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Roles of Rubella virus nonstructural proteins in RNA replication Wang, Xiaojie


Rubella virus (RV) is an enveloped, positive-strand RNA virus in the family Togaviridae. The 9759-nucleotide genomic RNA contains two long open reading frames (ORFs). Onethird of the 3'-proximal ORF encodes the three structural proteins required for virus assembly. The other two-thirds encode the nonstructural proteins (NSPs) that function in RNA replication. The primary NSP polyprotein p200 is cleaved by a viral encoded protease into pi50 and p90. The aim of my thesis project is to identify the functional roles of NSPs in RV replication with respect to individual NSPs required for plus-strand RNA synthesis and the effect of highly conserved GDD motif within p90 region on viral replication. Both protease-inactive (pBRM33/Cl 152S) and cleavage-defective (pBRM33/G1301S) mutants can initiate minus-strand RNA but not plus-strand RNA synthesis (Liang and Gillam, 2000). In this study, individual RV NSPs were expressed by either a recombinant vaccinia virus or by co-electroporation of helper genomes. Complementation experiments were carried out by providing individual RV NSPs with either pBRM33/C1152S or pBRM33/G1301S mutant as a template. Rescue of pBRM33/C1152S by either p200 or pi50 indicates that functional protease can be provided in trans. Rescue of pBRM33/G1301S by either p200 or pl50/p90 suggests that the pl50/p90 replication complex functions in plus-strand RNA synthesis. Site-directed mutagenesis was employed to investigate the effect of putative RV RNAdependent RNA polymerase GDD motif on viral replication. Substitution of glycine by alanine (G1966A) resulted in impaired virus infectivity, reduced by 1.7xl0⁴-fold. Alteration of either aspartate residue completely abolished virus replication. A revertant was isolated from the passaged G1966A virus and its sequence confirmed. These results are consistent with the prediction that p90 is the RdRp of RV.

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