Role of Region II of Sindbis virus capsid protein in specific encapsidation of genomic RNA and nucleocapsid core assembly
Abstract
Sindbis virus is an enveloped positive-sense RNA virus in the alphavirus genus. The nucleocapsid core contains the genomic RNA surrounded by 240 copies of a single capsid protein. The capsid protein is multifunctional, and its roles include functioning as a protease, controlling specificity of RNA that is encapsidated into nucleocapsid cores, interacting with viral glycoproteins to promote mature virus release, and release of the genomic RNA into the newly infected cell. The capsid protein region comprising amino acids 81-113 was previously implicated in two processes; encapsidation of the viral genomic RNA and stable accumulation of nucleocapsid cores in the cytoplasm of infected cells. In the present study, specific amino acids within this region responsible for encapsidation of the genomic RNA have been identified. The region that is responsible for nucleocapsid core accumulation has considerable overlap with the region that controls encapsidation specificity. The process of Sindbis virus nucleocapsid core assembly was also investigated in vitro using both analytical ultracentrifugation and surface plasmon resonance. Several N-terminal truncated versions of the purified capsid protein and their ability to form nucleocapsid core-like particles in vitro were characterized by both methods. The in vitro generated product was compared with the in vivo (cytoplasmic) nucleocapsid cores and the extent of the reaction was determined from calculated values of the loading concentration of core-like particles. The ratio of capsid protein to bound nucleic acid (single stranded DNA) was also characterized by analytical ultracentrifugation. Finally, the first steps in formation of core-like particles were investigated following the interaction of capsid protein with nucleic acid sequestered on an optically active chip by surface plasmon resonance.
Degree
Ph.D.
Advisors
Kuhn, Purdue University.
Subject Area
Virology
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