Showing papers by "Sean P. J. Whelan published in 2008"

A Conserved Motif in Region V of the Large Polymerase Proteins of Nonsegmented Negative-Sense RNA Viruses That Is Essential for mRNA Capping

Abstract: Nonsegmented negative-sense (NNS) RNA viruses cap their mRNA by an unconventional mechanism. Specifically, 5′ monophosphate mRNA is transferred to GDP derived from GTP through a reaction that involves a covalent intermediate between the large polymerase protein L and mRNA. This polyribonucleotidyltransferase activity contrasts with all other capping reactions, which are catalyzed by an RNA triphosphatase and guanylyltransferase. In these reactions, a 5′ diphosphate mRNA is capped by transfer of GMP via a covalent enzyme-GMP intermediate. RNA guanylyltransferases typically have a KxDG motif in which the lysine forms this covalent intermediate. Consistent with the distinct mechanism of capping employed by NNS RNA viruses, such a motif is absent from L. To determine the residues of L protein required for capping, we reconstituted the capping reaction of the prototype NNS RNA virus, vesicular stomatitis virus, from highly purified components. Using a panel of L proteins with single-amino-acid substitutions to residues universally conserved among NNS RNA virus L proteins, we define a new motif, GxxT[n]HR, present within conserved region V of L protein that is essential for this unconventional mechanism of mRNA cap formation.

Vesicular Stomatitis Virus

Abstract: Vesicular stomatitis virus (VSV) has been extensively studied as a prototype of the nonsegmented negative-strand RNA viruses The relative safety of the virus combined with its abundant replication in a broad range of cultured cells have favored the use of VSV in research laboratories Studies with VSV have led to understanding of fundamental host cell processes, molecular details of viral gene expression, and the pathogenesis of viral infection The demonstration that purified virus particles contain a functional RNA-dependent RNA polymerase that synthesizes mRNA in vitro has led to major advances in our understanding of viral gene expression The development of a robust reverse genetic system and, most recently, the determination of crystal structures of the N-RNA template, a portion of the P and M proteins, and the pre- and postfusion forms of the G protein, now permit the detailed structure–function analysis of much of the viral genome In addition to serving as a prototype of nonsegmented negative-strand viruses, studies of VSV have also been used to illustrate fundamental principles in evolutionary biology and population genetics Finally, VSV causes an economically important disease of livestock that is transmitted through insect vectors Studies on the ecology and evolution of VSV in nature have revealed details of transmission and an unusually broad host range