In vitro assembly of alphavirus nucleocapsid cores
Abstract
Sindbis virus (SINV) is the prototype alphavirus, a group of single stranded, positive sense RNA viruses. The alphavirus nucleocapsid core (NC) is a T = 4 icosahedron composed of 240 copies of a single monomeric capsid protein (CP) and the ∼12kB viral genome. In order to examine the mechanism of NC assembly, an in vitro NC assembly system has been developed using SINV CP purified from a bacterial expression system. The nucleic acid and protein requirements of NC assembly have been examined. Cross-linking analysis of truncated and mutant SINV CPs, which fail to generate detectable particles, has identified a possible assembly intermediate, a nucleic acid-bound protein dimer. Purified cross-linked dimers of either truncated or mutant CPs that failed to assemble were found to incorporate into assembled NCs in assembly reactions, suggesting the cross-linking traps an authentic intermediate in NC assembly. Mapping of the position of the cross-link indicated lysine 250 of one CP was cross-linked to lysine 250 on an adjacent CP. Examination of the position of the cross-link suggests the cross-linked species is a inter-capsomere contact between a pentamer and hexamer at the quasi-three fold axis or is an analogous contact between hexamers at the three-fold axis of the NC. Purified dimers of assembly defective CPs were found to be competent for particle assembly. Particles rescued from defective CPs by cross-linking appear to be identical to authentic NCs. The ability of cross-linking to rescue the assembly of defective CPs provides insights into the role of the amino terminus of the CP and the overall assembly mechanism. Analytical ultracentrifugation has identified the existence of the observed nucleic acid bound dimer in solution, and has provided evidence for larger assembly intermediates. The nature of these oligomeric species further suggest an assembly mechanism involving inter-capsomere polymerization of 120 dimeric capsid proteins.
Degree
Ph.D.
Advisors
Kuhn, Purdue University.
Subject Area
Biochemistry|Microbiology|Molecular biology
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