Poxvirus Gene Expression and DNA Replication

痘病毒基因表达和 DNA 复制

• 批准号: 10014017
• 负责人: Bernard Moss
• 金额: $ 77.3万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014017
• 关键词: A20 protein; Anabolism; Antiviral Agents; Antiviral Therapy; Area; Biochemical Genetics; Cells; Cleaved cell; Consensus; Cytoplasm; D4 protein; DNA; DNA Ligases; DNA Primase; DNA Viruses; DNA biosynthesis; DNA sequencing; DNA-Binding Proteins; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Enzymes; Family; Focal Infection; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic Transcription; Genome; Goals; Late Gene Transcriptions; Length; Maps; Messenger RNA; Nucleic Acids; Okazaki fragments; Phosphotransferases; Poly(A) Tail; Poxviridae; Process; Protein Analysis; Protein Biosynthesis; Protein Kinase; Protein Sequence Analysis; Proteins; RNA; RNA Viruses; Reagent; Regulation; Replication Origin; Role; Site; Structure; System; Transcript; Translations; Vaccine Design; Vaccinia virus; Viral; Viral Genome; Viral Proteins; Virion; Virus; Virus Assembly; Virus Diseases; genetic approach; helicase; improved; member; mutant; nucleoside triphosphatase; poxvirus vectors; prevent; promoter; protein protein interaction; prototype; repaired; tissue culture; transcription factor; uracil-DNA glycosylase; vector vaccine; viral DNA

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.

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。牛痘病毒是痘病毒家族的原型成员，它提供了一个独特的系统，用于结合生物化学和遗传学方法来研究基因调控、mRNA生物合成和DNA合成的机制。对牛痘病毒的研究表明，这些基因分为三个时间类别-早期、中期和晚期。每个基因类别都有一个共有的DNA启动子序列和相应的转录因子，与病毒编码的多亚基RNA聚合酶相互作用。早期基因的转录系统在感染性病毒颗粒组装期间被包装在感染性病毒颗粒内，而中期和晚期基因转录的因子在感染后连续合成并定位在细胞质工厂区域内。痘病毒还编码通过在5'端添加帽结构和在3'端添加聚（A）尾来修饰其mRNA的酶，这是有效翻译和稳定性所必需的。细胞蛋白质合成的关闭和病毒蛋白质合成的严格调节由切割帽结构的痘病毒酶调节。利用新一代DNA测序技术，我们绘制了牛痘病毒基因组的完整转录和翻译图谱，并确定了RNA起始位点和poly（A）尾附近的序列。这些研究揭示了许多以前未注释的转录本。此外，已经确定了牛痘病毒感染对宿主mRNA的影响。 牛痘病毒DNA在细胞质工厂内合成为多联体，所述多联体在病毒组装期间分解成单位长度的基因组并包装。对条件致死突变体的研究表明，DNA复制需要五种VACV早期蛋白：即E9 DNA聚合酶、D4尿嘧啶DNA糖基化酶、A20持续合成因子、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性合成，并具有3至5个引物的核酸外切活性。D4在DNA复制中的重要作用不依赖于其尿嘧啶DNA糖基化酶活性，推测其具有兼性修复功能。A20和D4蛋白相互作用并一起为DNA聚合酶提供持续合成能力。B1激酶最近被其他人证明磷酸化一种称为BAF的细胞DNA结合蛋白，并阻止后者阻断VACV DNA复制。D5的潜在作用来自于广泛的蛋白质序列分析，这表明90-kDa D5蛋白是AAA+类NTPases内的解旋酶超家族III的成员，其包括一些其他DNA和RNA病毒的复制解旋酶。我们最近发现，D5蛋白作为DNA引发酶具有第二个基本功能。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引发酶和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，我们证明了在基因组末端附近存在双向复制起点。另一个最近的发现是，预测痘病毒FEN 1样过去的一年，我们提供了与复制病毒DNA相关的病毒和宿主蛋白的第一次分析。 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，并有助于我们了解其他病毒和细胞中的这些过程。