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首页> 外文期刊>Journal of Molecular Biology >Mutant viral polymerase in the transition of virus to error catastrophe identifies a critical site for RNA binding.
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Mutant viral polymerase in the transition of virus to error catastrophe identifies a critical site for RNA binding.

机译：病毒向错误灾难过渡期间的突变型病毒聚合酶确定了RNA结合的关键位点。

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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.
机译：在向错误灾难过渡的过程中，从源自病毒唑处理病毒种群的分子克隆中获得了氨基酸替换为G118D，V239M和G373D（三重DMD突变体）的口蹄疫病毒（FMDV）聚合酶（3D）。 （通过致命诱变消灭病毒）。 DMD 3D在大肠杆菌中表达，纯化，其活性与通过定点诱变产生的替代G118D，G118A或D338A（后者影响催化基序YGDD）的野生型酶和突变型酶的活性相比。这些酶与FMDV聚合酶特异的单克隆抗体的相互作用没有发现差异。使用poly（A）/ oligo（dT）（15）和poly（A）/ VPg作为模板引物，在一些反应下，具有G118D或G118A的突变酶相对于野生型3D聚合活性降低了100倍条件。正如预期的那样，无法检测到D338A具有3D活性（<野生型3D值的0.01倍）。 DMD和G118突变体显示与模板引物RNA的结合受损，而D338A突变体显示与野生型3D相似的结合。用编码DMD 3D的FMDV RNA转染细胞导致选择仅保留V239M和G373D替代的逆转录病毒。一致地，当感染性转录本用G118D，G118A或D338A编码3D时，分离出具有恢复为野生型序列的病毒。该残基在FMDV聚合酶模板结合槽中的位置支持了G118在模板-引物结合中的作用。除了鉴定对聚合酶与RNA结合至关重要的氨基酸残基外，结果还证明了病毒向错误灾难的转变中存在有缺陷的基因组。

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