ORCID

https://orcid.org/0000-0002-8342-5316

Date of Award

Summer 8-15-2016

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Immunology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Noroviruses are a leading cause of epidemic gastroenteritis and a major health burden worldwide. One source for outbreaks is individuals who shed virus asymptomatically and persistently. Viral persistence is a successful strategy for viruses to spread, but the mechanisms and consequences of norovirus persistent infection are unknown. In this dissertation, we sought to determine the norovirus determinant(s) of persistence and explore the functions of the associated viral molecules.

To determine the viral determinants of persistent infection and tropism, we used the murine norovirus model system in mice. Using plasmid infectious clones for persistent strain CR6 and non-persistent strain CW3, we mapped the viral persistence determinant to the poorly understood non-structural gene NS1/2. The NS1 domain of NS1/2CR6 was necessary and sufficient for persistence. A single amino acid change, NS1/2D94E, conferred persistence on CW3. Viral persistence was restricted to replication and shedding in the intestine, and NS1/2 conferred intestinal tropism. In contrast, the capsid protein VP1 conferred acute replication in the spleen. Moreover, CW3 grew more rapidly in macrophages ex vivo, and this difference mapped to VP1. Therefore, NS1/2 and VP1 are the major determinants for persistence and tropism in vivo and ex vivo.

To determine a molecular function of NS1/2, we characterized its interaction with the host protein Vamp-Associated Protein A (VAPA). Murine norovirus replication was delayed in Vapa-/- cells and this was rescued by exogenous VAPA. Moreover, in Vapa-/- cells, NS1/2 protein levels were decreased early during viral infection as well as with electroporated viral RNA. The interaction of murine norovirus NS1/2 with VAPA occurred in a region within the poorly conserved NS1 domain of NS1/2. Investigations in the structural basis of NS1/2-VAPA interaction revealed sequence and functional mimicry between the VAPA binding region of NS1 and the host diphenylalanine-acidic-tract (FFAT)-motif that binds VAPA. The NS1/2-FFAT-mimic interacted with VAPA similarly to bona fide host FFAT motifs. Furthermore, mutations within NS1/2 that disrupted interaction with VAPA inhibited viral replication. Thus, VAPA is a pro-norovirus host factor interacting directly with a norovirus protein that functionally mimics FFAT motifs to co-opt VAPA function.

In conclusion, we mapped the norovirus determinants of persistence and tropism to NS1/2 and VP1. Furthermore, we determined that the NS1/2 interaction with VAPA enhanced murine norovirus infection. These are the first structural and functional studies to characterize NS1/2 in molecular detail. This work provides the basis for further exploration to identify the function of NS1/2 that contributes to persistent infection in mice.

Language

English (en)

Chair and Committee

Herbert W. Virgin

Committee Members

Gaya Amarasinghe, Jacco Boon, Michael Diamond, Andrzej Krezel, Wayne Yokoyama

Comments

Permanent URL: https://doi.org/doi:10.7936/K7M90718

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