The 3′-to-5′ Exonuclease Activity of Vaccinia Virus DNA Polymerase Is Essential and Plays a Role in Promoting Virus Genetic Recombination
The 3′-to-5′ Exonuclease Activity of Vaccinia Virus DNA Polymerase Is Essential and Plays a Role in Promoting Virus Genetic Recombination
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DOI：
10.1128/jvi.02255-08
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发表时间：
2009-05-01
影响因子：
5.4
通讯作者：
Evans, David H.
中科院分区：
文献类型：
--
作者：
Gammon, Don B.;Evans, David H.
痘病毒在感染过程中会经历异常高水平的基因重组，尽管催化这些反应的酶从未被鉴定出来。然而，很明显，病毒编码的 DNA 聚合酶在病毒重组中发挥着一些未知但关键的作用。使用一种新的、基于抗病毒药物的策略来剖析重组和复制反应，我们现在表明病毒 DNA 聚合酶的 3' 至 5' 校对核酸外切酶活性在促进重组反应中发挥着关键作用。制备线性 DNA 底物，其中包含掺入分子 3' 末端的 dCMP 类似物西多福韦 (CDV)。该药物在体外阻断了由痘苗病毒 DNA 聚合酶的校对活性催化的多联重组分子的形成。当将含有 CDV 的重组底物转染到感染野生型牛痘病毒的细胞中时，重组的形成也被阻断。如果将含有 CDV 的底物转染到感染 CDV 抗性 (CDVr) 病毒的细胞中，则可以克服这些抑制作用，但前提是抗性与病毒聚合酶 3' 至 5' 核酸外切酶结构域内的 A314T 取代突变映射有关。在聚合酶结构域中编码 CDVr 突变的病毒仍然表现出 CDV 诱导的重组缺陷。 A314T 取代还增强了该酶从双链 DNA 3' 端切除 CDV 分子并在体外重组这些 DNA 的能力，这一点从使用纯化的突变 DNA 聚合酶的实验中可以看出。 3'至5'核酸外切酶活性似乎是一种重要的病毒功能，我们的结果表明这可能是因为痘病毒利用它来促进基因交换。
Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3'-to-5' proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3' ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDVr) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3'-to-5' exonuclease domain of the viral polymerase. Viruses encoding a CDVr mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3' ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3'-to-5' exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.