Item Details

abstract

The rotavirus virion is comprised of three distinct protein layers, surrounding a segmented, double-stranded (ds) RNA genome. The innermost layer is a T=1 icosahedron formed by the core shell protein (VP2). Tethered beneath VP2 are 11-12 copies of the viral polymerase (VP1), which mediate genome replication within an assembling particle. Specifically, VP1 is bound by VP2 during early stages of rotavirus assembly, thereby (i) packaging the polymerase into nascent particles and (ii) triggering the polymerase to mediate intra-particle dsRNA synthesis. The VP2 N-terminal domain (NTD; residues 1-102) protrudes into the particle interior and plays key roles in polymerase interactions. Previous work has shown that a recombinant (r) VP2 protein lacking the first 10 NTD amino acids (delta10) maintains its ability to package rVP1 into virus-like particles (VLPs), but it is unable to activate the polymerase in the context of an in vitro RNA synthesis assay. In contrast, a mutant lacking the entire NTD (delta102) is defective for both polymerase packaging and activation. This work sought to delineate which of the extreme NTD residues (1-10) mediate polymerase activation and to map downstream NTD regions important for polymerase packaging. To do this, rVP2 NTD proteins were engineered with either alanine substitutions or internal deletions in their NTDs and were tested for the capacity to support in vitro dsRNA synthesis and to form VLPs. Results showed that a rVP2 mutant with alanine mutations at NTD residues R4, K5, and R6 supported very little dsRNA synthesis from rVP1. In contrast, rVP2 containing a deletion of residues 11-36 robustly activated rVP1 dsRNA synthesis. Both of these mutants formed VLPs that contained rVP1, suggesting that they are properly folded core shell proteins and capable of polymerase packaging. The rVP2 proteins were expressed in Spodoptera frugiperda insect cells using baculovirus vectors and purified using centrifugation. Vast experimental variation in rVP2 protein expression was observed as well as differing levels of co-purifying contaminant proteins in the preparations. Thus, through this project, rVP2 expression protocols were optimized by identifying new cell lines and baculovirus titering methods, thereby stream-lining future work on this project. Altogether, this work has helped inform a mechanistic understanding of VP1-VP2 interactions during rotavirus assembly and genome replication, as well as developed critical protocols for the McDonald Esstman Lab.

subject

Baculovirus

Core Shell

Polymerase

Protein Expression

RdRp

Rotavirus

contributor

Sullivan, Owen M (author)

Esstman, Sarah M (committee chair)

Zhang, Ke (committee member)

Alexander, Rebecca W (committee member)

Lyles, Douglas S (committee member)

Ornelles, David A (committee member)

date

2020-08-28T08:35:30Z (accessioned)

2020-08-28T08:35:30Z (available)

2020 (issued)

degree

Biology (discipline)

identifier

http://hdl.handle.net/10339/96956 (uri)

language

en (iso)

publisher

Wake Forest University

title

ROTAVIRUS CORE SHELL PROTEIN N-TERMINAL DOMAIN DETERMINANTS CRITICAL FOR POLYMERASE ACTIVATION

type

Thesis