Vesicular Stomatitis Virus RNA Research Articles

Locations and in situ structure of the polymerase complex inside the virion of vesicular stomatitis virus

The polymerase complex of nonsegmented negative-strand RNA viruses primarily consists of a large (L) protein and a phosphoprotein (P). L is a multifunctional enzyme carrying out RNA-dependent RNA polymerization and all other steps associated with transcription and replication, while P is the nonenzymatic cofactor, regulating the function and conformation of L. The structure of a purified vesicular stomatitis virus (VSV) polymerase complex containing L and associated P segments has been determined; however, the location and manner of the attachments of L and P within each virion are unknown, limiting our mechanistic understanding of VSV RNA replication and transcription and hindering engineering efforts of this widely used anticancer and vaccine vector. Here, we have used cryo-electron tomography to visualize the VSV virion, revealing the attachment of the ring-shaped L molecules to VSV nucleocapsid proteins (N) throughout the cavity of the bullet-shaped nucleocapsid. Subtomogram averaging and three-dimensional classification of regions containing N and the matrix protein (M) have yielded the in situ structure of the polymerase complex. On average, ∼55 polymerase complexes are packaged in each virion. The capping domain of L interacts with two neighboring N molecules through flexible attachments. P, which exists as a dimer, bridges separate N molecules and the connector and C-terminal domains of L. Our data provide the structural basis for recruitment of L to N by P in virus assembly and for flexible attachments between L and N, which allow a quick response of L in primary transcription upon cell entry.

Vesiculopolins, a New Class of Anti-Vesiculoviral Compounds, Inhibit Transcription Initiation of Vesiculoviruses

Vesicular stomatitis virus (VSV) represents a promising platform for developing oncolytic viruses, as well as vaccines against significant human pathogens. To safely control VSV infection in humans, small-molecule drugs that selectively inhibit VSV infection may be needed. Here, using a cell-based high-throughput screening assay followed by an in vitro transcription assay, compounds with a 7-hydroxy-6-methyl-3,4-dihydroquinolin-2(1H)-one structure and an aromatic group at position 4 (named vesiculopolins, VPIs) were identified as VSV RNA polymerase inhibitors. The most effective compound, VPI A, inhibited VSV-induced cytopathic effects and in vitro mRNA synthesis with micromolar to submicromolar 50% inhibitory concentrations. VPI A was found to inhibit terminal de novo initiation rather than elongation for leader RNA synthesis, but not mRNA capping, with the VSV L protein, suggesting that VPI A is targeted to the polymerase domain in the L protein. VPI A inhibited transcription of Chandipura virus, but not of human parainfluenza virus 3, suggesting that it specifically acts on vesiculoviral L proteins. These results suggest that VPIs may serve not only as molecular probes to elucidate the mechanisms of transcription of vesiculoviruses, but also as lead compounds to develop antiviral drugs against vesiculoviruses and other related rhabdoviruses.

RNA Synthesis and Capping by Non-segmented Negative Strand RNA Viral Polymerases: Lessons From a Prototypic Virus.

Non-segmented negative strand (NNS) RNA viruses belonging to the order Mononegavirales are highly diversified eukaryotic viruses including significant human pathogens, such as rabies, measles, Nipah, and Ebola. Elucidation of their unique strategies to replicate in eukaryotic cells is crucial to aid in developing anti-NNS RNA viral agents. Over the past 40 years, vesicular stomatitis virus (VSV), closely related to rabies virus, has served as a paradigm to study the fundamental molecular mechanisms of transcription and replication of NNS RNA viruses. These studies provided insights into how NNS RNA viruses synthesize 5′-capped mRNAs using their RNA-dependent RNA polymerase L proteins equipped with an unconventional mRNA capping enzyme, namely GDP polyribonucleotidyltransferase (PRNTase), domain. PRNTase or PRNTase-like domains are evolutionally conserved among L proteins of all known NNS RNA viruses and their related viruses belonging to Jingchuvirales, a newly established order, in the class Monjiviricetes, suggesting that they may have evolved from a common ancestor that acquired the unique capping system to replicate in a primitive eukaryotic host. This article reviews what has been learned from biochemical and structural studies on the VSV RNA biosynthesis machinery, and then focuses on recent advances in our understanding of regulatory and catalytic roles of the PRNTase domain in RNA synthesis and capping.

Global analysis of polysome-associated mRNA in vesicular stomatitis virus infected cells.

Infection of mammalian cells with vesicular stomatitis virus (VSV) results in the inhibition of cellular translation while viral translation proceeds efficiently. VSV RNA synthesis occurs entirely within the cytoplasm, where during transcription the viral polymerase produces 5 mRNAs that are structurally indistinct to cellular mRNAs with respect to their 5′ cap-structure and 3′-polyadenylate tail. Using the global approach of massively parallel sequencing of total cytoplasmic, monosome- and polysome-associated mRNA, we interrogate the impact of VSV infection of HeLa cells on translation. Analysis of sequence reads in the different fractions shows >60% of total cytoplasmic and polysome-associated reads map to the 5 viral genes by 6 hours post-infection, a time point at which robust host cell translational shut-off is observed. Consistent with an overwhelming abundance of viral mRNA in the polysome fraction, the reads mapping to cellular genes were reduced. The cellular mRNAs that remain most polysome-associated following infection had longer half-lives, were typically larger, and were more AU rich, features that are shared with the viral mRNAs. Several of those mRNAs encode proteins known to positively affect viral replication, and using chemical inhibition and siRNA depletion we confirm that the host chaperone heat shock protein 90 (hsp90) and eukaryotic translation initiation factor 3A (eIF3A)—encoded by 2 such mRNAs—support viral replication. Correspondingly, regulated in development and DNA damage 1 (Redd1) encoded by a host mRNA with reduced polysome association inhibits viral infection. These data underscore the importance of viral mRNA abundance in the shut-off of host translation in VSV infected cells and link the differential translatability of some cellular mRNAs with pro- or antiviral function.

Comparative Oncology Evaluation of Intravenous Recombinant Oncolytic Vesicular Stomatitis Virus Therapy in Spontaneous Canine Cancer.

Clinical translation of intravenous therapies to treat disseminated or metastatic cancer is imperative. Comparative oncology, the evaluation of novel cancer therapies in animals with spontaneous cancer, can be utilized to inform and accelerate clinical translation. Preclinical murine studies demonstrate that single-shot systemic therapy with a vesicular stomatitis virus (VSV)-IFNβ-NIS, a novel recombinant oncolytic VSV, can induce curative remission in tumor-bearing mice. Clinical translation of VSV-IFNβ-NIS therapy is dependent on comprehensive assessment of clinical toxicities, virus shedding, pharmacokinetics, and efficacy in clinically relevant models. Dogs spontaneously develop cancer with comparable etiology, clinical progression, and response to therapy as human malignancies. A comparative oncology study was carried out to investigate feasibility and tolerability of intravenous oncolytic VSV-IFNβ-NIS therapy in pet dogs with spontaneous cancer. Nine dogs with various malignancies were treated with a single intravenous dose of VSV-IFNβ-NIS. Two dogs with high-grade peripheral T-cell lymphoma had rapid but transient remission of disseminated disease and transient hepatotoxicity that resolved spontaneously. There was no shedding of infectious virus. Correlative pharmacokinetic studies revealed elevated levels of VSV RNA in blood in dogs with measurable disease remission. This is the first evaluation of intravenous oncolytic virus therapy for spontaneous canine cancer, demonstrating that VSV-IFNβ-NIS is well-tolerated and safe in dogs with advanced or metastatic disease. This approach has informed clinical translation, including dose and target indication selection, leading to a clinical investigation of intravenous VSV-IFNβ-NIS therapy, and provided preliminary evidence of clinical efficacy and potential biomarkers that correlate with therapeutic response. Mol Cancer Ther; 17(1); 316-26. ©2017 AACR.

Safety Studies in Tumor and Non-Tumor-Bearing Mice in Support of Clinical Trials Using Oncolytic VSV-IFNβ-NIS.

Oncolytic VSV-IFNβ-NIS is selectively destructive to tumors. Here, we present the IND enabling preclinical rodent studies in support of clinical testing of vesicular stomatitis virus (VSV) as a systemic therapy. Efficacy studies showed dose-dependent tumor regression in C57BL/KaLwRij mice bearing syngeneic 5TGM1 plasmacytomas after systemic VSV administration. In contrast, the virus was effective at all doses tested against human KAS6/1 xenografts in SCID mice. Intravenous administration of VSV-mIFNβ-NIS is well tolerated in C57BL/6 mice up to 5 × 10(10) TCID50 (50% tissue culture infective dose)/kg with no neurovirulence, no cytokine storm, and no abnormalities in tissues. Dose-limiting toxicities included elevated transaminases, thrombocytopenia, and lymphopenia. Inactivated viral particles did not cause hepatic toxicity. Intravenously administered VSV was preferentially sequestered by macrophages in the spleen and liver. Quantitative RT-PCR analysis for total viral RNA on days 2, 7, 21, and 58 showed highest VSV RNA in day 2 samples; highest in spleen, liver, lung, lymph node, kidney, gonad, and bone marrow. No infectious virus was recovered from tissues at any time point. The no observable adverse event level and maximum tolerated dose of VSV-mIFNβ-NIS in C57BL/6 mice are 10(10) TCID50/kg and 5 × 10(10) TCID50/kg, respectively. Clinical translation of VSV-IFNβ-NIS is underway in companion dogs with cancer and in human patients with relapsed hematological malignancies and endometrial cancer.

Structure and Function of the N-Terminal Domain of the Vesicular Stomatitis Virus RNA Polymerase.

Viruses have various mechanisms to duplicate their genomes and produce virus-specific mRNAs. Negative-strand RNA viruses encode their own polymerases to perform each of these processes. For the nonsegmented negative-strand RNA viruses, the polymerase is comprised of the large polymerase subunit (L) and the phosphoprotein (P). L proteins from members of the Rhabdoviridae, Paramyxoviridae, and Filoviridae share sequence and predicted secondary structure homology. Here, we present the structure of the N-terminal domain (conserved region I) of the L protein from a rhabdovirus, vesicular stomatitis virus, at 1.8-Å resolution. The strictly and strongly conserved residues in this domain cluster in a single area of the protein. Serial mutation of these residues shows that many of the amino acids are essential for viral transcription but not for mRNA capping. Three-dimensional alignments show that this domain shares structural homology with polymerases from other viral families, including segmented negative-strand RNA and double-stranded RNA (dsRNA) viruses. Negative-strand RNA viruses include a diverse set of viral families that infect animals and plants, causing serious illness and economic impact. The members of this group of viruses share a set of functionally conserved proteins that are essential to their replication cycle. Among this set of proteins is the viral polymerase, which performs a unique set of reactions to produce genome- and subgenome-length RNA transcripts. In this article, we study the polymerase of vesicular stomatitis virus, a member of the rhabdoviruses, which has served in the past as a model to study negative-strand RNA virus replication. We have identified a site in the N-terminal domain of the polymerase that is essential to viral transcription and that shares sequence homology with members of the paramyxoviruses and the filoviruses. Newly identified sites such as that described here could prove to be useful targets in the design of new therapeutics against negative-strand RNA viruses.

Primate-specific miR-576-3p sets host defense signalling threshold.

MicroRNAs (miRNAs) have been shown to regulate viral infection, but the miRNAs that target intracellular sensors and adaptors of innate immunity have not been fully uncovered. Here we conduct a miRNA mimic screen and validation with miRNA inhibitors in cells infected with vesicular stomatitis virus (VSV) to identify miRNAs that regulate viral-host interactions. We identify miR-576-3p as a robust regulator of infection by VSV and other RNA and DNA viruses. While a miR-576-3p mimic sensitizes cells to viral replication, inhibition of endogenous miR-576-3p prevents infection. miR-576-3p is induced by IRF3 concomitantly with interferon and targets STING, MAVS and TRAF3, which are critical factors for interferon expression. Interestingly, miR-576-3p and its binding sites are primate-specific and miR-576-3p levels are reduced in inflammatory diseases. These findings indicate that induction of miR-576-3p by IRF3 triggers a feedback mechanism to reduce interferon expression and set an antiviral response threshold to likely avoid excessive inflammation.

Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells.

The synthesis and subsequent genomic integration of DNA that is complementary to the genomes of non-retroviral RNA viruses are rarely observed. However, upon infection of various human cell lines and primary fibroblasts with the vesicular stomatitis virus (VSV), we detected DNA complementary to the VSV RNA. The VSV DNA was detected in the cytoplasm as single-stranded DNA fully complementary to the viral mRNA from the poly(A) region to the 7-methyl guanosine cap. The formation of this DNA was cell-dependent. Experimentally, we found that the transduction of cells that do not produce VSV DNA with the long interspersed nuclear element 1 and their infection with VSV could lead to the formation of VSV DNA. Viral DNA complementary to other RNA viruses was also detected in the respective infected human cells. Thus, the genetic information of the non-retroviral RNA virus genome can flow into the DNA of mammalian cells expressing LINE-1-like elements.

Smurf1 Protein Negatively Regulates Interferon-γ Signaling through Promoting STAT1 Protein Ubiquitination and Degradation

Interferons are important cytokines that mediate antiviral, antiproliferative, antitumor, and immunoregulatory activities. However, uncontrolled IFN signaling may lead to autoimmune diseases. Here we identified Smurf1 as a negative regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation. Smurf1 interacted with STAT1 through the WW domains of Smurf1 and the PY motif in STAT1 and catalyzed K48-linked polyubiquitination of STAT1. Interestingly, the Smurf1-mediated ubiquitination and degradation did not require STAT1 tyrosine and serine phosphorylation. Subsequently, overexpression of Smurf1 attenuated IFN-γ-mediated STAT1 activation and antiviral immune responses, whereas knockdown of Smurf1 enhanced IFN-γ-mediated STAT1 activation, expression of STAT1 target genes, and antiviral immune responses. Furthermore, IFN-γ stimulation led to enhanced expression of Smurf1. Therefore, our results demonstrate that Smurf1 is a negative feedback regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation.

RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses

Negative-strand (NS) RNA viruses comprise many pathogens that cause serious diseases in humans and animals. Despite their clinical importance, little is known about the host factors required for their infection. Using vesicular stomatitis virus (VSV), a prototypic NS RNA virus in the family Rhabdoviridae, we conducted a human genome-wide siRNA screen and identified 72 host genes required for viral infection. Many of these identified genes were also required for infection by two other NS RNA viruses, the lymphocytic choriomeningitis virus of the Arenaviridae family and human parainfluenza virus type 3 of the Paramyxoviridae family. Genes affecting different stages of VSV infection, such as entry/uncoating, gene expression, and assembly/release, were identified. Depletion of the proteins of the coatomer complex I or its upstream effectors ARF1 or GBF1 led to detection of reduced levels of VSV RNA. Coatomer complex I was also required for infection of lymphocytic choriomeningitis virus and human parainfluenza virus type 3. These results highlight the evolutionarily conserved requirements for gene expression of diverse families of NS RNA viruses and demonstrate the involvement of host cell secretory pathway in the process.

Molecular Mechanism of Signal Perception and Integration by the Innate Immune Sensor Retinoic Acid-inducible Gene-I (RIG-I)

RIG-I is a major innate immune sensor for viral infection, triggering an interferon (IFN)-mediated antiviral response upon cytosolic detection of viral RNA. Double-strandedness and 5'-terminal triphosphates were identified as motifs required to elicit optimal immunological signaling. However, very little is known about the response dynamics of the RIG-I pathway, which is crucial for the ability of the cell to react to diverse classes of viral RNA while maintaining self-tolerance. In the present study, we addressed the molecular mechanism of RIG-I signal detection and its translation into pathway activation. By employing highly quantitative methods, we could establish the length of the double-stranded RNA (dsRNA) to be the most critical determinant of response strength. Size exclusion chromatography and direct visualization in scanning force microscopy suggested that this was due to cooperative oligomerization of RIG-I along dsRNA. The initiation efficiency of this oligomerization process critically depended on the presence of high affinity motifs, like a 5'-triphosphate. It is noteworthy that for dsRNA longer than 200 bp, internal initiation could effectively compensate for a lack of terminal triphosphates. In summary, our data demonstrate a very flexible response behavior of the RIG-I pathway, in which sensing and integration of at least two distinct signals, initiation efficiency and double strand length, allow the host cell to mount an antiviral response that is tightly adjusted to the type of the detected signal, such as viral genomes, replication intermediates, or small by-products.

Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey

Two methods for the extraction of RNA of vesicular stomatitis virus (VSV) Indiana1 and New Jersey and their simultaneous amplification by one-step polymerase chain reaction using reverse transcriptase were evaluated. A guanidine-thiocyanate-based RNA extraction (Qiagen RNeasy Mini Kit, Qiagen, Valencia, CA ) followed by column-based purification coupled with one-step RT-PCR proved to be a simple, safe, practicable, and reliable tool for rapid, highly sensitive, and specific differential diagnosis of both types of VSV in cell lysate and spiked tissue samples as compared with the tri-phasic extraction method (Tri-reagent method). When RNA was extracted either from VSV cell culture stock or from VSV spiked bovine lymph nodes by using Qiagen RNeasy Mini Kit, the detection limit in the multiplex RT-PCR was as low as 0.505 to 2.84 TCID(50) for VSV-IND and VSV-NJ, respectively. The multiplex RT-PCR consistently detected VSV-IND and NJ RNA in as little as 0.1-1.0 fg of total RNA from spiked BHK-21 cell suspension when Qiagen RNeasy mini kit was used. The multiplex RT-PCR assay was capable of detecting both types of VSV in a one-step reaction tube. The minimum sensitivity of this assay in various experiments was 0.1683 TCID(50) (IND), 0.0946 TCID(50) (NJ), and 0.057 fg (IND and NJ) per 2 µl PCR sample, which is significantly more sensitive than reported previously (0.28-2.8 TCID50/1 µl). So the present study improved the sensitivity of previously reported multiplex RT-PCR for the detection and differentiation of VSV-IND and VSV-NJ in a single assay.

Molecular architecture of the vesicular stomatitis virus RNA polymerase

Nonsegmented negative-strand (NNS) RNA viruses initiate infection by delivering into the host cell a highly specialized RNA synthesis machine comprising the genomic RNA completely encapsidated by the viral nucleocapsid protein and associated with the viral polymerase. The catalytic core of this protein-RNA complex is a 250-kDa multifunctional large (L) polymerase protein that contains enzymatic activities for nucleotide polymerization as well as for each step of mRNA cap formation. Working with vesicular stomatitis virus (VSV), a prototype of NNS RNA viruses, we used negative stain electron microscopy (EM) to obtain a molecular view of L, alone and in complex with the viral phosphoprotein (P) cofactor. EM analysis, combined with proteolytic digestion and deletion mapping, revealed the organization of L into a ring domain containing the RNA polymerase and an appendage of three globular domains containing the cap-forming activities. The capping enzyme maps to a globular domain, which is juxtaposed to the ring, and the cap methyltransferase maps to a more distal and flexibly connected globule. Upon P binding, L undergoes a significant rearrangement that may reflect an optimal positioning of its functional domains for transcription. The structural map of L provides new insights into the interrelationship of its various domains, and their rearrangement on P binding that is likely important for RNA synthesis. Because the arrangement of conserved regions involved in catalysis is homologous, the structural insights obtained for VSV L likely extend to all NNS RNA viruses.

Protein Expression Redirects Vesicular Stomatitis Virus RNA Synthesis to Cytoplasmic Inclusions

Positive-strand and double-strand RNA viruses typically compartmentalize their replication machinery in infected cells. This is thought to shield viral RNA from detection by innate immune sensors and favor RNA synthesis. The picture for the non-segmented negative-strand (NNS) RNA viruses, however, is less clear. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we examined the location of the viral replication machinery and RNA synthesis in cells. By short-term labeling of viral RNA with 5′-bromouridine 5′-triphosphate (BrUTP), we demonstrate that primary mRNA synthesis occurs throughout the host cell cytoplasm. Protein synthesis results in the formation of inclusions that contain the viral RNA synthesis machinery and become the predominant sites of mRNA synthesis in the cell. Disruption of the microtubule network by treatment of cells with nocodazole leads to the accumulation of viral mRNA in discrete structures that decorate the surface of the inclusions. By pulse-chase analysis of the mRNA, we find that viral transcripts synthesized at the inclusions are transported away from the inclusions in a microtubule-dependent manner. Metabolic labeling of viral proteins revealed that inhibiting this transport step diminished the rate of translation. Collectively those data suggest that microtubule-dependent transport of viral mRNAs from inclusions facilitates their translation. Our experiments also show that during a VSV infection, protein synthesis is required to redirect viral RNA synthesis to intracytoplasmic inclusions. As viral RNA synthesis is initially unrestricted, we speculate that its subsequent confinement to inclusions might reflect a cellular response to infection.

Histidine-mediated RNA transfer to GDP for unique mRNA capping by vesicular stomatitis virus RNA polymerase

The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus, a prototype of nonsegmented negative-strand (NNS) RNA viruses, forms a covalent complex with a 5'-phosphorylated viral mRNA-start sequence (L-pRNA), a putative intermediate in the unconventional mRNA capping reaction catalyzed by the RNA:GDP polyribonucleotidyltransferase (PRNTase) activity. Here, we directly demonstrate that the purified L-pRNA complex transfers pRNA to GDP to produce the capped RNA (Gpp-pRNA), indicating that the complex is a bona fide intermediate in the RNA transfer reaction. To locate the active site of the PRNTase domain in the L protein, the covalent RNA attachment site was mapped. We found that the 5'-monophosphate end of the RNA is linked to the histidine residue at position 1,227 (H1227) of the L protein through a phosphoamide bond. Interestingly, H1227 is part of the histidine-arginine (HR) motif, which is conserved within the L proteins of the NNS RNA viruses including rabies, measles, Ebola, and Borna disease viruses. Mutagenesis analyses revealed that the HR motif is required for the PRNTase activity at the step of the enzyme-pRNA intermediate formation. Thus, our findings suggest that an ancient NNS RNA viral polymerase has acquired the PRNTase domain independently of the eukaryotic mRNA capping enzyme during evolution and PRNTase becomes a rational target for designing antiviral agents.

Mutations in the C-Terminal Loop of the Nucleocapsid Protein Affect Vesicular Stomatitis Virus RNA Replication and Transcription Differentially

The 2.9-A structure of the vesicular stomatitis virus nucleocapsid (N) protein bound to RNA shows the RNA to be tightly sequestered between the two lobes of the N protein. Domain movement of the lobes of the N protein has been postulated to facilitate polymerase access to the RNA template. We investigated the roles of individual amino acid residues in the C-terminal loop, involved in long-range interactions between N protein monomers, in forming functional ribonucleoprotein (RNP) templates. The effects of specific N protein mutations on its expression, interaction with the phosphoprotein, and formation of RNP templates that supported viral RNA replication and transcription were examined. Mutations introduced into the C-terminal loop, predicted to break contact with other residues in the loop, caused up to 10-fold increases in RNA replication without an equivalent stimulation of transcription. Mutation F348A, predicted to break contact between the C-terminal loop and the N-terminal arm, formed templates that supported wild-type levels of RNA replication but almost no transcription. These data show that mutations in the C-terminal loop of the N protein can disparately affect RNA replication and transcription, indicating that the N protein plays a role in modulating RNP template function beyond its structural role in RNA encapsidation.

Gene Therapy for Cancer

Gene Therapy for Cancer

The RAS/Raf1/MEK/ERK Signaling Pathway Facilitates VSV-mediated Oncolysis: Implication for the Defective Interferon Response in Cancer Cells

Vesicular stomatitis virus (VSV) can replicate in malignant cells more efficiently than in normal cells. Although the selective replication appears to be caused by defects in the interferon (IFN) system in malignant cells, the mechanisms which render these cells less responsive to IFN remain poorly understood. Here we present evidence that an activated RAS/Raf1/MEK/ERK pathway plays a critical role in the defects. NIH 3T3 or human primary cells stably expressing active RAS or Raf1 were rapidly killed by VSV. Although IFNalpha treatment no longer protected the RAS- or Raf1-overexpressing cells from VSV infection, responsiveness to IFNalpha was restored following treatment with the mitogen-activated protein kinase kinase (MEK) inhibitor U0126. Similarly, human cancer-derived cell lines became more responsive to IFNalpha in conjunction with U0126 treatment. Intriguingly, dual treatment with both IFNalpha and U0126 severely reduced the levels of viral RNAs in the infected cells. Moreover, cancer cells showed defects in inducing an IFNalpha-responsive factor, MxA, which is known to block VSV RNA synthesis, and U0126 restored the MxA expression. Our observations suggest that activation of the extracellular signal-regulated protein kinase (ERK) signaling leads to the defect in IFNalpha-mediated upregulation of MxA protein, which facilitates VSV oncolysis. In view of the fact that 30% of all cancers have constitutive activation of the RAS/Raf1/MEK/ERK pathway, VSV would be an ideal oncolytic virus for targeting such cancers.

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.