The role of the nucleocapsid serine proteinase in the formation of the Sindbis virus nucleocapsid protein
Abstract
The Sindbis virus (SINV) nucleocapsid protein (NCP) is a 264 amino acid protein that is involved in a variety of functions in the virus life cycle. In addition to its role as a viral structural protein and an RNA-binding protein, the NCP is also a serine proteinase that functions to cotranslationally cleave itself from the structural polyprotein. The serine proteinase domain of SINV NCP consists of residues 114–264. Genetic and biochemical analysis of the SINV NCP proteinase determined that the P1 site (W264), the P4 site (T261) and the P5 site (G260) are important for in vitro cleavage of the nucleocapsid protein from the structural polyprotein. Analysis of the X-ray structure of the SINV nucleocapsid protein showed that the P1 residue sits in a deep, but narrow, hydrophobic pocket of NCP. The sides of the S1 pocket are formed from the interactions of small hydrophobic residues contributed by the β strands of the NCP. The bottom of the pocket is formed by W191. The P4 residue sits in a shallow hydrophobic pocket and forms hydrophobic interactions with the residues that line the pocket. In addition to these specific interactions, the glycine residue in the P5 position (G260) appears to be necessary for proper folding of the C-terminal residues (260–264) of the nucleocapsid protein into the active site pocket. Substitutions at this site result in a noncleaving proteinase, in the 1 hr in vitro cleavage assay. In vivo analysis of these SINV NCP mutants revealed that the substitutions in P sites of the proteinase greatly affect the ability of the virus to replicate.
Degree
Ph.D.
Advisors
Kuhn, Purdue University.
Subject Area
Molecular biology|Microbiology
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