Mutational analysis of the rotavirus RNA-dependent RNA polymerase active site

Tuesday, October 09, 2012 — Poster Session I
1:00 p.m. – 3:00 p.m	Natcher Conference Center, Building 45	NIAID	VIROL-13

* FARE Award Winner

Authors

• K. M. Ogden
• H. N. Ramanathan
• J. T. Patton

Abstract

Rotaviruses are the primary cause of severe acute dehydrating gastroenteritis in infants. The rotavirus RNA-dependent RNA polymerase (RdRp; VP1) is a hollow enzyme that contains canonical RdRp motifs A-F and a priming loop (PL). In the absence of the rotavirus inner shell protein, VP1 is inactive, it fails to interact stably with divalent cations or nucleotides, and the PL bends away from the active site. To gain insight into functions of catalytic site residues, we aligned sequences of phylogenetically divergent rotaviruses and performed mutational analyses of VP1. We found that conserved aspartic acids in canonical RdRp motifs A and C were required for RNA synthesis. Mutants of conserved arginines in motif F exhibited significantly diminished RNA synthetic capacity. Mutation of several individual PL residues diminished levels of RNA synthesis, but for S495A and Q496A mutants, they were enhanced. These studies represent the first biochemical assessment of contributions of PL and motif F residues to RdRp function. They suggest that conserved aspartic acids in VP1 motifs A and C perform canonical functions, interactions of motif F with nucleotides are important for RNA synthesis, and the PL is a dynamic regulatory element, with some residues promoting and others hindering RNA synthesis.

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