Selective Degradation of mRNA by Herpes Simplex Virus 1

描述（由申请人提供）：普遍的观点是HSV-1 UL 41被膜蛋白通过在5'末端或其附近切割RNA介导病毒和细胞mRNA的非特异性降解，并且由于病毒基因的较高转录速率，病毒基因表达占优势。我们的研究构成了这项资助申请的基础，挑战了这种观点的几个方面。具体而言：（i）微阵列分析揭示了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（北方分析、实时PCR和免疫印迹）显示了4组mRNA。组1以IEX-1、c-fos、考克斯-2和Ikappabalpha mRNA为例，确实上调，但不产生蛋白质产物。这些mRNA以UL 41依赖性方式通过去腺苷酸化、内切核酸裂解和3'至5'进行性降解而降解。此外，部分降解的mRNA的5'结构域倾向于滞留，并且容易在细胞质提取物中检测到。第2组以tristetraprolin（TTP）mRNA为例，第3组以GADD 45 β mRNA为例，也上调，但稳定且确实翻译，再次均以UL 41依赖性方式！ 大量但未上调的肌动蛋白mRNA迅速降解。形成组2和组3的RNA含有A-U富集元件，其特征在于快速翻转mRNA，而肌动蛋白和GADD 45 β mRNA不具有这些元件。TTP是一种应激相关蛋白，通过将富含A-U的mRNA结合并转运至外泌体而参与这些mRNA的降解。实质上，由UL 41蛋白介导的mRNA降解不是不加区别的，而是高度选择性的。该应用程序有四个目标，即。(1)鉴定TTP mRNA中赋予野生型病毒感染细胞中mRNA选择性稳定性的序列; (2)确定TTP mRNA在野生型病毒感染细胞中免于降解的机制。(3)为了确定野生型病毒感染细胞中含有A-U丰富元件的mRNA与未感染细胞或感染细胞相比降解速率差异的基础？UL 41突变病毒，以及（4）确定现在与UL 41蛋白相关的许多功能是否是共变体，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.