Abstract 2592: Host dependency factors for binding and internalization of oncolytic Seneca Valley Virus

Abstract

Abstract Background Seneca Valley Virus (SVV-001), an experimental oncolytic picornavirus, is the prototype member of the genus Senecavirus of the family Picornaviridae. SVV-001 is remarkable for its exquisite tumor selectivity for human cancers with neuroendocrine features including small cell lung carcinoma (SCLC) and numerous pediatric cancers while being non-cytotoxic in normal human cells in vitro and in vivo. The mechanism of tropism and neuroendocrine tumor specificity of SVV-001 has not been elucidated previously. Identification of host factors involved in infection would have immediate benefit for ongoing clinical trials by providing potential biomarker candidates to predict efficacy. Here we present data implicating α4 integrin as a key component of SVV-001 infection and demonstrating that the virus is dependent on caveloae for internalization and trafficking. Results Microarray analysis comparing permissive with non-permissive cell lines and membrane sub-proteome analysis of permissive H446 SCLC cells predict α4 integrin is a potential receptor candidate for SVV-001. To test SVV-001 dependency on α4 integrin, stable shRNA knockdown cell lines were generated from the permissive parental SCLC cell line H446. Viral infection was studied by single virus particle tracking, GFP reporter virus infection, and FACS analysis. α4 integrin knockdown cells showed reduced viral internalization and intracellular motility and were resistant to infection. Heterologous expression of human α4 integrin in chinese hamster ovary (CHO) cells significantly increased binding of fluorescently-labeled virus to the cell surface, but did not result in productive infection. Tracking of fluorescently-labeled SVV-001 in permissive cells expressing either clathrin-GFP, caveolin-1-GFP, or flotillin-1-GFP revealed a striking and long-lasting colocalization of internalized viral particles with caveolin-rich vesicles. Dependency on caveolin-1 for internalization was supported by results from pharmacological inhibition experiments. Pre-treatment of cells with nystatin, a cholesterol sequestration drug, resulted in a reduction in virus-induced cytopathic effect when compared to untreated cells. Stable shRNA caveolin-1 knockdown cells showed significantly reduced internalization, reduced infection by recombinant GFP reporter virus and significant impairment in SVV-001 genomic RNA release. Conclusions Defining the mechanism of tropism for SVV-001 is a key factor in successful clinical application. α4 integrin and caveolin-1 represent the first host cell factors identified in the mechanism of tropism of SVV-001 leading to a basic understanding of how the virus binds and enters permissive cells. These findings may have direct relevance for current clinical trials of SVV-001 in adult and pediatric settings and for development of second-generation derivatives of this agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2592.