Mutant Viral Polymerase in the Transition of Virus to Error Catastrophe Identifies a Critical Site for RNA Binding

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• 作者：Armando Arias ; Rubé ; n Agudo ; Cristina Ferrer-Orta ; Rosa Pé ; rez-Luque ; Antero Airaksinen ; Emiliana Brocchi ; Esteban Domingo ; Nuria Verdaguer and Cristina Escarmí ; s
• 刊名：Journal of Molecular Biology
• 出版年：2005
• 出版时间：2005
• 年：2005
• 卷：353
• 期：5
• 页码：1021-1032
• 全文大小：625 K

文摘

A foot-and-mouth disease virus (FMDV) polymerase (3D) with amino acid replacements G118D, V239M and G373D (triple DMD mutant) was obtained from a molecular clone derived from a virus population treated with ribavirin, in the transition to error catastrophe (virus extinction through lethal mutagenesis). DMD 3D was expressed in Escherichia coli, purified, and its activity compared with that of wild-type enzyme and mutant enzymes with either replacement G118D, G118A or D338A (the latter affecting the catalytic motif YGDD), generated by site-directed mutagenesis. No differences among the enzymes were noted in their interaction with monoclonal antibodies specific for the FMDV polymerase. Mutant enzymes with G118D or G118A showed a 100-fold decrease in polymerization activity relative to wild-type 3D, using poly(A)/oligo(dT)₁₅ and poly(A)/VPg as template-primers, under several reaction conditions. As expected, the activity of 3D with D338A was undetectable (<0.01 times the value for wild-type 3D). DMD and the G118 mutants showed impaired binding to template-primer RNA whereas the D338A mutant showed a binding similar to wild-type 3D. Transfection of cells with FMDV RNA encoding DMD 3D resulted in selection of revertant viruses that maintained only substitutions V239M and G373D. Consistently, when infectious transcripts encoded 3D with either G118D, G118A or D338A, viruses with reversions to the wild-type sequence were isolated. The implication of G118 in template–primer binding is supported by the location of this residue in the template-binding groove of the FMDV polymerase. In addition to identifying an amino acid residue that is critical for the binding of polymerase to RNA, the results document the presence of defective genomes in the transition of virus to error catastrophe.