Arias Armando, Agudo Rubén, Ferrer-Orta Cristina, Pérez-Luque Rosa, Airaksinen Antero, Brocchi Emiliana, Domingo Esteban, Verdaguer Nuria, Escarmís Cristina
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Cantoblanco, E-28049 Madrid, Spain.
J Mol Biol. 2005 Nov 11;353(5):1021-32. doi: 10.1016/j.jmb.2005.09.022. Epub 2005 Sep 26.
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.
从经利巴韦林处理的病毒群体衍生的分子克隆中获得了一种口蹄疫病毒（FMDV）聚合酶（3D），其具有氨基酸替换G118D、V239M和G373D（三重DMD突变体），处于向错误灾难（通过致死诱变导致病毒灭绝）的转变过程中。DMD 3D在大肠杆菌中表达、纯化，并将其活性与野生型酶以及通过定点诱变产生的具有替换G118D、G118A或D338A（后者影响催化基序YGDD）的突变酶的活性进行比较。在与口蹄疫病毒聚合酶特异性单克隆抗体的相互作用中，未观察到这些酶之间存在差异。在几种反应条件下，使用聚（A）/寡聚（dT）15和聚（A）/VPg作为模板引物时，具有G118D或G118A的突变酶相对于野生型3D的聚合活性降低了100倍。正如预期的那样，具有D338A的3D的活性无法检测到（<野生型3D值的0.01倍）。DMD和G118突变体与模板引物RNA的结合受损，而D338A突变体表现出与野生型3D相似的结合。用编码DMD 3D的口蹄疫病毒RNA转染细胞导致选择出仅保留替换V239M和G373D的回复病毒。一致地，当感染性转录本编码具有G118D、G118A或D338A的3D时，分离出了恢复为野生型序列的病毒。FMDV聚合酶模板结合槽中该残基的位置支持了G118在模板引物结合中的作用。除了鉴定出对聚合酶与RNA结合至关重要的氨基酸残基外，结果还证明了在病毒向错误灾难转变过程中存在缺陷基因组。
