Valery Z. Grdzelishvili

Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus

Positive-strand RNA viruses are the largest virus class and include many pathogens such as hepatitis C virus and the severe acute respiratory syndrome coronavirus (SARS). Brome mosaic virus (BMV) is a representative positive-strand RNA virus whose RNA replication, gene expression, and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. By using traditional yeast genetics, host genes have been identified that function in controlling BMV translation, selecting BMV RNAs as replication templates, activating the replication complex, maintaining a lipid composition required for membrane-associated RNA replication, and other steps. To more globally and systematically identify such host factors, we used engineered BMV derivatives to assay viral RNA replication in each strain of an ordered, genome-wide set of yeast single-gene deletion mutants. Each deletion strain was transformed to express BMV replicase proteins and a BMV RNA replication template with the capsid gene replaced by a luciferase reporter. Luciferase expression, which is dependent on viral RNA replication and RNA-dependent mRNA synthesis, was measured in intact yeast cells. Approximately 4,500 yeast deletion strains (≈80% of yeast genes) were screened in duplicate and selected strains analyzed further. This functional genomics approach revealed nearly 100 genes whose absence inhibited or stimulated BMV RNA replication and/or gene expression by 3- to >25-fold. Several of these genes were shown previously to function in BMV replication, validating the approach. Newly identified genes include some in RNA, protein, or membrane modification pathways and genes of unknown function. The results further illuminate virus and cell pathways. Further refinement of virus screening likely will reveal contributions from additional host genes.

Vesicular stomatitis virus as a flexible platform for oncolytic virotherapy against cancer

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.

A Single Amino Acid Change in the L-Polymerase Protein of Vesicular Stomatitis Virus Completely Abolishes Viral mRNA Cap Methylation

ABSTRACT The vesicular stomatitis virus (VSV) RNA polymerase synthesizes viral mRNAs with 5′-cap structures methylated at the guanine-N7 and 2′-O-adenosine positions (7mGpppAm). 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 L-polymerase 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.

RESISTANCE OF PANCREATIC CANCER CELLS TO ONCOLYTIC VESICULAR STOMATITIS VIRUS: ROLE OF TYPE I INTERFERON SIGNALING

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.

Characterization of retinoic acid-inducible gene-I expression in primary murine glia following exposure to vesicular stomatitis virus.

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, we 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, we demonstrate that microglia and astrocytes constitutively express both RIG-I and MDA5 transcripts and protein. Importantly, we 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.

Vesicular Stomatitis Virus as an Oncolytic Agent against Pancreatic Ductal Adenocarcinoma

ABSTRACT Vesicular 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-ΔM51 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.

Vesicular stomatitis virus infects resident cells of the central nervous system and induces replication-dependent inflammatory responses.

Vesicular stomatitis virus (VSV) infection of mice via intranasal administration results in a severe encephalitis with rapid activation and proliferation of microglia and astrocytes. We have recently shown that these glial cells express RIG-I and MDA5, cytosolic pattern recognition receptors for viral RNA. However, it is unclear whether VSV can replicate in glial cells or if such replication is required for their inflammatory responses. Here we demonstrate that primary microglia and astrocytes are permissive for VSV infection and limited productive replication. Importantly, we show that viral replication is required for robust inflammatory mediator production by these cells. Finally, we have confirmed that in vivo VSV administration can result in viral infection of glial cells in situ. These results suggest that viral replication within resident glial cells might play an important role in CNS inflammation following infection with VSV and possibly other neurotropic nonsegmented negative-strand RNA viruses.

RIG-I mediates nonsegmented negative-sense RNA virus-induced inflammatory immune responses of primary human astrocytes.

While astrocytes produce key inflammatory mediators following exposure to neurotropic nonsegmented negative‐sense RNA viruses such as rabies virus and measles virus, the mechanisms by which resident central nervous system (CNS) cells perceive such viral challenges have not been defined. Recently, several cytosolic DExD/H box RNA helicases including retinoic acid‐inducible gene I (RIG‐I) have been described that function as intracellular sensors of replicative RNA viruses. Here, we demonstrate that primary human astrocytes constitutively express RIG‐I and show that such expression is elevated following exposure to a model neurotropic RNA virus, vesicular stomatitis virus (VSV). Evidence for the functional nature of RIG‐I expression in these cells comes from the observation that this molecule associates with its downstream effector molecule, interferon promoter stimulator‐1, following VSV infection and from the finding that a specific ligand for RIG‐I elicits astrocyte immune responses. Importantly, RIG‐I knockdown significantly reduces inflammatory cytokine production by VSV‐infected astrocytes and inhibits the production of soluble neurotoxic mediators by these cells. These findings directly implicate RIG‐I in the initiation of inflammatory immune responses by human glial cells and provide a potential mechanism underlying the neuronal cell death associated with acute viral CNS infections.

The use of a novel MUC1 antibody to identify cancer stem cells and circulating MUC1 in mice and patients with pancreatic cancer.

MUC1 is over‐expressed and aberrantly glycosylated in >60% of human pancreatic cancer (PC). Development of novel approaches for detection and/or targeting of MUC1 are critically needed and should be able to detect MUC1 on PC cells (including cancer stem cells) and in serum.

Cell Type Mediated Resistance of Vesicular Stomatitis Virus and Sendai Virus to Ribavirin

Ribavirin (RBV) is a synthetic nucleoside analog with broad spectrum antiviral activity. Although RBV is approved for the treatment of hepatitis C virus, respiratory syncytial virus, and Lassa fever virus infections, its mechanism of action and therapeutic efficacy remains highly controversial. Recent reports show that the development of cell-based resistance after continuous RBV treatment via decreased RBV uptake can greatly limit its efficacy. Here, we examined whether certain cell types are naturally resistant to RBV even without prior drug exposure. Seven different cell lines from various host species were compared for RBV antiviral activity against two nonsegmented negative-strand RNA viruses, vesicular stomatitis virus (VSV, a rhabdovirus) and Sendai virus (SeV, a paramyxovirus). Our results show striking differences between cell types in their response to RBV, ranging from virtually no antiviral effect to very effective inhibition of viral replication. Despite differences in viral replication kinetics for VSV and SeV in the seven cell lines, the observed pattern of RBV resistance was very similar for both viruses, suggesting that cellular rather than viral determinants play a major role in this resistance. While none of the tested cell lines was defective in RBV uptake, dramatic variations were observed in the long-term accumulation of RBV in different cell types, and it correlated with the antiviral efficacy of RBV. While addition of guanosine neutralized RBV only in cells already highly resistant to RBV, actinomycin D almost completely reversed the RBV effect (but not uptake) in all cell lines. Together, our data suggest that RBV may inhibit the same virus via different mechanisms in different cell types depending on the intracellular RBV metabolism. Our results strongly point out the importance of using multiple cell lines of different origin when antiviral efficacy and potency are examined for new as well as established drugs in vitro.