Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of human-, animal-, and plant-infecting (ϩ)RNA viruses, has been studied as a model for viral RNA replication, encapsidation, recombination, and other processes (3). BMV has three genomic RNAs. RNAs 1 and 2 encode the interacting, multifunctional 1a helicase-like and 2a polymerase RNA replication factors (4, 5), which form endoplasmic reticulum (ER) membrane-associated RNA replication complexes with functional similarities to the replicative cores of retrovirus and double-strand (ds)RNA virus virions (6). RNA3 encodes protein 3a that enables infection spread between cells in natural hosts. The negative-strand [(Ϫ) RNA]3 replication intermediate also serves as a template for synthesis of a subgenomic (sg) mRNA, RNA4, which encodes the viral coat protein (Fig. 1A).The yeast Saccharomyces cerevisiae has proven a valuable model for normal and disease processes in human and other cells. The unusual ability of BMV to direct its genomic RNA replication, gene expression, encapsidation, and other processes in this yeast (7,8) has allowed traditional yeast mutagenic analyses that have identified host genes involved in multiple steps of BMV RNA replication and gene expression. Such host genes encode a wide variety of functions and contribute to diverse replication steps, including supporting and regulating viral translation, selecting and recruiting viral RNAs as replication templates, activating the RNA replication complex through chaperones, and providing a lipid profile compatible with membrane-associated viral RNA replication (9-14; reviewed in refs. 2 and 15).Here, we sought to develop a more rapid, global method to systematically identify yeast host factors with effects on BMV RNA replication by using an ordered array of yeast deletion strains (16) to assay virus replication in the absence of each of Ϸ4,500 yeast factors, which is Ϸ80% of the yeast genome. We describe screening this deletion array by using a whole-cell assay based on BMV-directed Renilla luciferase (Rluc) expression by pathways dependent on viral RNA replication and viral RNAdirected sg mRNA synthesis. The assay identified nearly 100 host genes whose absence repressed or enhanced BMV-directed Rluc expression by 3-to 25-fold. The results provide a significantly expanded view of virus-host interactions and should advance understanding of virus and cell pathways. Materials and MethodsYeast. YMI04 and ded1i yeast were described (11). Strains BY4743 (WT; ref. 17) and the homozygous diploid deletion series (BY4743 strain background; ref. 16) were from Research Genetics (Huntsville, AL). Standard yeast techniques were used (18), except for 96-well transformations, which were based on a one-step procedure (19). Briefly, yeast were grown to saturation overnight at 30°C in 96-well plates (1.2 ml per well), pelleted, suspended in 100 l of transformation mix (0.18 M LiAc, pH 5.5, 36% polyethylene glycol-3350, 90 mM DTT, 0.5 mg͞ml sheared salmon sperm DNA, and 20 g͞ml of each plasmid), incubate...

Oncolytic virus (OV) therapy takes advantage of common cancer characteristics, such as defective type I interferon (IFN) signaling, to preferentially infect and kill cancer cells with viruses. Our recent study (Murphy et al., 2012, J. Virol., 86: 3073-87) found human pancreatic ductal adenocarcinoma (PDA) cells were highly heterogeneous in their permissiveness to vesicular stomatitis virus (VSV) and suggested at least some resistant cell lines retained functional type I IFN responses. Here we examine cellular responses to infection by the oncolytic VSV recombinant VSV-ΔM51-GFP by analyzing a panel of 11 human PDA cell lines for expression of 33 genes associated with type I IFN pathways. Although all cell lines sensed infection by VSV-ΔM51-GFP and most activated IFN-α and β expression, only resistant cell lines displayed constitutive high-level expression of the IFN-stimulated antiviral genes MxA and OAS. Inhibition of JAK/STAT signaling decreased levels of MxA and OAS and increased VSV infection, replication and oncolysis, further implicating IFN responses in resistance. Unlike VSV, vaccinia and herpes simplex virus infectivity and killing of PDA cells was independent of the type I IFN signaling profile, possibly because these two viruses are better equipped to evade type I IFN responses. Our study demonstrates heterogeneity in the type I IFN signaling status of PDA cells and suggests MxA and OAS as potential biomarkers for PDA resistance to VSV and other OVs sensitive to type I IFN responses.

Oncolytic virus (OV) therapy is an emerging anti-cancer approach that utilizes viruses to preferentially infect and kill cancer cells, while not harming healthy cells. Vesicular stomatitis virus (VSV) is a prototypic non-segmented, negative-strand RNA virus with inherent OV qualities. Antiviral responses induced by type I interferon pathways are believed to be impaired in most cancer cells, making them more susceptible to VSV than normal cells. Several other factors make VSV a promising OV candidate for clinical use, including its well-studied biology, a small, easily manipulated genome, relative independence of a receptor or cell cycle, cytoplasmic replication without risk of host-cell transformation, and lack of pre-existing immunity in humans. Moreover, various VSV-based recombinant viruses have been engineered via reverse genetics to improve oncoselectivity, safety, oncotoxicity and stimulation of tumour-specific immunity. Alternative delivery methods are also being studied to minimize premature immune clearance of VSV. OV treatment as a monotherapy is being explored, although many studies have employed VSV in combination with radiotherapy, chemotherapy or other OVs. Preclinical studies with various cancers have demonstrated that VSV is a promising OV; as a result, a human clinical trial using VSV is currently in progress.

Vesicular stomatitis virus (VSV) is a prototypic nonsegmented negative-strand RNA virus. VSV’s broad cell tropism makes it a popular model virus for many basic research applications. In addition, a lack of preexisting human immunity against VSV, inherent oncotropism and other features make VSV a widely used platform for vaccine and oncolytic vectors. However, VSV’s neurotropism that can result in viral encephalitis in experimental animals needs to be addressed for the use of the virus as a safe vector. Therefore, it is very important to understand the determinants of VSV tropism and develop strategies to alter it. VSV glycoprotein (G) and matrix (M) protein play major roles in its cell tropism. VSV G protein is responsible for VSV broad cell tropism and is often used for pseudotyping other viruses. VSV M affects cell tropism via evasion of antiviral responses, and M mutants can be used to limit cell tropism to cell types defective in interferon signaling. In addition, other VSV proteins and host proteins may function as determinants of VSV cell tropism. Various approaches have been successfully used to alter VSV tropism to benefit basic research and clinically relevant applications.

bVesicular stomatitis virus (VSV) is a promising oncolytic agent against a variety of cancers. However, it has never been tested in any pancreatic cancer model. Pancreatic ductal adenocarcinoma (PDA) is the most common and aggressive form of pancreatic cancer. In this study, the oncolytic potentials of several VSV variants were analyzed in a panel of 13 clinically relevant human PDA cell lines and compared to conditionally replicative adenoviruses (CRAds), Sendai virus and respiratory syncytial virus. VSV variants showed oncolytic abilities superior to those of other viruses, and some cell lines that exhibited resistance to other viruses were successfully killed by VSV. However, PDA cells were highly heterogeneous in their susceptibility to virus-induced oncolysis, and several cell lines were resistant to all tested viruses. Resistant cells showed low levels of very early VSV RNA synthesis, indicating possible defects at initial stages of infection. In addition, unlike permissive PDA cell lines, most of the resistant cell lines were able to both produce and respond to interferon, suggesting that intact type I interferon responses contributed to their resistance phenotype. Four cell lines that varied in their permissiveness to VSV-⌬M51 and CRAd dl1520 were tested in mice, and the in vivo results closely mimicked those in vitro. While our results demonstrate that VSV is a promising oncolytic agent against PDA, further studies are needed to better understand the molecular mechanisms of resistance of some PDAs to oncolytic virotherapy.

The vesicular stomatitis virus (VSV) RNA polymerase synthesizes viral mRNAs with 5-cap structures methylated at the guanine-N7 and 2-O-adenosine positions (7mGpppA m ). Previously, our laboratory showed that a VSV host range (hr) and temperature-sensitive (ts) mutant, hr1, had a complete defect in mRNA cap methylation and that the wild-type L protein could complement the hr1 defect in vitro. Here, we sequenced the L, P, and N genes of mutant hr1 and found only two amino acid substitutions, both residing in the Lpolymerase protein, which differentiate hr1 from its wild-type parent. These mutations (N505D and D1671V) were introduced separately and together into the L gene, and their effects on VSV in vitro transcription and in vivo chloramphenicol acetyltransferase minigenome replication were studied under conditions that are permissive and nonpermissive for hr1. Neither L mutation significantly affected viral RNA synthesis at 34°C in permissive (BHK) and nonpermissive (HEp-2) cells, but D1671V reduced in vitro transcription and genome replication by about 50% at 40°C in both cell lines. Recombinant VSV bearing each mutation were isolated, and the hr and ts phenotypes in infected cells were the result of a single D1671V substitution in the L protein. While the mutations did not significantly affect mRNA synthesis by purified viruses, 5-cap analyses of product mRNAs clearly demonstrated that the D1671V mutation abrogated all methyltransferase activity. Sequence analysis suggests that an aspartic acid at amino acid 1671 is a critical residue within a putative conserved S-adenosyl-L-methionine-binding domain of the L protein.Vesicular stomatitis virus (VSV, a rhabdovirus) is a prototypic nonsegmented negative-strand (NNS) RNA virus belonging to the order Mononegavirales, whose members share a similar genome organization and common mechanisms of genome replication and gene expression. This order includes many medically important pathogens, including the lethal rabies, Ebola, Marburg, Nipah, and Hendra viruses. The RNA-dependent RNA polymerase (RdRp) of NNS RNA viruses is packaged into mature virions and consists of two viral subunits, the phosphoprotein (P) and the large (L) protein. The RNA genome of NNS viruses is tightly encapsidated by the nucleocapsid (N) protein, and the resulting nucleocapsid serves as the template for the sequential transcription of monocistronic mRNAs and for genome replication. Recent studies on VSV suggest that two separate RdRp complexes, which differ in their protein content, are involved in genome replication versus mRNA transcription (14, 47).The VSV RdRp produces mRNA transcripts modified at their 5Ј end by capping and cap methylation (71). The mechanism of mRNA 5Ј capping in VSV and other NNS RNA viruses is unusual, where, in contrast to cellular cap structures, both the ␣ and ␤ phosphates in the GpppA triphosphate bridge are derived from a GDP donor (2, 5, 23). The cytoplasmic localization of virus transcription and the unusual mechanism of capping suggest that the guanylyltransferas...

Oncolytic virus (OV) therapy is an anti-cancer approach that uses viruses that preferentially infect, replicate in and kill cancer cells. Vesicular stomatitis virus (VSV, a rhabdovirus) is an OV that is currently being tested in the USA in several phase I clinical trials against different malignancies. Several factors make VSV a promising OV: lack of pre-existing human immunity against VSV, a small and easy to manipulate genome, cytoplasmic replication without risk of host cell transformation, independence of cell cycle and rapid growth to high titres in a broad range of cell lines facilitating large-scale virus production. While significant advances have been made in VSV-based OV therapy, room for improvement remains. Here we review recent studies (published in the last 5 years) that address 'old' and 'new' challenges of VSV-based OV therapy. These studies focused on improving VSV safety, oncoselectivity and oncotoxicity; breaking resistance of some cancers to VSV; preventing premature clearance of VSV; and stimulating tumour-specific immunity. Many of these approaches were based on combining VSV with other therapeutics. This review also discusses another rhabdovirus closely related to VSV, Maraba virus, which is currently being tested in Canada in phase I/II clinical trials.

Vesicular stomatitis virus (VSV) is a negative-sense single-stranded RNA virus that closely resembles its deadly cousin, rabies virus. In mice, VSV elicits a rapid and severe T cell–independent encephalitis, indicating that resident glial cells play an important role in the initiation of central nervous system (CNS) inflammation. Recently, retinoic acid–inducible gene I (RIG-I)-like helicases have been shown to function as intracellular pattern recognition receptors for replicative viral RNA motifs. In the present study, the authors demonstrate that the expression of two members of this RIG-I–like receptor family (RLR), RIG-I and melanoma differentiation-associated antigen 5 (MDA5), are elevated in mouse brain tissue following intranasal administration of VSV. Using isolated cultures of primary murine glial cells, the authors demonstrate that microglia and astrocytes constitutively express both RIG-I and MDA5 transcripts and protein. Importantly, the authors show that such expression is elevated following challenge with VSV or another negative-sense RNA virus, Sendai virus. The authors provide evidence that such induction is indirect and secondary to the production of soluble mediators by infected cells. Circumstantial evidence for the functional nature of RLR expression in glial cells comes from the observation that microglia express the RLR downstream effector molecule, interferon promoter stimulator-1, and demonstrate diminished levels of the negative RLR regulator, laboratory of genetics and physiology 2, following viral challenge. These findings raise the exciting possibility that RLR molecules play important roles in the detection of viral CNS pathogens and the initiation of protective immune responses or, alternatively, the progression of damaging inflammation within the brain.