All ETDs from UAB

Advisory Committee Chair

Ming Luo

Advisory Committee Members

Terje Dokland

Donald Muccio

Kirill Popov

Peter Prevelige, Jr

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

All negative stranded RNA viruses (NSRV) have genomes encapsidated by oligomerized nucleoprotein (N). The N-RNA complex acts as the template for both transcription and replication. The crystal structure of the N protein from the model NSRV vesicular stomatitis virus (VSV) established the structural basis for the oligomerization of N-protein and the encapsidation of RNA. To determine how the structure of N protein changes in response to specific sequences of RNA, we developed a strategy to digest the random RNA encapsidated in the recombinant N-protein and reencapsidated specific RNA sequences. Poly-rA, poly-rC, poly-rU, and poly-rG sequences were encapsidated and co-crystallized with the N-protein. The structures suggest that the N protein may coordinate unique base stacking of viral RNA sequences that could be used by the viral polymerase to stably bind to and initiate transcription or replication. Additionally, poly-rU showed no specific interactions with the N protein and weak base stacking. This could be important in the polyadenlyation and termination of transcription at uridine tracts in nonsegmented NSRV. Influenza virus is a segmented NSRV that causes seasonal epidemics and has the potential to cause pandemics. There have been four major pandemics since 1918, caused by the reassortment of avian influenza virus genome segments with human influenza virus genome segments. Vaccination will not be protective against a novel influenza virus so antivirals are needed to control influenza virus until a new vaccine formulation can be manufactured. Neuraminidase inhibitors are the only effective antiviral treatment available for influenza virus infection but cases of resistance in patients have been reported. If resistance were to become more prevalent, an alternative to neuraminidase inhibitors would be essential. We discovered a potent influenza virus inhibitor, 136. Here we show that 136 inhibits influenza virus and VSV in plaque reduction assays. Using a number of entry assays, we show that 136 blocks influenza virus fusion at the lipid mixing step. Unlike other fusion inhibitors being developed, 136 does not stabilize the neutral pH form of HA. 136 binds at or very near the viral membrane and alters the properties of the viral membrane necessary for fusion to cellular membranes.

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