNA.

By immunoaffinity column chromatography, we have purified two RNA polymerase complexes, the transcriptase and replicase, from vesicular stomatitis virus-infected baby hamster kidney cells. The transcriptase is a multiprotein complex, containing the virus-encoded RNA polymerase L and P proteins, and two cellular proteins, translation elongation factor-1alpha and heat-shock protein 60. In addition, the complex contains a submolar amount of cellular mRNA cap guanylyltransferase. The replicase, on the other hand, is a complex containing the viral proteins, L, P, and the nucleocapsid (N), but lacking elongation factor-1alpha, heat-shock protein 60, and guanylyltransferase. The transcriptase complex synthesizes capped mRNAs and initiates transcription at the first gene (N) start site, whereas the replicase complex initiates RNA synthesis at the precise 3' end of the genome RNA and synthesizes encapsidated replication products in the presence of the N-P complex. We propose that two RNA polymerase complexes that differ in their content of virally and host-encoded proteins are separately responsible for transcription and replication of vesicular stomatitis virus genome RNA.

Identification of a novel tripartite complex involved in replication of vesicular stomatitis virus genome RNA.

Our laboratory's recent observations that transcriptionally inactive phosphoprotein (P) mutants can efficiently function in replicating vesicular stomatitis virus (VSV) defective interfering particle in a three-plasmid-based (L, P, and N) reverse genetics system in vivo (A. K. Pattnaik, L. Hwang, T. Li, N. Englund, M. Mathur, T. Das, and A. K. Banerjee, J. Virol. 71:8167-8175, 1997) led us to propose that a tripartite complex consisting of L-(N-P) protein may represent the putative replicase for synthesis of the full-length genome RNA. In this communication we demonstrate that such a complex is indeed detectable in VSV-infected BHK cells. Furthermore, coexpression of L, N, and P proteins in Sf21 insect cells by recombinant baculovirus containing the respective genes also resulted in the formation of a tripartite complex, as shown by immunoprecipitation with specific antibodies. A basic amino acid mutant of P protein, P260A, previously shown to be inactive in transcription but active in replication (T. Das, A. K. Pattnaik, A. M. Takacs, T. Li, L. N. Hwang, and A. K. Banerjee, Virology 238:103-114, 1997) was also capable of forming the mutant [L-(N-Pmut)] complex in both insect cells and BHK cells. Sf21 extract containing either the wild-type P protein or the mutant P protein along with the L and N proteins was capable of synthesizing 42S genome-sense RNA in an in vitro replication reconstitution reaction. Addition of N-Pmut or wild-type N-P complex further stimulated the synthesis of the genome-length RNA. These results indicate that the transcriptase and replicase complexes of VSV are possibly two distinct entities involved in carrying out capped mRNAs and uncapped genome and antigenome RNAs, respectively.

Carboxy-Terminal Five Amino Acids of the Nucleocapsid Protein of Vesicular Stomatitis Virus Are Required for Encapsidation and Replication of Genome RNA

The encapsidation of vesicular stomatitis virus (VSV) genome RNA, a prerequisite step to the replication process by the nucleocapsid protein (N) was studied by its ability to package VSV leader RNA in vitro in a RNase-resistant form. The VSV leader RNA was derived from the SP6 transcription vector while the N protein was made in rabbit reticulocyte lysate. The in vitro encapsidation was carried out by translating N mRNA in the presence of 32P-labeled presynthesized leader RNA. The RNA encapsidation property of the N protein was completely abrogated when the C-terminal five amino acids (VEFDK-COOH) were deleted. Systematic mutational analyses within the C-terminal five amino acid regions reveal that the RNA encapsidation activity was lost in all mutants except K → A and K → R, indicating that C-terminal five amino acids, in particular the lysine residue play critical role in genome RNA encapsidation. To correlate the in vitro encapsidation abilities of these mutant N proteins with genome RNA replication, we have used a full-length cDNA clone of VSV genome RNA to rescue infectious virions from cells expressing L, P, and wt or mutant N proteins and measured the recovery of plaque forming units. The results indicate that the N mutants that are defective in in vitro encapsidation of leader RNA do not support replication, establishing the requirement of C-terminal five amino acids of the N protein in viral replication.

Basic Amino Acid Residues at the Carboxy-Terminal Eleven Amino Acid Region of the Phosphoprotein (P) Are Required for Transcription but Not for Replication of Vesicular Stomatitis Virus Genome RNA

The phosphoprotein (P) of vesicular stomatitis virus (VSV) serotypes New Jersey [P(NJ)] and Indiana [P(I)] contains a highly conserved carboxy-terminal domain which is required for binding to the cognate N-RNA template as well as to form a soluble complex with the nucleocapsid protein Nin vivo.We have shown that the deletion of 11 amino acids from the C-terminal end of the P(I) protein abolishes both the template binding and the complex forming activity with the N protein. Within this region, there are conserved basic amino acid residues (R260 and K262) that are potential candidates for such interactions. We have generated mutant P proteins by substitution of these basic amino acid residues with alanine and studied their role in both transcription and replication. We have found that the R260A mutant failed to bind to the N-RNA template, whereas the K262A mutant bound efficiently as the wild-type protein. The R260A mutant, as expected, was unable to support mRNA synthesisin vitroin a transcription reconstitution reaction as well as transcriptionin vivoof a minigenome using a reverse genetic approach. However, the K262A mutant supported low level of transcription (12%) bothin vitroandin vivo,suggesting that direct template binding of P protein through the C-terminal domain is necessary but not sufficient for optimal transcription. Using a two-hybrid system we have also shown that both R260A and K262A mutants interact inefficiently with the L protein, suggesting further that the two point mutants display differential phenotype with respect to binding to the template. In addition, both R260A and K262A mutants were shown to interact efficiently with the N proteinin vivo,indicating that these mutants form N-P complexes which are presumably required for replication. This contention is further supported by the demonstration that these mutants support efficient replication of a DI RNAin vivo.Since the transcription defective P mutants can support efficient replication, we propose that the transcriptase and the replicase are composed of two distinct complexes containing (L-P2-3) and L-(N-P), respectively.

Fas-mediated cytotoxicity induces degradation of vesicular stomatitis virus RNA transcripts and reduces viral titer

Several investigators have recently examined the effect of Fas (CD95)-mediated apoptotic cell death on target cells (TC). The effect of Fas-mediated death on viral RNA within the TC, however, has not been explored. In this study, we investigated the ability of the Fas pathway to mediate pre-lytic degradation of vesicular stomatitis virus (VSV) RNA and TC RNA. We show that engagement of Fas antigen on VSV-infected Jurkat cells induces pre-lytic degradation of VSV RNA transcripts, whereas full-length VSV genome RNA, known to be tightly associated with viral proteins, is not degraded. Cellular RNA, including β-actin and glyceraldehyde-3-phosphate-dehydrogenase mRNAs, is also degraded by Fas-mediated cytotoxicity. In addition, Fas-mediated cytotoxicity reduced the yield of VSV plaque-forming units (PFU) from Jurkat by an average of 82.0%. An antiFas blocking Ab inhibited the RNA degradation and restored the number of VSV PFU to near control levels. These data indicate that the Fas lytic pathway could play a role in the elimination of viruses through degradation of intracellular viral RNA.

Gene expression of vesicular stomatitis virus genome RNA

It is just over 20 years since the landmark discovery was made by Baltimore et al. (1970) that vesicular stomatitis virus (VSV) packages within the mature virion the vital RNA-dependent RNA polymerase that transcribes the negative sense (anti-message sense) genome RNA into messenger RNAs. This unique observation provided the window of opportunity to study the regulation of VSV gene expression in the test tube and made VSV an early paradigm for RNA viral gene expression, a position that it has maintained ever since. In the past two decades, availability of information pertaining to the mechanism of gene expression of VSV has burgeoned, providing a better understanding of the structure and function of not only the VSV genome and its products but also those of negative-strand RNA viruses in general. The complete nucleotide sequence of the genome is now known (Rose and Schubert, 1987) and hence the sequence of the mRNAs and the deduced amino acid sequences of the viral proteins. All virus-coded proteins have been identified and their possible functions in the viral life cycle assigned. Notwithstanding this progress, the precise mechanisms by which the linear, single-strand RNA genome is transcribed into mRNAs and eventually replicated still remain a challenge to molecular virologists. VSV, a rhabdovirus, is thus a prototype model of all nonsegmented negative-strand RNA viruses, such as rabies, measles, mumps, parainfluenza, respiratory syncytial, and Sendai, to mention a few. While rabies is a rhabdovirus, all the other examples belong to the Paramyxoviridae family. The gene order and overall mechanism of gene expression in these two families of viruses are generally expected to be very similar. Available evidence has in fact demonstrated a commonality in their modes of transcription and replication. However, there are specific differences between VSV and the paramyxoviruses (as there are among the individual members of the Paramyxoviridae family) that warrant

Initiation and replication of vesicular stomatitis virus genome RNA in a cell-free system.

A system for studying the in vitro replication of the RNA genomes of both wild-type vesicular stomatitis virus (VSV) and its defective interfering particle MS-T has been developed. After lysolecithin treatment of cells infected with VSV or VSV plus MS-T, a cell-free cytoplasmic extract is prepared which will support VSV mRNA synthesis and the synthesis of the 42S wild-type or 19S MS-T genome RNAs. The genome-length RNAs synthesized in vitro are assembled into RNase-resistant nucleocapsids. The level of 42S RNA synthesis in vitro (6-13% of total RNA synthesis) reflects the level of replication in vivo. Although the extracts of VSV-infected cells can also support the synthesis of VSV proteins, RNA replication is not dependent on de novo protein synthesis but utilizes the preformed soluble proteins present in the infected cell at the time the extract is prepared. The initiation of genomic RNA during in vitro replication can be demonstrated because detergent-disrupted, purified MS-T particles will replicate their RNA when added to either a total cytoplasmic extract from VSV-infected cells or the soluble protein fraction derived from such an extract.

Expression of a recombinant DNA gene coding for the vesicular stomatitis virus nucleocapsid protein.

A cDNA clone containing the entire vesicular stomatitis virus nucleocapsid gene was assembled by fusing portions of two partial clones. When the cDNA clone was inserted into a new general-purpose eucaryotic expression vector and introduced into appropriate host cells, abundant N-protein synthesis ensued. The expressed protein was indistinguishable from authentic N protein produced during vesicular stomatitis virus infections. The recombinant N protein was recognized by a polyclonal antibody and two different monoclonal antibodies and could not be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from authentic N. Our results suggest that the recombinant N protein produced in transfected cells rapidly aggregates into high-molecular-weight complexes in the absence of vesicular stomatitis virus genomic RNA.

Covalent attachment of psoralen to a single site on vesicular stomatitis virus genome RNA blocks expression of viral genes

The transcribing nucleocapsid of vesicular stomatitis virus contains a linear genome RNA encapsidated by the N protein and two other dissociable minor proteins L and NS. Photoreaction of 4′-substituted psoralen modifies the genome RNA and inhibits mRNA synthesis in vitro by the virion-associated RNA polymerase. By using 3H-labeled psoralen we have shown that photoreaction occurs with a cytosine residue within the N gene near the 3′ end of the viral genome RNA. Removal of the L and the NS proteins from the nucleocapsid does not expose additional photoreacting sites. The RNA within the nucleocapsid of a defective interfering particle derived from the 5′ end of the viral genome failed to react with psoralen under the same conditions. It appears that the entire genome RNA is protected by the N protein except for a site on the N gene that affects transcription of other viral genes. The mechanism of RNA transcription is discussed in the light of these observations.

In vitro transcription of vesicular stomatitis virus. Incorporation of deoxyguanosine and deoxycytidine, and formation of deoxyguanosine caps.

Detergent-disrupted vesicular stomatitis virus carried out in vitro transcription at a reduced rate in the presence of deoxyguanosine triphosphate. The transcripts annealed completely to excess vesicular stomatitis virus genomic RNA and consistent of the short leader RNA and even polyadenylated messenger RNA. Incubation with RNase T1 showed that the transcripts were resistant to cleavage. Nearest-neighbor analysis demonstrated that approximately two-thirds of the guanosine residues had been replaced by deoxyguanosine. The hybrid "deoxyguanosine-RNAs" carried cap structures which contained deoxyguanosine instead of guanosine. Competition experiments using both guanosine- and deoxyguanosine triphosphates indicated that GpppA and dGpppA cap structures were synthesized in approximately equal amounts at a ratio of 20 microM guanosine triphosphate to 50 microM deoxyguanosine triphosphate. Deoxyguanosine triphosphate was accepted by the polymerases of various strains and serotypes of vesicular stomatitis virus, demonstrating that its incorporation was a common characteristic. Deoxycytidine triphosphate could also substitute for cytidine triphosphate but to a lesser degree. Deoxyadenine, deoxyuridine-, and thymidine triphosphates were not or were very poorly accepted even at concentrations of 2 MM.

Alteration of the 5′ terminal caps of the mRNAs of vesicular stomatitis virus by cycloleucine in vivo

Cycloleucine, a methionine analog, which inhibits methylation in animal cells also inhibits the reproduction of vesicular stomatitis virus (VSV) in infected cells. The replication of the VSV genome RNA is abolished and the steady state levels of viral mRNAs are greatly reduced. These effects of cycloleucine in vivo appear to be due to the role of the drug as an inhibitor of S-adenosylmethionine formation, since RNA synthesis and methylation in vitro with purified virus in the presence of added S-adenosylmethionine are unaffected by the drug. Pulse label experiments show that VSV mRNAs are synthesized in infected cells at nearly normal rates in the presence of cycloleucine, however, the majority of the transcripts appear to be unstable and are rapidly degraded. The limited amounts of VSV mRNAs which do accumulate during a 2 hr label in vivo contain the undermethylated 5′-terminal caps GpppA and 7mGpppA. No GpppA m capped or pppA initiated RNAs were detected. These last observations suggest that the pathway of viral RNA methylation within the infected cell occurs in the order GpppA → 7mGpppA → 7mGpppA m, instead of the reverse order which was proposed based on transcription studies in vitro. [ Testa, D., and Banerjee, A. K. (1977). J. Virol. 24, 786–793]. At least some of the undermethylated mRNAs are functional since there is a low level of synthesis in vivo of the virus proteins G, N, NS, and M.

In vivo transcription of the 5'-terminal extracistronic region of vesicular stomatitis virus RNA.

In vivo transcription and polyadenylation at the junction of the L cistron and the 5'-terminal extracistronic region of vesicular stomatitis virus RNA was investigated. Annealing of 5'32P-labeled RNA representing the 5'-terminal noncoding 77 nucleotides of vesicular stomatitis virus genomic RNA to L gene mRNA resulted in specific duplex formation. Two specific RNase T1- and RNase A resistant duplexes, 66 and 77 nucleotides long, bound to oligodeoxythymidylic acid cellulose. The specific sizes of the duplexes and their selection by oligodeoxythymidylic acid cellulose chromatography demonstrated that they were covalently linked to the polyadenylic acid tail of L gene mRNA. These data strongly suggest that the viral polymerase polyadenylates L gene mRNA in vivo by using the stretch of seven uridine residues at the end of the L cistron and that the polymerase can resume transcribing the 5'-terminal extracistronic region, resulting in a covalent linkage of the transcript to the polyadenylic acid tail of L gene mRNA.

Characterization and mapping of RNase III cleavage sites in VSV genome RNA.

Ribonuclease III cleaves the genome RNA of vesicular stomatitis virus (VSV) to yield an array of fragments which range in size from 3.5 to 0.1 x 10(6) daltons under partial digestion conditions. The locations of the RNase III cleavage sites which give rise to these fragments have been ordered relative to the 3' end of the virion RNA by digestion of 3' end-labeled RNA. Based on a map of the cleavage sites we predicted that fragments having the same size could be generated which contain information from each gene. Annealing of individual VSV mRNA probes to Northern blots of the separated RNase III-generated fragments confirmed that fragments having the same size are, in fact, generated which contain information from each coding region of the VSV genome. Analysis of maps of partial digestion products indicates that fragments having the same size arise repeatedly along the 3' half of the genome. The cleavage of VSV RNA by RNase III can be detected only if the nuclease treated molecules are denatured. This suggest that the structure features in VSV RNA which signal cleavage involve areas of higher order RNA structure.

Characterization of the non-permissive infection of rabbit cornea cells by vesicular stomatitis virus.

Rabbit cornea cells (RC-60) restrict the reproduction of vesicular stomatitis virus (VSV) (Thacore & Youngner, 1975). In cells infected with VSV alone, an inhibition in the synthesis of VSV genome RNA is observed. A number of parameters which could affect virus RNA synthesis have been examined: virus transcription and post-transcriptional modification, translation, modification of proteins and migration of the G protein to the surface of the cell; they all appear to be normal, although somewhat diminished, in the restricted system. In these cells, therefore, it is the replication of VSV RNA that is directly inhibited, although limited synthesis of both (+) and (-) strand genome length RNA does occur. When the cells are co-infected with rabbit poxvirus (RPV) as a helper virus, however, VSV production is normal. Our studies suggest that RPV plays a role in the maturation as well as in the replication of VSV RNA in RC-60 cells. Certain mutants of RPV have been found to lack the helper function and are unable to convert the RC-60 cells into a permissive host for VSV. These mutants should facilitate the elucidation of the mechanism by which RPV is able to overcome the restriction in these cells.

Site on the vesicular stomatitis virus genome specifying polyadenylation and the end of the L gene mRNA.

The 5'-terminal nucleotide sequence from positions 50 to 130 of vesicular stomatitis virus RNA was determined indirectly by using a defective interfering particle RNA which contains covalently linked genomic minus and antigenomic plus sense RNAs. The last 18 nucleotides of the L gene coding for in the viral polymerase were identified and isolated by specific duplex formation between 5' terminally labeled oligonucleotides from a small single-stranded defective interfering particle RNA and L gene mRNA. The L gene ends at position 60 from the 5' terminus of the vesicular stomatitis genome. The data demonstrated that the first seven adenine residues in the polyadenylic acid tail of L gene mRNA may be coded for in the genome and suggested that the viral transcriptase itself may carry out polyadenylation, possibly by chattering at the uridine-rich sequence at the end of the L gene. Analysis of the 5'-terminal sequence of vesicular stomatitis virus genomic RNA revealed that it might fold into a complex secondary structure with possibly 62% of the bases paired.

Intervening polyadenylate sequences in RNA transcripts of vesicular stomatitis virus

Purified and partially resolved vesicular stomatitis virus (VSV) messenger RNA has been annealed to the VSV genomic RNA and visualized in the electron microscope under conditions in which duplex regions have a wider image width than single-stranded RNA. The locations of the intercistronic boundaries between the messages have been mapped on the VSV genome. The contour of the double-stranded regions is occasionally interrupted by looped-out single-stranded RNA. The loops are comprised of post-transcriptionally synthesized polyadenylate. Most of these structures are found at the intercistronic boundaries and covalently bridge adjacent message sequences. In this paper, we discuss the possible significance of these loops.

A unique RNA species involved in initiation of vesicular stomatitis virus RNA transcription in vitro

Purified virions of vesicular stomatitis virus (VSV) are capable of synthesizing two distinct types of virus-specific RNA in vitro. The first consists of several viral mRNAs which have been previously shown to contain the blocked 5′ terminal sequence GpppApApCpApGp and 3′ terminal poly(A). The second type of RNA has an unblocked 5′ terminus and does not contain poly(A) stretches long enough to bind to oligo(dT)-cellulose columns. It migrates in 20% polyacrylamide gels as a single homogeneous peak with an estimated chain length of 68 nucleotides. Base analysis demonstrated that this small RNA molecule is composed of 48% AMP, 20% CMP, 11% GMP, and 21% UMP. The 5′ terminal sequence of the small RNA is ppApCpGp, which appears to be complementary to the 3′ terminal sequence of the VSV genome RNA (…PypGpU). These results indicate that this small RNA molecule probably represents the initiated lead-in RNA segment which is removed during formation of VSV mRNAs by a possible processing mechanism.

3′-Terminal sequence of vesicular stomatitis virus genome RNA

The 3′-terminal sequence of vesicular stomatitis virus genome RNA is …PypGpU. The implication of this result in relation to the mode of transcription of vesicular stomatitis virus RNA in vitro is discussed.