Date of Award

Spring 5-7-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Dr. Margo A. Brinton

Second Advisor

Dr. Susanna F. Greer

Third Advisor

Dr. Zhi-Ren Liu

Abstract

Two cellular innate responses, the dsRNA protein kinase (PKR) pathway and the 2'-5' oligoadenylate synthetase (OAS)/RNase L pathway, are activated by dsRNAs produced by viruses and reduce translation of host and viral mRNAs. PKR activation results in eIF2a phosphorylation. As a consequence of eIF2a phosphorylation, stress granules (SGs) are formed by the aggregation of stalled SG proteins with pre-initiation complexes and mRNA. West Nile virus (WNV) infections do not induce eIF2a phosphorylation despite upregulation of PKR mRNA and protein suggesting an active suppression of PKR activation. Assessment of the mechanism of suppression of PKR activation in WNV-infected cells indicated that WNV infections do not induce PKR phosphorylation so that active suppression is not required.

In contrast to infections with "natural" strains of WNV, infections with the chimeric W956 infectious clone (IC) virus efficiently induce SGs in infected cells. After two serial passages, the IC virus generated a mutant (IC-P) that does not induce SGs efficiently but does induce the formation of NS3 granules that persist throughout the infection. This mutant was characterized.

2'-5' oligoadenylate synthetases (OAS) are activated by viral dsRNA to produce 2-5A oligos that activate RNase L to digest viral and cellular RNAs. Resistance to flavivirus-induced disease in mice is conferred by the full-length 2'-5' oligoadenylate synthetase 1b (Oas1b) protein. Oas1b is an inactive synthetase that is able to suppress the in vitro synthetase activity of the active synthetase Oas1a. The ability of Oas1b to inhibit Oas1a synthetase activity in vivo and to form a heteromeric complex with Oas1a was investigated. Oas1b suppressed 2-5A production in vivo. Oas1a and Oas1b overexpressed in mammalian cells co-immunoprecipitated indicating the formation of heteromeric complexes by these proteins.

Unlike mice, humans encode a single OAS1 gene that generates alternatively spliced transcripts encoding different isoforms. Synthetase activity has previously been reported for only three of the isoforms. The in vitro synthetase activity of additional OAS1 isoforms was analyzed. All tested isoforms synthesized higher order 2-5A oligos. However, p44A only produced 2-5A dimers which inhibit RNase L.

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Biology Commons

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