Mechanisms of KSHV posttranscriptional gene regulation

 描述(由申请人提供)：有效的病毒复制需要在受感染的宿主细胞中表达病毒基因。通过研究涉及病毒基因表达的宿主-病毒相互作用，揭示了病原体和宿主的潜在生物学。卡波西肉瘤相关疱疹病毒(KSHV)是一种双链核DNA病毒，是引起多种淋巴增生性疾病的重要人类病原体。KSHV ORF57(MTA)蛋白在疱疹病毒中是保守的，是病毒复制所必需的，因此阐明其机制有助于我们理解KSHV的生命周期。ORF57被认为在从合成到加工到翻译的几乎每一个阶段的mRNA表达中发挥作用，但新出现的数据表明，ORF57的一个核心功能是增加病毒RNA在细胞核中的稳定性。我们最近描述了一个涉及聚(A)结合蛋白PABPN1和聚(A)聚合酶α/γ的细胞核RNA衰退途径，初步研究强烈表明ORF57保护病毒RNA免受这一衰退途径的影响。这里提出的工作试图揭示KSHV ORF57和宿主介导的核RNA衰变之间的机制联系。特定目的1使用ORF57缺失的KSHV BACmid克隆来测试ORF57介导的抑制核RNA衰退在裂解再激活背景下的重要性。在这个目标中，我们进一步测试这种活性是否在疱疹病毒上保守。特异性目标2将确定ORF57失活这一细胞RNA衰退途径的分子机制。特定目的3试图定义使某些RNA ORF57具有反应性而其他转录本相对不受ORF57影响的顺式和反式作用因子。ORF57对于复制是绝对必需的，但其机制在很大程度上仍不清楚，因此这项工作通过定义一个重要的KSHV因子的活动来为KSHV生物学提供信息。此外，PABPN1介导的衰退是最近发现的一种宿主途径，其重要性才刚刚开始意识到，但它似乎是广泛存在的。因此，拟议的研究将显著影响目前对病毒和人类细胞基因调控分子机制的理解。

 DESCRIPTION (provided by applicant): Productive viral replication requires the expression of viral genes in the context of an infected host cell. By studying host-virus interactions involved i viral gene expression, the underlying biology of both the pathogen and the host are uncovered. The Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded nuclear DNA virus and an important human pathogen that causes several lymphoproliferative disorders. The KSHV ORF57 (Mta) protein is conserved among herpesviruses and is essential for viral replication, so elucidation of its mechanisms informs our understanding of the KSHV life cycle. ORF57 has been proposed to function in nearly every stage of mRNA expression from synthesis to processing to translation, but emerging data show that a central function of ORF57 is to increase the stability of viral RNAs in the nucleus. We recently described a cellular nuclear RNA decay pathway that involves the poly (A)-binding protein PABPN1 and poly (A) polymerases PAPα/γ, and preliminary studies strongly suggest that ORF57 protects viral RNAs from this decay pathway. The work proposed here seeks to uncover the mechanistic links between KSHV ORF57 and host- mediated nuclear RNA decay. Specific Aim 1 uses an ORF57-null KSHV BACmid clone to test the importance of ORF57-mediated inhibition of nuclear RNA decay in the context of lytic reactivation. In this aim, we further test whether this activity is conserved amon herpesviruses. Specific Aim 2 will determine the molecular mechanism by which ORF57 inactivates this cellular RNA decay pathway. Specific Aim 3 seeks to define the cis- and trans-acting factors that make certain RNAs ORF57-respononsive while other transcripts are relatively unaffected by ORF57. ORF57 is absolutely required for replication, but its mechanisms remain largely unknown, so this work informs KSHV biology by defining the activities of an essential KSHV factor. In addition, PABPN1-mediated decay is a recently described discovered host pathway whose importance is only beginning to be realized, but it appears to be widespread. As such, the proposed studies will significantly impact the current understanding of the molecular mechanisms of gene regulation for both virus and human cells.