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Molecular characterization of rubella virus nonstructural proteins and viral RNA synthesis Liang, Yuying

Abstract

Rubella virus (RV) is a single-strand positive RNA virus. Its 9762-nucleotide (nt) genomic RNA contains two large open readings frames (ORFs) at the 5'- and 3'-termini, encoding nonstructural and structural proteins, respectively. The nonstructural proteins (NSPs) are first translated as a 200-kDa polyprotein (p200), which undergoes a single proteolytic cleavage into pl50 and p90 by an intrinsic protease, the RV nonstructural protease (NS-pro). The purpose of this thesis work is to characterize RV NSPs and viral RNA synthesis at the molecular level, including the characterization of the RV NS-pro domains, the roles of NSPs in viral RNA synthesis, and the regulatory mechanism for viral RNA synthesis. RV NS-pro is an M-group papain-like cysteine protease (PCP) with both trans- and cis-cleavage activities. Employing an in vitro translation system, I mapped the regions of NS-pro required for trans- and cis- activities, and demonstrated the importance of an X domain in rrarcs-cleavage activity. RV NS-pro catalytic region was also predicted to have a papain-like folding from primary and secondary structure analyses. The time course of RV RNA synthesis was precisely determined. Synthesis of negative-strand RNA stops after 10 h postinfection (hpi), and subsequently switches to highly efficient synthesis of positive-strand RNAs (genomic and subgenomic RNA). The roles of respective NSPs and the underlying regulatory mechanism for RV RNA synthesis were investigated by mutational analysis and complementation experiments. Processing of p200 was found to be crucial for virus replication. Uncleaved p200 was shown to be functional in negative-strand RNA synthesis but not for positive-strand RNA. In contrast, the complex formed by the cleavage products p150/p90 is not an active replicase for negative-strand RNA, but is required for the efficient generation of positive-strand RNAs. Syntheses of both negative- and positive-strand RV RNAs were found to be cis-preferential from complementation experiments. A mechanism for RV NSP translation, processing and RV RNA replication was proposed. Newly translated p200 functions in cis to synthesize a full-length negative-strand RNA. The subsequent processing of p200 into p150/p90 converts this replicase into one with positive-strand RNA specificity, which functions in cis to produce genomic and subgenomic RNA efficiently.

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