Poxvirus DNA Replication

痘病毒在受感染细胞的细胞质内编码复制其基因组所需的酶和因子。痘苗病毒是痘病毒家族的原型成员，为研究DNA复制机制提供了一种结合生化和遗传方法的独特系统。DNA被合成为串联体，在病毒组装过程中被分解成单位长度的基因组和包装。对条件致死性突变体的研究表明，DNA复制需要5种早期蛋白：E9 DNA聚合酶、D4尿嘧啶DNA糖基化酶、A20加工因子、B1蛋白激酶和D5核苷三磷酸酶（NTPase）。DNA聚合酶催化引物和模板依赖的合成，并具有3至5的外切核活性。D4在DNA复制中的重要作用独立于其尿嘧啶DNA糖基酶活性，可能具有兼性修复功能。A20和D4蛋白相互作用，共同为DNA聚合酶提供加工能力。最近发现B1激酶磷酸化细胞DNA结合蛋白BAF，并阻止后者阻断VACV DNA复制。条件致死性D5突变体的快速停止DNA复制表型表明在复制叉上有功能。D5也与A20相互作用并形成多路定时器。D5的潜在作用来自于广泛的蛋白质序列分析，这表明90 kda的D5蛋白是AAA+类NTPases中解旋酶超家族III的成员，该家族包括一些其他DNA和RNA病毒的复制解旋酶。我们最近发现D5蛋白作为DNA引物酶还有另一个重要功能。此外，我们发现，如果细胞DNA连接酶被抑制，病毒DNA连接酶是必不可少的。DNA引物酶和DNA连接酶的两个重要作用的发现表明痘病毒DNA复制可能涉及冈崎片段。最近的另一项发现是预测的痘病毒fen1样核酸酶是同源重组、双链断裂修复和全尺寸基因组形成所必需的。

Poxviruses encode enzymes and factors needed for replication of their genomes within the cytoplasm of infected cells. Vaccinia virus, the prototypic member of the poxvirus family, provides a unique system for combining biochemical and genetic approaches for investigating mechanisms of DNA replication. The DNA is synthesized as concatemers that are resolved into unit length genomes and packaged during virus assembly. Studies with conditional lethal mutants indicate that five VACV early proteins are required for DNA replication: namely E9 DNA polymerase, D4 uracil DNA glycosylase, A20 processivity factor, B1 protein kinase and D5 nucleoside triphosphatase (NTPase). The DNA polymerase catalyzes primer- and template-dependent synthesis and possesses 3 to 5 exonucleolytic activity. The essential role of D4 in DNA replication is independent of its uracil DNA glycosylase activity, which presumably has a facultative repair function. The A20 and D4 proteins interact and together provide processivity for the DNA polymerase. The B1 kinase was recently shown to phosphorylate a cellular DNA-binding protein called BAF and prevent the latter from blocking VACV DNA replication. The fast stop DNA replication phenotype of conditional lethal D5 mutants suggested a function at the replication fork. D5 also interacts with A20 and forms multimers. Potential roles for D5 have come from extensive protein sequence analyses, which indicate that the 90-kDa D5 protein is a member of the helicase superfamily III within the AAA+ class of NTPases, which includes the replicative helicases of some other DNA and RNA viruses. We recently showed that the D5 protein has a second essential function as a DNA primase. In addition, we found that the viral DNA ligase was essential if cellular DNA ligase was inhibited. The two findings of a DNA primase and an essential role for a DNA ligase suggest that poxvirus DNA replication may involve Okazaki fragments. Another recent finding is that a predicted poxvirus FEN1-like nuclease is required for homologous recombination, double-strand break repair and full-size genome formation.