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1.  Prophylactic Alpha Interferon Treatment Increases the Therapeutic Index of Oncolytic Vesicular Stomatitis Virus Virotherapy for Advanced Hepatocellular Carcinoma in Immune-Competent Rats 
Journal of Virology  2005;79(21):13705-13713.
Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with intrinsic oncolytic specificity due to substantially attenuated antiviral responses in many tumors. We have recently reported that recombinant VSV vector can be used as an effective oncolytic agent to safely treat multifocal hepatocellular carcinoma (HCC) in the livers of immune-competent rats via hepatic artery infusion. When administered at doses above the maximum tolerated dose (MTD), however, the animals suffered from neurotoxicity and/or acute lethal hepatotoxicity. Since VSV is extremely sensitive to the antiviral actions of alpha/beta interferon (IFN-α/β) in normal cells, we tested if prophylactic treatment with rat IFN-α would enhance VSV safety without compromising treatment efficacy in tumor-bearing rats. We found that VSV retained its replication potential in human and rat HCC cells after preincubation with relatively high doses of rat and human IFN-α in vitro, and its MTD in tumor-bearing rats treated systemically with rat IFN-α at 66 IU/g body weight (BW), equivalent to a human IFN-α dose that is currently prescribed for patients with viral hepatitis, was elevated by more than 1/2 log unit. Furthermore, we demonstrate that intratumoral replication of VSV was not attenuated by administration of 66 IU/g BW rat IFN-α, as tumor response and survival advantage in VSV-treated rats in the presence or absence of rat IFN-α were equivalent. The results suggest that prophylactic rat IFN-α treatment elevates the therapeutic index of hepatic arterial VSV therapy for multifocal HCC in rats. Since human IFN-α is currently in clinical use, its prophylactic application should be considered in future clinical translational protocols for VSV-mediated oncolytic virotherapy as a novel therapeutic modality in patients with advanced HCC, as well as other types of cancer.
doi:10.1128/JVI.79.21.13705-13713.2005
PMCID: PMC1262571  PMID: 16227290
2.  Expression of Interferon-β Enhances Both Efficacy and Safety of Oncolytic Vesicular Stomatitis Virus for Therapy of Mesothelioma 
Cancer research  2009;69(19):7713-7720.
Our pre-clinical and clinical trials using a replication-defective adenoviral vector expressing IFN-β have shown promising results for the treatment of malignant mesothelioma. Based on the hypotheses that a replication-competent Vesicular Stomatitis Virus (VSV) oncolytic vector would transduce more tumor cells in vivo, that co-expression of the immunostimulatory IFN-β gene would enhance the immune-based effector mechanisms associated both with regression of mesotheliomas and with VSV-mediated virotherapy, and that virus-derived IFN-β would add further safety to the VSV platform, we tested the use of IFN-β as a therapeutic transgene expressed from VSV as a novel treatment for mesothelioma. VSV-IFN-β showed significant therapy against AB12 murine mesotheliomas in the context of both local and loco-regional viral delivery. Biologically active IFN-β expressed from VSV added significantly to therapy compared to VSV alone, dependent in part upon host CD8+ T-cell responses. Immune monitoring suggested that these anti-tumor T-cell responses may be due to a generalised T-cell activation rather than the priming of tumor antigen-specific T-cell responses. Finally, IFN-β also added considerable extra safety to the virus by providing protection from off-target viral replication in non-tumor tissues and protected SCID mice from developing lethal neurotoxicity. The enhanced therapeutic index provided by the addition of IFN-β to VSV therefore provides a powerful justification for the development of this virus for future clinical trials.
doi:10.1158/0008-5472.CAN-09-1013
PMCID: PMC3891512  PMID: 19773437
VSV; interferon-β; mesothelioma; oncolytic virotherapy
3.  Development of Recombinant Vesicular Stomatitis Viruses That Exploit Defects in Host Defense To Augment Specific Oncolytic Activity 
Journal of Virology  2003;77(16):8843-8856.
Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus normally sensitive to the antiviral actions of alpha/beta interferon (IFN-α/β). Recently, we reported that VSV replicates to high levels in many transformed cells due, in part, to susceptible cells harboring defects in the IFN system. These observations were exploited to demonstrate that VSV can be used as a viral oncolytic agent to eradicate malignant cells in vivo while leaving normal tissue relatively unaffected. To attempt to improve the specificity and efficacy of this system as a potential tool in gene therapy and against malignant disease, we have genetically engineered VSV that expresses the murine IFN-β gene. The resultant virus (VSV-IFNβ) was successfully propagated in cells not receptive to murine IFN-α/β and expressed high levels of functional heterologous IFN-β. In normal murine embryonic fibroblasts (MEFs), the growth of VSV-IFNβ was greatly reduced and diminished cytopathic effect was observed due to the production of recombinant IFN-β, which by functioning in a manner involving autocrine and paracrine mechanisms induced an antiviral effect, preventing virus growth. However, VSV-IFNβ grew to high levels and induced the rapid apoptosis of transformed cells due to defective IFN pathways being prevalent and thus unable to initiate proficient IFN-mediated host defense. Importantly, VSV expressing the human IFN-β gene (VSV-hIFNβ) behaved comparably and, while nonlytic to normal human cells, readily killed their malignant counterparts. Similar to our in vitro observations, following intravenous and intranasal inoculation in mice, recombinant VSV (rVSV)-IFNβ was also significantly attenuated compared to wild-type VSV or rVSV expressing green fluorescent protein. However, VSV-IFNβ retained propitious oncolytic activity against metastatic lung disease in immunocompetent animals and was able to generate robust antitumor T-cell responses. Our data indicate that rVSV designed to exploit defects in mechanisms of host defense can provide the basis for new generations of effective, specific, and safer viral vectors for the treatment of malignant and other disease.
doi:10.1128/JVI.77.16.8843-8856.2003
PMCID: PMC167243  PMID: 12885903
4.  Antiviral protection by vesicular stomatitis virus-specific antibodies in alpha/beta interferon receptor-deficient mice. 
Journal of Virology  1995;69(4):2153-2158.
The role of innate, alpha/beta interferon (IFN-alpha/beta)-dependent protection versus specific antibody-mediated protection against vesicular stomatitis virus (VSV) was evaluated in IFN-alpha/beta receptor-deficient mice (IFN-alpha/beta R0/0 mice). VSV is a close relative to rabies virus that causes neurological disease in mice. In contrast to normal mice, IFN-alpha/beta R0/0 mice were highly susceptible to infection with VSV because of ubiquitous high viral replication. Adoptive transfer experiments showed that neutralizing antibodies against the glycoprotein of VSV (VSV-G) protected these mice efficiently against systemic infection and against peripheral subcutaneous infection but protected only to a limited degree against intranasal infection with VSV. In contrast, VSV-specific T cells or antibodies specific for the nucleoprotein of VSV (VSV-N) were unable to protect IFN-alpha/beta R0/0 mice against VSV. These results demonstrate that mice are extremely sensitive to VSV if IFN-alpha/beta is not functional and that under these conditions, neutralizing antibody responses mediate efficient protection, but apparently only against extraneuronal infection.
PMCID: PMC188883  PMID: 7884863
5.  Variable Deficiencies in the Interferon Response Enhance Susceptibility to Vesicular Stomatitis Virus Oncolytic Actions in Glioblastoma Cells but Not in Normal Human Glial Cells▿  
Journal of Virology  2006;81(3):1479-1491.
With little improvement in the poor prognosis for humans with high-grade glioma brain tumors, alternative therapeutic strategies are needed. As such, selective replication-competent oncolytic viruses may be useful as a potential treatment modality. Here we test the hypothesis that defects in the interferon (IFN) pathway could be exploited to enhance the selective oncolytic profile of vesicular stomatitis virus (VSV) in glioblastoma cells. Two green fluorescent protein-expressing VSV strains, recombinant VSV and the glioma-adapted recombinant VSV-rp30a, were used to study infection of a variety of human glioblastoma cell lines compared to a panel of control cells, including normal human astrocytes, oligodendrocyte precursor cells, and primary explant cultures from human brain tissue. Infection rate, cell viability, viral replication, and IFN-α/β-related gene expression were compared in the absence and presence of IFN-α or polyriboinosinic polyribocytidylic acid [poly(I:C)], a synthetic inducer of the IFN-α/β pathway. Both VSV strains caused rapid and total infection and death of all tumor cell lines tested. To a lesser degree, normal cells were also subject to VSV infection. In contrast, IFN-α or poly(I:C) completely attenuated the infection of all primary control brain cells, whereas most glioblastoma cell lines treated with IFN-α or poly(I:C) showed little or no sign of protection and were killed by VSV. Together, our results demonstrate that activation of the interferon pathway protects normal human brain cells from VSV infection while maintaining the vulnerability of human glioblastoma cells to viral destruction.
doi:10.1128/JVI.01861-06
PMCID: PMC1797501  PMID: 17108037
6.  RESISTANCE OF PANCREATIC CANCER CELLS TO ONCOLYTIC VESICULAR STOMATITIS VIRUS: ROLE OF TYPE I INTERFERON SIGNALING 
Virology  2012;436(1):221-234.
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.
doi:10.1016/j.virol.2012.11.014
PMCID: PMC3544977  PMID: 23246628
7.  Variation in Susceptibility of Human Malignant Melanomas to Oncolytic Vesicular Stomatitis Virus 
Surgery  2012;153(3):333-343.
Background
Vesicular stomatitis virus (VSV) is a novel, anti-cancer therapy that selectively targets cancer cells with defective antiviral responses; however, not all malignant cells are sensitive to the oncolytic effects of VSV. Herein, we explore the mechanistic determinants of mutant M protein VSV (M51R-VSV) susceptibility in malignant melanoma cells.
Methods
Cell viability after VSV infection was measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) viability assay in a panel of melanoma cell lines. VSV infectability, viral protein synthesis and viral progeny production were quantified by flow cytometry, 35S-methionine electrophoresis, and viral plaque assays, respectively. Interferon (IFN) responsiveness was determined using MTS assay after β-IFN pre-treatment. Xenografts were established in athymic nude mice and treated with intratumoral M51R-VSV.
Results
Cell viability after M51R-VSV infection at a multiplicity of infection (MOI) of 10 pfu/mL, 48 hours post-infection) ranged between 0±1 and 59±9% (mean ± standard deviation). Sensitive cell lines supported VSV infection, viral protein synthesis, and viral progeny production. In addition, when pre-treated with β-IFN, sensitive cells became resistant to M51R-VSV, suggesting that IFN-mediated antiviral signaling is defective in these cells. In contrast, resistant melanoma cells do not support VSV infection, viral protein synthesis, or viral replication, indicating that anti-viral defenses remain intact. In a murine xenograft model, intratumoral M51R-VSV treatment decreased tumor growth relative to controls after 26 days in SK-Mel 5 (−21±19% vs. 2100±770%, p<0.0001) and SK-Mel 3 (2000±810% vs 7000±3000%, p=0.008) established tumors.
Conclusions
M51R-VSV is a viable, anti-cancer therapy, but susceptibility varies among melanomas. Future work will exploit specific mechanisms of resistance to expand the therapeutic efficacy of M51R-VSV.
doi:10.1016/j.surg.2012.09.003
PMCID: PMC3561511  PMID: 23102637
8.  Vesicular stomatitis virus modified with single chain IL-23 exhibits oncolytic activity against tumor cells in vitro and in vivo 
Viruses are potentially attractive agents for development as novel oncolytic agents. Reverse genetic approaches allow for the attenuation of candidate viruses and can enhance their ability to exploit inherent cellular and molecular properties of tumors, including deficiencies in interferon (IFN) signaling. Vesicular stomatitis virus (VSV) is a promising oncolytic agent for exactly these reasons. VSV infection of immunocompetent mice is usually rapidly cleared due to the virus’ sensitivity to type I IFN responses. However, in tumors that are unable to activate the IFN response, VSV is able to replicate without inhibition, resulting in cell destruction. Unfortunately, when VSV is introduced into mice intranasally or systemically via therapeutic doses into tumor-bearing rodents, hosts may develop fatal encephalitis. We have previously found that a recombinant VSV expressing the pro-inflammatory cytokine interleukin-23 (IL-23) is significantly attenuated in the central nervous system (CNS). As a result of this, we hypothesized that attenuation in the CNS is partially a result of enhanced NO response as a result of IL-23 signaling. Infection of the CNS with this virus (designated VSV23) is characterized by decreased viral replication, morbidity, and mortality. We have now extended those studies which reveal that VSV23 maintains oncolytic capacity in vitro in multiple cell lines including NB41A3 neuroblastomas, L929 adipose-derived cells, immortalized BHK-21 cells, and the murine mammary derived JC cells. Additionally, in vivo VSV23 infection results in JC tumor destruction and induces enhanced memory responses against tumor cells.
PMCID: PMC2885733  PMID: 20556219
oncolysis; IL-23; vesicular stomatitis virus; tumor cells; oncolytic activity
9.  Mutations in the Glycoprotein of Vesicular Stomatitis Virus Affect Cytopathogenicity: Potential for Oncolytic Virotherapy▿§ 
Journal of Virology  2011;85(13):6513-6520.
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 G6R 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 G6R 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.
doi:10.1128/JVI.02484-10
PMCID: PMC3126483  PMID: 21561919
10.  A Confocal and Electron Microscopic Comparison of Interferon β–Induced Changes in Vesicular Stomatitis Virus Infection of Neuroblastoma and Nonneuronal Cells 
DNA and cell biology  2010;29(3):103-120.
Vesicular stomatitis virus (VSV) replication is highly sensitive to interferon (IFN)-induced antiviral responses. Pretreatment of sensitive cultured cells with IFNβ results in a 104-fold reduction in the release of infectious VSV particles. However, differences exist between the mechanisms of reduced infectious particle titers in cell lines of neuroblastoma and nonneuronal lineage. In L929-fibroblast-derived cells, using immunofluorescence confocal microscopy, infection under control conditions reveals the accumulation of VSV matrix, phosphoprotein (P), and nucleocapsid (N) proteins over time, with induced cellular morphological changes indicative of cytopathic effects (CPEs). Upon observing L929 cells that had been pretreated with IFNβ, neither detectable VSV proteins nor CPEs were seen, consistent with type I IFN antiviral protection. When using the same techniques to observe VSV infections of NB41A3 cells, a neuroblastoma cell line, aside from similar viral progression in the untreated control cells, IFNβ-treated cells illustrated a severely attenuated VSV infection. Attenuated VSV progression was observed through detection of VSV matrix, P, and N proteins in isolated cells during the first 8 h of infection. However, by 18–24 h postinfection all neuroblastomas had succumbed to the viral infection. Finally, upon closer inspection of IFNβ-treated NB41A3 cells, no detectable changes in VSV protein localization were identified compared with untreated, virally infected neuroblastomas. Next, to extend our study to test our hypothesis that virion assembly is compromised within type I IFN-treated neuroblastoma cells we employed electron microscopy to examine our experimental conditions at the ultrastructural level. Using VSV-specific antibodies in conjunction with immuno-gold reagents, we observed several similarities between the two cell lines, such as identification of viroplasmic regions containing VSV N and P proteins and signs of stress-induced CPEs of VSV-infected cells, which had either been mock-treated or pretreated with interferon-β (IFNβ). One difference we observed between nonneuronal and neuroblastoma cells was more numerous actively budding VSV virions across untreated L929 plasma membranes, compared with untreated NB41A3 cells. Additionally, IFNβ-treated, VSV-infected L929 cells exhibited neither cytoplasmic viroplasm nor viral protein expression. In contrast, IFNβ-treated, VSV-infected NB41A3 cells showed evidence of VSV infection at a very low frequency as well as small-scale viroplasmic regions that colocalized with viral N and P proteins. Finally, we observed that VSV viral particles harvested from untreated VSV-infected L929 and NB41A3 cells were statistically similar in size and shape. A portion of VSV virions from IFNβ-treated, virally infected NB41A3 cells were similar in size and shape to virus from both untreated cell types. However, among the sampling of virions, pleomorphic viral particles that were identified from IFNβ-treated, VSV-infected NB41A3 cells were different enough to suggest a mis-assembly mechanism as part of the IFNβ antiviral state in neuroblastoma cells.
doi:10.1089/dna.2009.0963
PMCID: PMC2833218  PMID: 20113203
11.  A Confocal and Electron Microscopic Comparison of Interferon β–Induced Changes in Vesicular Stomatitis Virus Infection of Neuroblastoma and Nonneuronal Cells 
DNA and Cell Biology  2010;29(3):103-120.
Vesicular stomatitis virus (VSV) replication is highly sensitive to interferon (IFN)-induced antiviral responses. Pretreatment of sensitive cultured cells with IFNβ results in a 104-fold reduction in the release of infectious VSV particles. However, differences exist between the mechanisms of reduced infectious particle titers in cell lines of neuroblastoma and nonneuronal lineage. In L929-fibroblast-derived cells, using immunofluorescence confocal microscopy, infection under control conditions reveals the accumulation of VSV matrix, phosphoprotein (P), and nucleocapsid (N) proteins over time, with induced cellular morphological changes indicative of cytopathic effects (CPEs). Upon observing L929 cells that had been pretreated with IFNβ, neither detectable VSV proteins nor CPEs were seen, consistent with type I IFN antiviral protection. When using the same techniques to observe VSV infections of NB41A3 cells, a neuroblastoma cell line, aside from similar viral progression in the untreated control cells, IFNβ-treated cells illustrated a severely attenuated VSV infection. Attenuated VSV progression was observed through detection of VSV matrix, P, and N proteins in isolated cells during the first 8 h of infection. However, by 18–24 h postinfection all neuroblastomas had succumbed to the viral infection. Finally, upon closer inspection of IFNβ-treated NB41A3 cells, no detectable changes in VSV protein localization were identified compared with untreated, virally infected neuroblastomas. Next, to extend our study to test our hypothesis that virion assembly is compromised within type I IFN-treated neuroblastoma cells, we employed electron microscopy to examine our experimental conditions at the ultrastructural level. Using VSV-specific antibodies in conjunction with immuno-gold reagents, we observed several similarities between the two cell lines, such as identification of viroplasmic regions containing VSV N and P proteins and signs of stress-induced CPEs of VSV-infected cells, which had either been mock-treated or pretreated with interferon-β (IFNβ). One difference we observed between nonneuronal and neuroblastoma cells was more numerous actively budding VSV virions across untreated L929 plasma membranes compared with untreated NB41A3 cells. Additionally, IFNβ-treated, VSV-infected L929 cells exhibited neither cytoplasmic viroplasm nor viral protein expression. In contrast, IFNβ-treated, VSV-infected NB41A3 cells showed evidence of VSV infection at a very low frequency as well as small-scale viroplasmic regions that colocalized with viral N and P proteins. Finally, we observed that VSV viral particles harvested from untreated VSV-infected L929 and NB41A3 cells were statistically similar in size and shape. A portion of VSV virions from IFNβ-treated, virally infected NB41A3 cells were similar in size and shape to virus from both untreated cell types. However, among the sampling of virions, pleomorphic viral particles that were identified from IFNβ-treated, VSV-infected NB41A3 cells were different enough to suggest a misassembly mechanism as part of the IFNβ antiviral state in neuroblastoma cells.
doi:10.1089/dna.2009.0963
PMCID: PMC2833218  PMID: 20113203
12.  Pseudotyping Vesicular Stomatitis Virus with Lymphocytic Choriomeningitis Virus Glycoproteins Enhances Infectivity for Glioma Cells and Minimizes Neurotropism▿† 
Journal of Virology  2011;85(11):5679-5684.
Vesicular stomatitis virus (VSV)-based oncolytic virotherapy has the potential to significantly improve the prognosis of aggressive malignancies such as brain cancer. However, VSV's inherent neurotoxicity has hindered clinical development so far. Given that this neurotropism is attributed to the glycoprotein VSV-G, VSV was pseudotyped with the nonneurotropic envelope glycoprotein of the lymphocytic choriomeningitis virus (LCMV-GP→VSV-GP). Compared to VSV, VSV-GP showed enhanced infectivity for brain cancer cells in vitro while sparing primary human and rat neurons in vitro and in vivo, respectively. In conclusion, VSV-GP has a much wider therapeutic window than VSV and is thus more suitable for clinical applications, especially in the brain.
doi:10.1128/JVI.02511-10
PMCID: PMC3094995  PMID: 21450833
13.  IFN-β-Induced Alteration of VSV Protein Phosphorylation in Neuronal Cells 
Viral immunology  2009;22(6):353-369.
Vesicular stomatitis virus (VSV) replication is highly sensitive to interferon (IFN)-induced antiviral responses. VSV infection of well-known cell lines pretreated with IFN-β results in a 104-fold reduction in the release of infectious particles, with a concomitant abrogation in viral transcript and/or protein levels. However, in cell lines of neuronal lineage only a threefold reduction in viral transcript and protein levels was observed, despite the same 104-fold reduction in released infectious virions, suggesting an assembly defect. Examination of VSV matrix (M) protein ubiquitination yielded no differences between mock- and IFN-β-treated neuronal cells. Further analysis of potential post-translational modification events, by scintillation and two-dimensional electrophoretic methods, revealed IFN-β-induced alterations in M protein and phosphoprotein (P) phosphorylation. Hypophosphorylated P protein was demonstrated by reduced 32P counts, normalized by 35S-cysteine/methionine incorporation, and by a shift in isoelectric focusing. Hypophosphorylation of VSV P protein was found to occur in neuronal cell lysates, but not within budded virions from the same IFN-β-treated cells. In contrast, hyperphosphorylation of VSV M protein was observed in both cell lysates and viral particles from IFN-β-treated neuronal cells. Hyperphosphorylated M protein was demonstrated by increased 32P counts relative to 35S-cysteine/methionine normalization, and by altered isoelectric focusing in protein populations from cell and viral lysates. Hyperphosphorylated VSV M protein was found to inhibit its association with VSV nucleocapsid, suggesting a possible mechanism for type I IFN-mediated misassembly through disruption of the interactions between ribonucleoprotein cores, and hyperphosphorylated M protein bound to the plasma membrane inner leaflet.
doi:10.1089/vim.2009.0057
PMCID: PMC2812813  PMID: 19951173
14.  IFN-β-Induced Alteration of VSV Protein Phosphorylation in Neuronal Cells 
Viral Immunology  2009;22(6):353-369.
Abstract
Vesicular stomatitis virus (VSV) replication is highly sensitive to interferon (IFN)-induced antiviral responses. VSV infection of well-known cell lines pretreated with IFN-β results in a 104-fold reduction in the release of infectious particles, with a concomitant abrogation in viral transcript and/or protein levels. However, in cell lines of neuronal lineage only a threefold reduction in viral transcript and protein levels was observed, despite the same 104-fold reduction in released infectious virions, suggesting an assembly defect. Examination of VSV matrix (M) protein ubiquitination yielded no differences between mock- and IFN-β-treated neuronal cells. Further analysis of potential post-translational modification events, by scintillation and two-dimensional electrophoretic methods, revealed IFN-β-induced alterations in M protein and phosphoprotein (P) phosphorylation. Hypophosphorylated P protein was demonstrated by reduced 32P counts, normalized by 35S-cysteine/methionine incorporation, and by a shift in isoelectric focusing. Hypophosphorylation of VSV P protein was found to occur in neuronal cell lysates, but not within budded virions from the same IFN-β-treated cells. In contrast, hyperphosphorylation of VSV M protein was observed in both cell lysates and viral particles from IFN-β-treated neuronal cells. Hyperphosphorylated M protein was demonstrated by increased 32P counts relative to 35S-cysteine/methionine normalization, and by altered isoelectric focusing in protein populations from cell and viral lysates. Hyperphosphorylated VSV M protein was found to inhibit its association with VSV nucleocapsid, suggesting a possible mechanism for type I IFN-mediated misassembly through disruption of the interactions between ribonucleoprotein cores, and hyperphosphorylated M protein bound to the plasma membrane inner leaflet.
doi:10.1089/vim.2009.0057
PMCID: PMC2812813  PMID: 19951173
15.  Oncolytic Vesicular Stomatitis Virus in an Immunocompetent Model of MUC1-Positive or MUC1-Null Pancreatic Ductal Adenocarcinoma 
Journal of Virology  2013;87(18):10283-10294.
Vesicular stomatitis virus (VSV) is a promising oncolytic agent against various malignancies. Here, for the first time, we tested VSV in vitro and in vivo in a clinically relevant, immunocompetent mouse model of pancreatic ductal adenocarcinoma (PDA). Our system allows the study of virotherapy against PDA in the context of overexpression (80% of PDA patients) or no expression of human mucin 1 (MUC1), a major marker for poor prognosis in patients. In vitro, we tested three VSV recombinants, wild-type VSV, VSV-green fluorescent protein (VSV-GFP), and a safe oncolytic VSV-ΔM51-GFP, against five mouse PDA cell lines that either expressed human MUC1 or were MUC1 null. All viruses demonstrated significant oncolytic abilities independent of MUC1 expression, although VSV-ΔM51-GFP was somewhat less effective in two PDA cell lines. In vivo administration of VSV-ΔM51-GFP resulted in significant reduction of tumor growth for tested mouse PDA xenografts (+MUC1 or MUC1 null), and antitumor efficacy was further improved when the virus was combined with the chemotherapeutic drug gemcitabine. The antitumor effect was transient in all tested groups. The developed system can be used to study therapies involving various oncolytic viruses and chemotherapeutics, with the goal of inducing tumor-specific immunity while preventing premature virus clearance.
doi:10.1128/JVI.01412-13
PMCID: PMC3754007  PMID: 23864625
16.  Some Attenuated Variants of Vesicular Stomatitis Virus Show Enhanced Oncolytic Activity against Human Glioblastoma Cells relative to Normal Brain Cells▿  
Journal of Virology  2009;84(3):1563-1573.
Vesicular stomatitis virus (VSV) has been shown in laboratory studies to be effective against a variety of tumors, including malignant brain tumors. However, attenuation of VSV may be necessary to balance the potential toxicity toward normal cells, particularly when targeting brain tumors. Here we compared 10 recombinant VSV variants resulting from different attenuation strategies. Attenuations included gene shifting (VSV-p1-GFP/RFP), M protein mutation (VSV-M51), G protein cytoplasmic tail truncations (VSV-CT1/CT9), G protein deletions (VSV-dG-GFP/RFP), and combinations thereof (VSV-CT9-M51). Using in vitro viability and replication assays, the VSV variants were grouped into three categories, based on their antitumor activity and non-tumor-cell attenuation. In the first group, wild-type-based VSV-G/GFP, tumor-adapted VSV-rp30, and VSV-CT9 showed a strong antitumor profile but also retained some toxicity toward noncancer control cells. The second group, VSV-CT1, VSV-dG-GFP, and VSV-dG-RFP, had significantly diminished toxicity toward normal cells but showed little oncolytic action. The third group displayed a desired combination of diminished general toxicity and effective antitumor action; this group included VSV-M51, VSV-CT9-M51, VSV-p1-GFP, and VSV-p1-RFP. A member of the last group, VSV-p1-GFP, was then compared in vivo against wild-type-based VSV-G/GFP. Intranasal inoculation of young, postnatal day 16 mice with VSV-p1-GFP showed no adverse neurological effects, whereas VSV-G/GFP was associated with high lethality (80%). Using an intracranial tumor xenograft model, we further demonstrated that attenuated VSV-p1-GFP targets and kills human U87 glioblastoma cells after systemic application. We concluded that some, but not all, attenuated VSV mutants display a favorable oncolytic profile and merit further investigation.
doi:10.1128/JVI.02040-09
PMCID: PMC2812324  PMID: 19906910
17.  Role of de novo protein synthesis in target cells recognized by cytotoxic T lymphocytes specific for vesicular stomatitis virus. 
Journal of Virology  1991;65(12):6856-6861.
The requirements for viral and host protein synthesis in the generation of target antigens for cytotoxic T lymphocytes (CTL) was evaluated by using vesicular stomatitis virus (VSV) inactivated by UV irradiation (UV-VSV). EL4 target cells incubated with UV-VSV were recognized and lysed by anti-VSV CTL, indicating that de novo synthesis of viral proteins was not required for the generation of antigens recognized by antiviral CTL. Anti-VSV CTL from H-2b mice primarily recognize determinants derived from the VSV N protein bound to the class I major histocompatibility complex (MHC) antigen H-2Kb. Comparison of a cloned CTL line representing this specificity and a heterogeneous population of anti-VSV CTL showed that determinants other than that recognized by the cloned CTL were generated more efficiently from UV-VSV. By using vaccinia virus recombinants that express deletion fragments of the N protein, it was shown that these additional determinants were probably derived from VSV proteins other than the N protein. The protein synthesis inhibitor emetine was used to determine whether newly synthesized host proteins were required for antigen generation. The addition of emetine to target cells prior to or at the time of the addition of UV-VSV inhibited lysis by anti-VSV CTL. This inhibition could be due to depletion of newly synthesized MHC molecules from intracellular membranes. This hypothesis was supported by using brefeldin A to delay membrane protein transport in target cells during the time of incubation with emetine and UV-VSV, which resulted in partial reversal of the effect of emetine. These results suggest that newly synthesized class I MHC molecules are required for the generation of antigens recognized by anti-VSV CTL.
PMCID: PMC250782  PMID: 1658379
18.  Sensitivity of Ribonucleic Acid and Deoxyribonucleic Acid Viruses to Different Species of Interferon in Cell Cultures 
Journal of Virology  1972;10(2):171-178.
Although two deoxyribonucleic acid (DNA) viruses, pseudorabies (PsRV) and vaccinia, are as susceptible as a ribonucleic acid (RNA) virus, vesicular stomatitis (VSV), to interferon when tested in chicken or mouse cells, they are refractory to inhibition in interferon-treated primary rabbit kidney cells and in a continuous line (RK-13) of rabbit kidney cells. Superinfection with VSV of RK-13 cells first infected with PsRV completely blocks the replication of PsRV with no effect on VSV yield. When the same experiment is carried out in RK-13 cells pretreated with 1,000 units of interferon, VSV replication is inhibited, which permits PsRV to replicate normally. These findings demonstrate that in the same cell one virus (PsRV) can be refractory to interferon and a second virus (VSV) can be susceptible. These experiments show that rabbit kidney cell cultures are deficient in the synthesis of resistance factors active against the DNA viruses tested and raise the possibility that separate resistance factors may exist for RNA and DNA viruses. In the case of sequential infection of interferon-treated RK-13 cells with vaccinia and VSV, it was found that not only was vaccinia replication refractory to inhibition by interferon, but also that prior infection with vaccinia was able to partially reverse the effect of the inhibitor on the replication of the VSV used for superinfection. On the basis of these and other data it is postulated that a vaccinia virion component or a replication product of vaccinia virus, or both, enables VSV to escape the inhibiting action of interferoninduced resistance factors.
PMCID: PMC356447  PMID: 4342236
19.  Peripheral Immunization Blocks Lethal Actions of Vesicular Stomatitis Virus within the Brain▿  
Journal of Virology  2009;83(22):11540-11549.
Vesicular stomatitis virus (VSV) is the prototype virus for 75 or more negative-strand RNA viruses in the rhabdovirus family. Some of these viruses, including VSV, can cause neurological impairment or death upon brain infection. VSV has shown promise in the prevention and treatment of disease as a vaccine vector and an oncolytic virus, but infection of the brain remains a concern. Three VSV variants, the wild-type-related VSV-G/GFP and two attenuated viruses, VSV-CT1 and VSV-CT9-M51, were compared for neuroinvasiveness and neuromorbidity. In nonimmunized mice, direct VSV-G/GFP injection into the brain invariably resulted in lethal encephalitis; in contrast, partial survival was seen after direct injection of the attenuated VSV strains. In addition, both attenuated VSV strains showed significantly reduced neuroinvasiveness after intranasal inoculation of young postnatal day 16 mice. Of the three tested variants, VSV-CT9-M51 generated the lowest degree of neuropathology. Despite its attenuated state, peripheral inoculations of VSV-CT9-M51 targeted and killed human glioblastoma implanted into the mouse brain. Importantly, we show here that intranasal or intramuscular immunization prevents the lethal effects of subsequent VSV-G/GFP, VSV-CT1, and VSV-CT9-M51 injections into the brain. These results indicate that attenuated recombinant viruses show reduced neurovirulence and that peripheral immunization blocks the lethal actions of all VSVs tested.
doi:10.1128/JVI.02558-08
PMCID: PMC2772672  PMID: 19726512
20.  Inhibition of vesicular stomatitis viral mRNA synthesis by interferons. 
Journal of Virology  1987;61(3):653-660.
Interferon (IFN) treatment inhibited the replication of vesicular stomatitis virus (VSV) in human GM2767 and mouse JLSV-11 cells. The replication of this virus in either human RD-114 or mouse A402 cells was insensitive to IFN treatment. We analyzed various steps in the VSV life cycle as they occurred under different conditions of IFN treatment to identify the point(s) at which IFN was exerting its inhibitory effect. IFN treatment led to strong inhibition of viral protein synthesis and accumulation of viral RNA in both lines of IFN-sensitive cells. No such effect was observed in the IFN-resistant cells. Using a temperature-sensitive mutant (tsG41) and wild-type VSV that were not undergoing protein synthesis, we determined that the major site of action of IFN against VSV replication in JLSV-11 and GM2767 cells was at the level of primary viral transcription. The accumulation of primary viral transcripts was strongly inhibited in these cells by IFN treatment. This effect was not a consequence of any effect of IFN on virus entry and uncoating. Thus, it appears that IFN exerts a direct effect on the VSV transcriptional process in GM2767 and JLSV-11 cells.
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PMCID: PMC254003  PMID: 3027394
21.  Refined Methods for Propagating Vesicular Stomatitis Virus Vectors that are Defective for G Protein Expression 
Journal of virological methods  2009;164(1-2):43-50.
Propagation-defective vesicular stomatitis virus (VSV) vectors that encode a truncated G protein (VSV-Gstem) or lack the G gene entirely (VSV-ΔG) are attractive vaccine vectors because they are immunogenic, cannot replicate and spread after vaccination, and do not express many of the epitopes that elicit neutralizing anti-VSV immunity. To consider advancing nonpropagating VSV vectors towards clinical assessment, scalable technology that is compliant with human vaccine manufacturing must be developed to produce clinical trial material. Accordingly, two propagation methods were developed for VSV-Gstem and VSV-ΔG vectors encoding HIV gag that have the potential to support large-scale production. One method is based on transient expression of G protein after electroporating plasmid DNA into Vero cells and the second is based on a stable Vero cell line that contains a G gene controlled by a heat shock-inducible transcription unit. Both methods reproducibly supported production of 1×107 to 1×108 infectious units (I.U.s) of vaccine vector per ml. Results from these studies also showed that optimization of the G gene is necessary for abundant G protein expression from electroporated plasmid DNA or from DNA integrated in the genome of a stable cell line, and that the titers of VSV-Gstem vectors generally exceeded VSV-ΔG.
doi:10.1016/j.jviromet.2009.11.023
PMCID: PMC2837098  PMID: 19941901
Negative-strand RNA virus; VSV; Vero cells; Electroporation; Heat shock; Non-propagating
22.  Vesicular Stomatitis Virus Variants Selectively Infect and Kill Human Melanomas but Not Normal Melanocytes 
Journal of Virology  2013;87(12):6644-6659.
Metastatic malignant melanoma remains one of the most therapeutically challenging forms of cancer. Here we test replication-competent vesicular stomatitis viruses (VSV) on 19 primary human melanoma samples and compare these infections with those of normal human melanocyte control cells. Even at a low viral concentration, we found a strong susceptibility to viral oncolysis in over 70% of melanomas. In contrast, melanocytes displayed strong resistance to virus infection and showed complete protection by interferon. Several recombinant VSVs were compared, and all infected and killed most melanomas with differences in the time course with increasing rates of melanoma infection, as follows: VSV-CT9-M51 < VSV-M51 < VSV-G/GFP < VSV-rp30. VSV-rp30 sequencing revealed 2 nonsynonymous mutations at codon positions P126 and L223, both of which appear to be required for the enhanced phenotype. VSV-rp30 showed effective targeting and infection of multiple subcutaneous and intracranial melanoma xenografts in SCID mice after tail vein virus application. Sequence analysis of mutations in the melanomas used revealed that BRAF but not NRAS gene mutation status was predictive for enhanced susceptibility to infection. In mouse melanoma models with specific induced gene mutations including mutations of the Braf, Pten, and Cdkn2a genes, viral infection correlated with the extent of malignant transformation. Similar to human melanocytes, mouse melanocytes resisted VSV-rp30 infection. This study confirms the general susceptibility of the majority of human melanoma types for VSV-mediated oncolysis.
doi:10.1128/JVI.03311-12
PMCID: PMC3676084  PMID: 23552414
23.  Posttranslational Modification of Vesicular Stomatitis Virus Glycoprotein, but Not JNK Inhibition, Is the Antiviral Mechanism of SP600125 
Journal of Virology  2012;86(9):4844-4855.
Vesicular stomatitis virus (VSV), a negative-sense single-stranded-RNA rhabdovirus, is an extremely promising oncolytic agent for cancer treatment. Since oncolytic virotherapy is moving closer to clinical application, potentially synergistic combinations of oncolytic viruses and molecularly targeted antitumor agents are becoming a meaningful strategy for cancer treatment. Mitogen-activated protein kinase (MAPK) inhibitors have been shown to impair liver cell proliferation and tumor development, suggesting their potential use as therapeutic agents for hepatocellular carcinoma (HCC). In this work, we show that the impairment of MAPK in vitro did not interfere with the oncolytic properties of VSV in HCC cell lines. Moreover, the administration of MAPK inhibitors did not restore the responsiveness of HCC cells to alpha/beta interferon (IFN-α/β). In contrast to previous reports, we show that JNK inhibition by the inhibitor SP600125 is not responsible for VSV attenuation in HCC cells and that this compound acts by causing a posttranslational modification of the viral glycoprotein.
doi:10.1128/JVI.06649-11
PMCID: PMC3347359  PMID: 22345438
24.  Transduction Efficiency of Pantropic Retroviral Vectors Is Controlled by the Envelope Plasmid to Vector Plasmid Ratio 
Biotechnology progress  2005;21(1):274-282.
Pantropic retroviral vectors pseudotyped with vesicular stomatitis virus envelope G protein (VSV-G) are typically produced by transient transfection of the VSV-G expression plasmid because constitutive expression of VSV-G is cytotoxic. To produce pantropic vectors, the VSV-G expression plasmid and the vector plasmid are cotransfected into a packaging cell line, such as 293-gag-pol. Typically, the ratio of VSV-G plasmid to the vector plasmid ranges from 0.33 to 1.0. However, it is not clear that this range is optimal for vector production. In this study we have systematically examined the effect of the ratio of VSV-G plasmid (pVSV-G) to vector plasmid on vector production. For this, 293-gag-pol stable packaging cells were cotransfected with pVSV-G and an enhanced green fluorescent protein- (EGFP-) expressing retroviral vector plasmid (pLTR-EGFP) by use of lipofectamine. Vector was collected following transfection and used to transduce three target cell lines, namely, 3T3 fibroblasts, telomerase-immortalized human diploid fibroblasts (HDF), and the human hepatoma cell line HuH7. Transduction efficiency was evaluated for vectors produced at different pVSV-G:pLTR-EGFP ratios such that the total amount of plasmid transfected into 293-gag-pol cells was kept constant. Our results indicate that transduction efficiency is greatest when the pVSV-G:pLTR-EGFP ratio is substantially below 1.0. For 3T3 and HDF cells, the maximum transduction efficiency was obtained when a ratio of pVSV-G:pLTR-EGFP ranging from 0.053 to 0.2 was used for transfection. The relative magnitude of this effect was greater for lower transduction efficiencies in control cultures. For HuH7 cells, the beneficial effects were smaller than those observed when HDF or 3T3 cells were used. The difference in transduction efficiency for vector produced under various pVSV-G:pLTR-EGFP ratios was not due to differences in the proliferation of packaging cells or target cells. Further characterization showed that the amount of vector RNA relative to p30gag decreased as the ratio of pVSV-G:pLTR-EGFP increased. These results indicate that transduction efficiency increases with increasing levels of vector RNA as long as a minimally sufficient level of pantropic envelope protein is expressed.
doi:10.1021/bp049865x
PMCID: PMC2913131  PMID: 15903266
25.  Oncolytic Vesicular Stomatitis Virus Induces Apoptosis in U87 Glioblastoma Cells by a Type II Death Receptor Mechanism and Induces Cell Death and Tumor Clearance In Vivo▿ 
Journal of Virology  2011;85(12):5708-5717.
Vesicular stomatitis virus (VSV) is a potential oncolytic virus for treating glioblastoma multiforme (GBM), an aggressive brain tumor. Matrix (M) protein mutants of VSV have shown greater selectivity for killing GBM cells versus normal brain cells than VSV with wild-type M protein. The goal of this research was to determine the contribution of death receptor and mitochondrial pathways to apoptosis induced by an M protein mutant (M51R) VSV in U87 human GBM tumor cells. Compared to controls, U87 cells expressing a dominant negative form of Fas (dnFas) or overexpressing Bcl-XL had reduced caspase-3 activation following infection with M51R VSV, indicating that both the death receptor pathway and mitochondrial pathways are important for M51R VSV-induced apoptosis. Death receptor signaling has been classified as type I or type II, depending on whether signaling is independent (type I) or dependent on the mitochondrial pathway (type II). Bcl-XL overexpression inhibited caspase activation in response to a Fas-inducing antibody, similar to the inhibition in response to M51R VSV infection, indicating that U87 cells behave as type II cells. Inhibition of apoptosis in vitro delayed, but did not prevent, virus-induced cell death. Murine xenografts of U87 cells that overexpress Bcl-XL regressed with a time course similar to that of control cells following treatment with M51R VSV, and tumors were not detectable at 21 days postinoculation. Immunohistochemical analysis demonstrated similar levels of viral antigen expression but reduced activation of caspase-3 following virus treatment of Bcl-XL-overexpressing tumors compared to controls. Further, the pathological changes in tumors following treatment with virus were quite different in the presence versus the absence of Bcl-XL overexpression. These results demonstrate that M51R VSV efficiently induces oncolysis in GBM tumor cells despite deregulation of apoptotic pathways, underscoring its potential use as a treatment for GBM.
doi:10.1128/JVI.02393-10
PMCID: PMC3126314  PMID: 21450822

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