Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Paul Bates

Abstract

Bunyaviruses are a large family of enveloped RNA viruses that are distributed globally and include many important human and agricultural pathogens. Compared with other pathogenic viruses, bunyaviruses are relatively understudied. Currently, there are no licensed vaccines or antivirals to treat bunyavirus infections in the United States. To better understand bunyavirus interactions with their mammalian hosts in the hopes of uncovering novel therapeutic targets, we utilized a forward genetic screening approach in a human haploid cell line (HAP1). We performed insertional mutagenesis of the HAP1 cells with a retroviral gene-trap vector and subsequently challenged the cells with recombinant vesicular stomatitis viruses (rVSV) encoding heterologous glycoproteins from representative bunyaviruses, severe fever with thrombocytopenia syndrome virus (SFTSV) and Andes virus (ANDV). The use of rVSV-SFTSV and rVSV-ANDV allows us to specifically interrogate the early steps of bunyavirus entry, as replication of VSV is uniformly cytotoxic. Selection of cells resistant to rVSV-SFTSV or rVSV-ANDV and sequencing of mutagenic integrations sites identified genes important for virus entry. For SFTSV, the synthesis of the glycolipid glucosylceramide was found to play a role in the proper trafficking and/or fusion of incoming virus particles. Selection with rVSV-ANDV uncovered a profound dependence upon cholesterol synthesis and a requirement for cholesterol in membranes for efficient internalization and fusion. In addition to discovering novel aspects of bunyavirus biology, both of these cellular pathways have FDA approved pharmacological inhibitors and future studies aim to determine whether these factors can serve as targets for antiviral intervention.

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