Date of Award

January 2015

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological Science

First Advisor

Richard Kuhn

Committee Member 1

Richard Kuhn

Committee Member 2

Cynthia Stauffacher

Committee Member 3

Jason Lanman

Abstract

The flavivirus non-structural protein 1 (NS1) is translocated into the endoplasmic reticulum (ER), glycosylated, and secreted from the infected cell. Among its various functions, a role of NS1 in RNA synthesis has been reported. The NS1 has three domains; “β-roll”, “wing” and “β-ladder (spaghetti loop)” domain. The characterization of the three domains of NS1 may help us to understand the multiple functions of NS1 in the context of specific regions of the protein. In this study, we have constructed chimeric Dengue/West Nile (DENV/WNV) viruses, which each contain one of the three domains of NS1 from WNV in a DENV genetic background. The chimeras were characterized with respect to viral RNA synthesis, trans-complementation and virion assembly. It was observed that the β-roll chimera was impaired in RNA synthesis, leading to decreased production of viral particles. The reciprocal chimera, WNV/DENV β-roll chimera also showed a reduced level of replication. The spaghetti loop chimera was defective in viral RNA synthesis, and formed small plaques. The wing domain chimera did not show viral RNA synthesis. Remarkably this chimera was rescued by mutations in the wing domain. Collectively, the results indicate that these three domains have a role in viral RNA synthesis. The wing domain is not interchangeable between DENV and WNV for DENV RNA replication, while β-roll and spaghetti loop chimeras could support RNA synthesis.

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