Mechanisms regulating poxvirus post-replicative protein synthesis

项目摘要 虽然天花已经被根除，但病毒可能从未知的库存或从头开始的可能性 合成仍然是一种可信的生物疗法。此外，其他痘病毒导致严重的发病率和死亡率。 人类和重要的经济动物；痘病毒正被广泛开发为疫苗和 蛋白质表达载体和抗癌药物。痘病毒的基因表达可以说是不可替代的 在阐明痘病毒复制的基本机制和开发基于痘病毒的实用程序方面。 痘苗病毒(VACV)是痘病毒的原型。VACV复制后基因(在病毒DNA之后表达的基因 复制)数量超过90个，主要编码病毒粒子相关蛋白。它们的翻译效率更高 而不是细胞内的mRNAs，并且高水平表达。这个项目的目标是阐明分子 驱动VACV复制后mRNAs选择性翻译的机制，所有这些都有一个不寻常的 特点--5‘-多聚(A)领先者。在VACV感染的复制后阶段，Poly(A)先导似乎 提供翻译优势。这个项目的中心假设是5‘-聚(A)领导人坐标 用特定的病毒和细胞因子促进VACV复制后mRNAs的翻译。支持这一点 假说，D9和D10，两种VACV编码的解帽酶，已知促进细胞和病毒 MRNA降解，是VACV复制后mRNAs有效翻译所必需的。外源 D10在未感染细胞中的表达促进了5‘-聚(A)先导的mRNA翻译。这是惊人的 鉴于细胞解帽解酶的激活会导致翻译抑制。此外，La相关蛋白 4(LARP4)是一种细胞内Poly(A)结合蛋白和mRNA翻译调节子，是VACV POST所必需的。 复制型蛋白质合成。LARP4在病毒工厂中也高度浓缩，在那里病毒复制后的mRNA 翻译就发生了。将实施三个具体目标来测试这些因素如何选择性地促进 VACV复制后mRNAs的翻译。在目标1中，痘病毒的作用和分子决定因素 解帽酶在促进病毒复制后信使核糖核酸翻译中的作用将被定义。在目标2中， VACV解离酶促进病毒复制后mRNA翻译的机制将是 已澄清。在目标3中，将确定LARP4如何调控VACV复制后的mRNA翻译。这个 结果将提供一个完整的新范例，说明病毒转录本中的固有序列如何， 特定的病毒和细胞因子协调VACV复制后mRNAs的选择性翻译，进而， VACV复制。这些信息可用于调整或阻止痘病毒复制，并提高 VACV载体在疫苗和肿瘤溶解治疗中的应用。此外，这些发现还可以提供知识 了解真核生物翻译的基本机制。

Project Summary Although smallpox had been eradicated, the chance the virus could arise from unknown stocks or de novo synthesis remains a credible biothreat. Moreover, other poxviruses cause significant morbidity and mortality in humans and economically important animals; and poxviruses are widely being developed as vaccine and protein expression vectors, and anti-cancer agents. Poxvirus gene expression is an arguably irreplaceable area in elucidating fundamental mechanisms of poxvirus replication and developing poxvirus-based utilities. Vaccinia virus (VACV) is the prototype poxvirus. VACV post-replicative genes (those expressed after viral DNA replication) number over 90 and encode mainly virion-associated proteins. They are more efficiently translated than cellular mRNAs and expressed at high levels. The objective of this project is to elucidate molecular mechanisms driving selective translation of VACV post-replicative mRNAs, all of which have an unusual feature—a 5'-poly(A) leader. During the post-replicative stage of VACV infection, the poly(A) leader appears to confer a translational advantage. The central hypothesis of this project is that the 5'-poly(A) leader coordinates with specific viral and cellular factors to promote translation of VACV post-replicative mRNAs. Supporting this hypothesis, D9 and D10, two VACV-encoded decapping enzymes that are known to promote cellular and viral mRNA degradation, are required for efficient translation of VACV post-replicative mRNAs. Exogenous expression of D10 in uninfected cells promotes translation of mRNA with a 5'-poly(A) leader. This is striking given that activation of cellular decapping enzymes causes translation repression. Moreover, La-related protein 4 (LARP4), a cellular poly (A)-binding protein and mRNA translation regulator, is required for VACV post- replicative protein synthesis. LARP4 is also highly enriched in viral factories, where viral post-replicative mRNA translation occurs. Three Specific Aims will be carried out to test how these factors promote selectively translation of VACV post-replicative mRNAs. In Aim 1, the role and molecular determinants of poxvirus decapping enzymes in promoting viral post-replicative mRNA translation will be defined. In Aim 2, the mechanisms by which VACV decapping enzymes promote viral post-replicative mRNA translation will be elucidated. In Aim 3, how LARP4 regulates VACV post-replicative mRNA translation will be determined. The results will provide an integrated new paradigm illustrating how an intrinsic sequence in viral transcripts, specific viral and cellular factors orchestrate selective translation of VACV post-replicative mRNAs, and in turn, VACV replication. The information can be used to tune or block poxvirus replication and improve the efficacy of VACV vectors used in vaccine and oncolytic cancer therapy. Moreover, these findings can provide knowledge for understanding fundamental mechanisms of eukaryotic translation.