Mutations in the GDD motif of rubella virus putative RNA-dependent RNA polymerase affect virus replication.
Rubella virus (RV) nonstructural proteins are translated as a p200 polyprotein that undergoes proteolytic cleavage into p150 and p90. From conserved amino acid sequence motifs in polypeptides, p90 has been proposed to be the RV RNA-dependent RNA polymerase (RdRp). To test whether the conserved GDD motif is involved in RdRp catalytic activity, three different alanine substitutions were introduced into it. Substitution of glycine by alanine (G1966A) resulted in impaired virus infectivity. Alteration of either aspartate residue completely abolished virus replication. A fully infectious variant was isolated from the G1966A mutant. Sequencing analysis showed that the alanine residue substituted in G1966A mutant had reverted to glycine in this variant. Complementation experiments were carried out to rescue the replication-defective RNA carrying G1966A, D1967A, or D1968A mutations. The defective RNA with G1966A mutation in p90 replicated efficiently when the helper genome that supplied a wild-type p90 was provided in trans. However, the replication-defective RNA with D1967A or D1968A was not rescued by supplementation of p90 in trans. Our studies support the idea that the GDD motif is critical for RV replication and p90 function as RV RdRp.
Wang
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Department of Pathology and Laboratory Medicine, University of British Columbia, 950 W28th Avenue, Vancouver, British Columbia, V5Z 4H4, Canada.
Gillam
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D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D016297:Mutagenesis, Site-Directed; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D012412:Rubella virus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D014709:Vero Cells; D014779:Virus Replication / Q000502:physiology*
D012324:RNA-Dependent RNA Polymerase