Selective Degradation of mRNA by Herpes Simplex Virus 1

描述（由申请人提供）：一种普遍的观点认为，HSV-1 UL41被膜蛋白通过在5'端或附近的RNA切割介导病毒和细胞mrna的非特异性降解，并且由于病毒基因的转录率较高，病毒基因表达普遍存在。我们的研究构成了这项拨款申请的基础，挑战了这种观点的几个方面。具体来说：(i)微阵列分析显示，与模拟感染细胞相比，感染细胞中数百个基因的上调。验证研究（Northern分析、Real-Time PCR和免疫印迹）显示了4组mrna。以IEX-1、c-fos、cox-2和Ikappabalpha mrna为例的第1组确实上调了，但没有产生蛋白产物。这些mrna以UL41依赖的方式通过死基化、核内裂解和3‘到5’的过程降解被降解。此外，部分降解的mrna的5'结构域倾向于停留，并且很容易在细胞质提取物中检测到。以tristetrprolin (TTP) mRNA为例的第2组和以GADD45beta mRNA为例的第3组也上调，但稳定且确实翻译，再次以UL41依赖的方式！！大量但未上调的肌动蛋白mRNA迅速降解。形成第2组和第3组的rna含有富A-U元素，具有快速翻转mrna的特征，而肌动蛋白和gadd45 β mrna不含这些元素。TTP是一种应激相关蛋白，通过结合并将这些mrna转移到外泌体，参与富含a - u mrna的降解。从本质上讲，UL41蛋白介导的mRNA降解不是不加区分的，而是高度选择性的。该应用程序有4个目的，即(1)鉴定在野生型病毒感染细胞中TTP mRNA中赋予mRNA选择性稳定性的序列；(2)确定野生型病毒感染细胞中TTP mRNA免于降解的机制。(3)确定野生型病毒感染细胞中含有A-U富元素的mrna与未感染细胞或感染？(4)确定目前与UL41蛋白相关的众多功能是否共变，以及这些功能在HSV-1生物学中的作用。

DESCRIPTION (provided by applicant): A prevailing view is that the HSV-1 UL41 tegument protein mediates nonspecific degradation of both viral and cellular mRNAs by cleavage of the RNA at or near the 5' terminus and that viral gene expression prevails because of a higher rate of transcription of viral genes. Our studies that form the basis of this grant application challenge several aspects of this view. Specifically: (i) Microarray analyses revealed the up regulation of several hundred genes in infected cells as compared to mock-infected cells. Validation studies (Northern analyses, Real-Time PCR and immunoblots) revealed 4 groups of mRNAs. Group 1 exemplified by IEX-1, c-fos, cox-2 and Ikappabalpha mRNAs were indeed up regulated but the protein products were not made. These mRNAs were degraded in a UL41 dependent manner by deadenylation, endonucleolytic cleavage and 3' to 5' processive degradation. Moreover, the 5' domains of the partially degraded mRNAs tended to linger and were readily detected in cytoplasmic extracts. Group 2 exemplified by tristetraprolin (TTP) mRNA and group 3 exemplified by GADD45beta mRNA were also up regulated but were stable and indeed translated, again all in a UL41 dependent manner!! Actin mRNA, abundant but not up regulated was rapidly degraded. The RNAs forming groups 2 and 3 contain A-U rich elements characteristic of rapidly turning over mRNAs whereas actin and GADD45beta mRNAs do not have these elements. TTP is a stress-related protein involved in the degradation of A-U rich mRNAs by binding and translocating these mRNAs to exosomes. In essence, the degradation of mRNA mediated by the UL41 protein is not indiscriminant but highly selective. This application has 4 aims i.e.(1) To identify the sequences in TTP mRNA that confer selective stability to the mRNA in wild-type virus infected cells;.(2) To define the mechanism by which the TTP mRNA is spared from degradation in wild-type virus infected cells.(3) To define the basis for the differential rates of degradation of mRNAs containing A-U rich elements in wild-type virus infected cells as compared to uninfected cells or cells infected with ?UL41 mutant virus, and (4) to determine whether the numerous functions now associated with the UL41 protein are co-variant and the role of these functions in the biology of HSV-1.