Generation of VSV pseudotypes using recombinant ΔG-VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines

Vesicular stomatitis virus (VSV) has been widely used to characterize cellular processes, viral resistance, and cytopathogenicity. Recently, VSV has also been used for oncolytic virotherapy due to its capacity to selectively lyse tumor cells. Mutants of the matrix (M) protein of VSV have generally been preferred to the wild-type virus for oncolysis because of their ability to induce type I interferon (IFN) despite causing weaker cytopathic effects. However, due to the large variability of tumor types, it is quite clear that various approaches and combinations of multiple oncolytic viruses will be needed to effectively treat most cancers. With this in mind, our work focused on characterizing the cytopathogenic profiles of four replicative envelope glycoprotein (G) VSV mutants. In contrast to the prototypic M mutant, VSV G mutants are as efficient as wild-type virus at inhibiting cellular transcription and host protein translation. Despite being highly cytopathic, the mutant G 6R triggers type I interferon secretion as efficiently as the M mutant. Importantly, most VSV G mutants are more effective at killing B16 and MC57 tumor cells in vitro than the M mutant or wild-type virus through apoptosis induction. Taken together, our results demonstrate that VSV G mutants retain the high cytopathogenicity of wild-type VSV, with G 6R inducing type I IFN secretion at levels similar to that of the M mutant. VSV G protein mutants could therefore prove to be highly valuable for the development of novel oncolytic virotherapy strategies that are both safe and efficient for the treatment of various types of cancer.

Section: Discussionmentioning

confidence: 99%

Mutations in the Glycoprotein of Vesicular Stomatitis Virus Affect Cytopathogenicity: Potential for Oncolytic Virotherapy

Janelle

Brassard

Lapierre

et al. 2011

“…The ability to recover fully replicationcompetent VSV from suitably engineered plasmid DNA (3,4) has enabled the generation of modified recombinant versions of VSV (rVSV), some of which are currently under active investigation for their therapeutic potential as replicating or nonreplicating vaccine vectors (5)(6)(7)(8) and as oncolytic agents for the treatment of a number of different types of human cancer (9)(10)(11)(12).…”

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confidence: 99%

Highly Attenuated Recombinant Vesicular Stomatitis Virus VSV-12′GFP Displays Immunogenic and Oncolytic Activity

Pol

Davis

2013

Vesicular stomatitis virus (VSV) has shown considerable promise both as an immunization vector and as an oncolytic virus. In both applications, an important concern is the safety profile of the virus. To generate a highly attenuated virus, we added two reporter genes to the 3= end of the VSV genome, thereby shifting the NPMGL genes from positions 1 to 5 to positions 3 to 7. The resulting virus (VSV-12=GFP) was highly attenuated, generating smaller plaques than four other attenuated VSVs. In one-step growth curves, VSV-12=GFP displayed the slowest growth kinetics. The mechanism of attenuation appears to be due to reduced expression of VSV genes downstream of the reporter genes, as suggested by a 10.4-fold reduction in L-protein RNA transcript. Although attenuated, VSV-12=GFP was highly effective at generating an immune response, indicated by a high-titer antibody response against the green fluorescent protein (GFP) expressed by the virus. Although VSV-12=GFP was more attenuated than other VSVs on both normal and cancer cells, it nonetheless showed a greater level of infection of human cancer cells (glioma and melanoma) than of normal cells, and this effect was magnified in glioma by interferon application, indicating selective oncolysis. Intravenous VSV-12=GFP selectively infected human gliomas implanted into SCID mice subcutaneously or intracranially. All postnatal day 16 mice given intranasal VSV-12=GFP survived, whereas only 10% of those given VSV-G/GFP survived, indicating reduced neurotoxicity. Intratumoral injection of tumors with VSV-12=GFP dramatically suppressed tumor growth and enhanced survival. Together these data suggest this recombinant virus merits further study for its oncolytic and vaccine potential. V esicular stomatitis virus (VSV) is an enveloped nonsegmented negative-strand RNA virus of the Rhabdoviridae family with a simply organized genome of 11.2 kb that encodes just five genes (N, P, M, G, and L) (1, 2). The ability to recover fully replicationcompetent VSV from suitably engineered plasmid DNA (3, 4) has enabled the generation of modified recombinant versions of VSV (rVSV), some of which are currently under active investigation for their therapeutic potential as replicating or nonreplicating vaccine vectors (5-8) and as oncolytic agents for the treatment of a number of different types of human cancer (9-12).In nature, VSV is a pathogen of livestock, such as horses, cattle, and swine, with infection of humans being relatively rare and resulting typically in subclinical or mild flu-like symptoms (13,14). Although encephalitis is not a characteristic of natural VSV infection (13), experimental infection of brain cells has been found in animal models (15)(16)(17)(18). We have previously shown that the use of recombinant attenuated VSV and peripheral immunization reduced or blocked the ability of VSV to infect central nervous system (CNS) cells (12,19). Further refinement of recombinant VSVs for therapeutic application, particularly within the brain, may benefit from additional viral attenuati...

“…To ascertain if PCBP2 affects VSV growth at the level of virus budding, we utilized VSV-PeGFP-⌬G virus, which lacks the G coding sequence in the viral genome. Infectious ⌬G viruses can be generated from transfected cells by complementing with G protein (65). Since VSV G protein is required for virus budding and infectivity (8,43), the ⌬G virus is defective in producing infectious progeny virions and undergoes only a single round of infection.…”

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confidence: 99%

Antagonistic Effects of Cellular Poly(C) Binding Proteins on Vesicular Stomatitis Virus Gene Expression

Dinh

Beura

Panda³

et al. 2011

Immunoprecipitation and subsequent mass spectrometry analysis of the cellular proteins from cells expressing the vesicular stomatitis virus (VSV) P protein identified the poly(C) binding protein 2 (PCBP2) as one of the P protein-interacting proteins. To investigate the role of PCBP2 in the viral life cycle, we examined the effects of depletion or overexpression of this protein on VSV growth. Small interfering RNA-mediated silencing of PCBP2 promoted VSV replication. Conversely, overexpression of PCBP2 in transfected cells suppressed VSV growth. Further studies revealed that PCBP2 negatively regulates overall viral mRNA accumulation and subsequent genome replication. Coimmunoprecipitation and immunofluorescence microscopic studies showed that PCBP2 interacts and colocalizes with VSV P protein in virus-infected cells. The P-PCBP2 interaction did not result in reduced levels of protein complex formation with the viral N and L proteins, nor did it induce degradation of the P protein. In addition, PCBP1, another member of the poly(C) binding protein family with homology to PCBP2, was also found to interact with the P protein and inhibit the viral mRNA synthesis at the level of primary transcription without affecting secondary transcription or genome replication. The inhibitory effects of PCBP1 on VSV replication were less pronounced than those of PCBP2. Overall, the results presented here suggest that cellular PCBP2 and PCBP1 antagonize VSV growth by affecting viral gene expression and highlight the importance of these two cellular proteins in restricting virus infections. Vesicular stomatitis virus (VSV)is an enveloped RNA virus in the family Rhabdoviridae. The negative-sense RNA genome of VSV is 11,161 nucleotides in length and encodes 5 proteins: the nucleocapsid protein (N), the phosphoprotein (P), the matrix (M), the glycoprotein (G), and the large protein (L). Inside the virion, the viral genome is tightly encapsidated with the N protein to form the ribonucleocapsid complex (RNP or NC) with which the viral RNA-dependent RNA polymerase (RdRp) complex of P-L proteins (41) is associated. The G protein forms spikes on the surface of the VSV virion and mediates virus attachment as well as entry by clathrin-mediated endocytosis. Based on the low-pH environment of the endosome, the VSV G protein mediates fusion of the viral envelope with the endosomal membrane, releasing the viral RNP into the cytoplasm of the infected cell. Once inside the cytosol, the RNP undergoes primary transcription by the associated RdRp activity of the L protein along with its cofactor P protein. Translation of the transcripts by the cellular translation machinery generates viral proteins, which are used for further steps in the infection process, including genome replication, secondary transcription, assembly, and budding (53).The viral P is a multifunctional protein that modulates the association of viral replicative proteins and is indispensable for viral growth. It is required for viral genome transcription and replication (18) and also pl...