Analysis of the essential human cytomegalovirus protein, pUL34

描述（由申请人提供）：在我们对基本病毒蛋白pUL 34对人巨细胞病毒（HCMV）复制的贡献的理解方面存在根本性的差距。这一空白仍然是一个重要的问题，因为直到它被填补，在了解HCMV感染对人类宿主的影响和新的治疗药物的设计仍然存在挑战。长期目标是开发针对基本UL 34蛋白的特异性新抗病毒化合物。本申请的目的是定义UL 34蛋白的功能及其对病毒复制的贡献，这是开发靶向抗病毒化合物的必要前体。中心假设是UL 34蛋白（pUL 34）与HCMV基因组中的pUL 34结合位点以及与细胞蛋白细丝蛋白A（FlnA）的相互作用构成了UL 34基因的基本功能。这一假设是根据申请人实验室的工作提出的。这项研究的基本原理是，确定UL 34蛋白的功能对于理解HCMV复制至关重要。UL 34编码在整个病毒复制周期中表达的序列特异性DNA结合蛋白。在HCMV基因组（AD 169）内的14个pUL 34结合位点中，3个位于裂解复制起点附近，6个位于蛋白质编码区内，其余位于非必需病毒基因的调控区。在强有力的初步数据的指导下，该假设将通过追求三个特定目标进行测试：1）确定pUL 34- DNA相互作用对病毒DNA复制的影响; 2）确定UL 34-DNA相互作用对必需基因UL 32，UL 37和UL 54表达的影响; 3）确定细丝蛋白A对pUL 34功能和病毒复制的贡献。对于第一个具体目的，将突变申请人已经显示结合pUL 34的裂解复制起点内的位点，并确定对病毒DNA复制的影响。将使用现有的质粒和HCMV-BAC。对于具体目的2，在申请人进行的初步研究中，已显示包含在必需病毒基因内的pUL 34结合位点降低基因表达。将使用RNA分析鉴定导致基因表达降低的分子机制。对于具体目标3，申请人已经鉴定细丝蛋白A作为pUL 34的结合配偶体。细胞内共定位 分析pUL 34和细丝蛋白A的模式;确定siRNA介导的细丝蛋白A表达敲低对病毒复制的影响。这项研究具有创新性，因为它将定义独特的UL 34蛋白在病毒复制和基因表达中的额外作用，包括pUL 34与细丝蛋白A的新型相互作用。这项研究意义重大，因为它将推进和扩大我们对导致病毒复制和相关疾病的HCMV基因调控复杂模式的理解。最终，这些知识有可能有助于对基因调控的普遍理解，并有助于开发新的治疗药物。

DESCRIPTION (provided by applicant): There is a fundamental gap in our understanding of the contributions of the essential viral protein, pUL34 to human cytomegalovirus (HCMV) replication. This gap remains an important problem, because until it is filled, challenges in understanding the effects of HCMV infection on the human host, and the design of new therapeutic agents remain. The long term goal is to develop specific, new antiviral compounds that target the essential UL34 proteins. The objective of this application is to define functions o UL34 proteins and their contributions to viral replication, a necessary precursor to developing targeted antiviral compounds. The central hypothesis is that UL34 protein (pUL34) interactions with the pUL34-binding sites in the HCMV genome and with the cellular protein filamin A (FlnA) comprise the essential functions of the UL34 gene. This hypothesis has been formulated based on work produced in the applicant's laboratory. The rationale for the proposed research is that defining the functions of the UL34 proteins is essential for understanding HCMV replication. UL34 encodes sequence-specific DNA binding proteins that are expressed throughout the viral replication cycle. Of the 14 pUL34-binding sites within the HCMV genome (AD169), 3 are located near the origin for lytic replication, 6 are within protein-coding regions, and the remainder are located in the regulatory regions of non-essential viral genes. Guided by strong preliminary data, the hypothesis will be tested by pursuing three specific aims: 1) Identify the effects of pUL34- DNA interactions on viral DNA replication; 2) Define the effects of UL34-DNA interactions on the expression of the essential genes UL32, UL37 and UL54; and 3) Determine the contribution of filamin A to pUL34 function and viral replication. For the first specific aim, sites within the origin for lytic replication that have been shown by the applicant to bind pUL34, will be mutated and the effects on viral DNA replication will be determined. The plasmids and the HCMV-BAC that are already on hand will be used. For specific aim 2, a pUL34-binding site contained within an essential viral gene has been shown to decrease gene expression in preliminary studies performed by the applicant. The molecular mechanism that results in decreased gene expression will be identified using RNA analyses. For specific aim 3, the applicant has identified filamin A as a binding partner for pUL34. The intracellular colocalization patterns of pUL34 and filamin A will be analyzed; the effect of siRNA-mediated knockdown of filamin A expression on viral replication will be determined. The research proposed here is innovative, because it will define the additional roles of the unique UL34 proteins in viral replication and gene expression, including the novel interaction of pUL34 with filamin A. The proposed research is significant because it is will advance and expand our understanding of the complex patterns of HCMV gene regulation that result in viral replication and associated diseases. Ultimately, such knowledge has the potential to contribute to the general understanding of gene regulation, and to the development of new therapeutic agents.