Poxvirus Gene Expression and DNA Replication

痘病毒编码在感染细胞的细胞质内转录和复制其基因组所需的酶和因子。痘苗病毒是痘病毒家族的典型成员，它为研究基因调控、信使核糖核酸生物合成和DNA合成机制提供了一种独特的生化和遗传方法相结合的系统。对痘苗病毒的研究表明，这些基因分为三个时间类别-早期、中期和晚期。每个基因类都有一个一致的DNA启动子序列和相应的转录因子，这些转录因子与病毒编码的多亚单位RNA聚合酶相互作用。早期基因的转录系统在组装过程中被包装在感染性病毒颗粒内，而中晚期基因转录因子在感染后相继合成并定位于细胞质工厂区。痘病毒还编码酶，通过在5‘端增加一个帽子结构和在3’端增加一个聚(A)尾巴来修改其mRNA，这是有效翻译和稳定所必需的。细胞蛋白质合成的关闭和病毒蛋白质合成的严格调节由裂解帽结构的痘病毒酶调节。利用新一代DNA测序技术，我们已经制作了痘苗病毒基因组的完整转录和翻译图谱，并确定了RNA起始点和PolyA尾部附近的序列。这些研究揭示了许多以前没有注释的成绩单。此外，还明确了痘苗病毒感染对宿主mRNAs的影响。 痘苗病毒DNA是在细胞质工厂中合成的，作为串联体被分解成单位长度的基因组，并在病毒组装过程中包装。对条件致死突变体的研究表明，VACV的DNA复制需要5种早期蛋白：E9 DNA聚合酶、D4尿嘧啶DNA糖基酶、A20加工因子、B1蛋白激酶和D5核苷三磷酸酶(NTPase)。该DNA聚合酶催化依赖于引物和模板的合成，并具有3-5个最高的核酸外切活性。D4在DNA复制中的重要作用不依赖于其尿嘧啶DNA糖基酶活性，该酶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，其他研究人员发现，B1K可以磷酸化一种名为BAF的细胞DNA结合蛋白，并阻止后者阻止VACV DNA复制。D5的潜在作用来自广泛的蛋白质序列分析，这些分析表明，90 kDa的D5蛋白是NTPase AAA+类中解旋酶超家族III的成员，包括其他一些DNA和RNA病毒的复制型解旋酶。我们最近发现，D5蛋白还有第二个基本功能，即DNA启动酶。此外，我们还发现，如果细胞DNA连接酶被抑制，病毒DNA连接酶是必不可少的。DNA启动酶和DNA连接酶的重要作用的两个发现表明，痘病毒DNA复制可能涉及冈崎片段。最近，我们证明了基因组末端附近存在双向复制起点。另一个最近的发现是，在过去的一年里，我们预测了一种类似于痘病毒FEN1的病毒，我们首次对与复制病毒DNA相关的病毒和宿主蛋白进行了分析。 了解痘病毒基因表达和DNA复制的调控将有助于设计疫苗和确定抗病毒治疗的靶点，并将有助于我们了解其他病毒和细胞中的这些过程。

Poxviruses encode enzymes and factors needed for transcription and 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 gene regulation, mRNA biosynthesis and DNA synthesis. Studies with vaccinia virus indicated that the genes are divided into three temporal classes - early, intermediate and late. Each gene class has a consensus DNA promoter sequence and corresponding transcription factors that interact with the virus-encoded multisubunit RNA polymerase. The transcription system for early genes is packaged within the infectious virus particle during its assembly, whereas the factors for intermediate and late gene transcription are synthesized successively after infection and localize within cytoplasmic factory areas. Poxviruses also encode enzymes that modify their mRNA by adding a cap structure to the 5' end and a poly(A) tail to the 3' end, which are necessary for efficient translation and stability. The shut down of cellular protein synthesis and the tight regulation of viral protein synthesis are regulated by poxvirus enzymes that cleave the cap structure. Using new generation DNA sequencing, we have made a complete transcription and translation map of the vaccinia virus genome and defined the RNA start sites and the sequences adjacent to the poly(A) tail. These studies have revealed numerous previously unannotated transcripts. In addition, the effects of vaccinia virus infection on host mRNAs have been defined. Vaccinia virus DNA is synthesized within cytoplasmic factories 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 prime 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 by others to phosphorylate a cellular DNA-binding protein called BAF and prevent the latter from blocking VACV DNA replication. 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. Recently, we demonstrated the presence of a bidirectional replication origin near the ends of the genome. Another recent finding is that a predicted poxvirus FEN1-like past present year, we provided the first analysis of the viral and host proteins that are associated with replicating viral DNA. An understanding of the regulation of poxvirus gene expression and DNA replication will help to design vaccines and identify targets for antiviral therapy and will contribute to our understanding of these processes in other viruses and cells.