Veterinary and Biomedical Sciences, Department of
Date of this Version
Summer 8-2011
Abstract
Viruses usurp host cell pathways for different stages of their infection. Understanding virus-host interaction will be invaluable to elucidate molecular mechanisms of virus infection and to identify drug targets. In order to identify such critical cellular genes in vesicular stomatitis virus (VSV, a model non-segmented negative strand RNA virus) infection, we developed a stable cell line constitutively expressing replication proteins of VSV. Attempts to establish a cell line replicating a sub-genomic replicon was not successful because of induction of interferon response by replication of viral genomic analog. Subsequently, we used siRNA technology and conducted a genome-wide siRNA screen in HeLa cells to identify host factors regulating VSV infection. A total of 23,000 human genes were knocked down individually, and their effect on viral infection was interrogated using a high-throughput cell-based assay. Our study identified several previously unknown host proteins required for VSV infection. Bioinformatics analysis predicted enrichment of several biological functions among these proteins and some of them are commonly utilized by other pathogens such as human immune deficiency virus (HIV), hepatitis C virus (HCV) and Influenza virus. We also noted that 35% of these genes (25 out of 72) are required for lymphocytic choriomeningitis virus (LCMV) and human parainfluenza virus type 3 (HPIV3) infection, suggesting evolutionary conserved mechanisms of virus-host interactions. Further studies focusing on host coatomer complex 1 (COPI) identified a role of COPI in early stage of VSV infection. The effect of COPI is mediated at the level of viral RNA synthesis. COPI functions are required not only for VSV but also for LCMV and HPIV. ADP ribosylation factor 1 (ARF1), the immediate upstream modulator of COPI was found as a required factor for VSV RNA synthesis. ARF1 is activated by the Golgi-associated brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1) which was found to be a critical determinant of VSV RNA synthesis. These studies suggested that the components of the cellular secretory pathway are required for VSV RNA synthesis.
Adviser: Asit K. Pattnaik
Comments
A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillments of Requirements For the Degree of Doctor of Philosophy, Major: Integrative Biomedical Sciences, Under the Supervision of Professor Asit K. Pattnaik. Lincoln, Nebraska: August, 2011
Copyright 2011 Debasis Panda