Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Robert W. Doms

Abstract

Hantaviruses are a genus of rodent-borne viruses within the family Bunyaviridae. Humans are a dead end host, wherein Hantavirus infection can cause severe, potentially fatal clinical disease. The specific mechanisms of Hantavirus infection are poorly understood. In particular, the host proteins and entry pathways that mediate Hantavirus entry are not yet known. We set out to identify these factors using two separate, but potentially complementary methods, focusing our efforts on a representative Hantavirus - Andes Virus (ANDV). First, using a high-throughput, luciferase-based assay with ANDV pseudotypes, we screened a siRNA library of ~9000 genes to identify host factors involved in ANDV infection. We ultimately identified 9 genes important for Hantavirus entry including SREBF2, a transcriptional regulator of cholesterol uptake. At the same time, a separate genetic screen performed by a collaborator independently identified SREBF2 and other members of the same cholesterol regulatory pathway (SCAP, S1P, S2P). Further experiments using siRNAs and small molecule inhibitors with both pseudotypes and live ANDV, identified an important role for cholesterol in ANDV internalization. Second, we attempted to use the highly characterized NCI-60 panel of cell lines to identify genes whose expression is correlated with ANDV entry. We infected a panel of 32 cell lines with ANDV pseudovirions in a FACS-based assay to measure relative permissiveness and then correlated permissiveness with gene expression using cDNA microarray data for these cell lines. Using this analysis we identified a list of 33 genes that potentially play a role in ANDV entry. Although initial follow-up experiments have failed to validate a subset of these genes, further experiments will be needed to fully test the entire list of candidate genes. Further analysis and experiments combining this list of genes with the cholesterol-dependence of ANDV entry may lead to further identification of specific pathways and factors utilized by Hantaviruses for infection and possibly potential targets for therapy.

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