Selective Degradation of mRNA by Herpes Simplex Virus 1

描述(申请人提供)：一种流行的观点认为，HSV-1 UL41被膜蛋白通过裂解5‘末端或附近的RNA来介导病毒和细胞mRNAs的非特异性降解，并且病毒基因的表达占优势，因为病毒基因的转录速度更快。我们的研究构成了这项赠款申请的基础，对这一观点的几个方面提出了挑战。具体地说：(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上调。验证研究(Northern分析、实时聚合酶链式反应和免疫印迹)揭示了4组mRNAs。以IEX-1为代表的第1组，c-fos、COX-2和Ikappabpha的mRNAs表达上调，但蛋白产物不表达。这些mRNAs通过去烯化、内切和3‘-5’过程性降解以UL41依赖的方式降解。此外，部分降解的mRNAs的5‘端结构域倾向于残留，并在细胞质提取液中很容易检测到。以TTP(tristetraprolin，TTP)为代表的第2组和以GADD45beta为代表的第3组也表达上调，但都是稳定的，而且确实是翻译的，同样都是以UL41依赖的方式！表达丰富但不上调的肌动蛋白mRNA迅速降解。形成基团2和3的RNA含有富含A-U的元素，具有快速翻转mRNAs的特征，而肌动蛋白和GADD45beta mRNAs则不含有这些元素。TTP是一种应激相关蛋白，通过结合和转运富含A-U的mRNAs到外体而参与这些mRNAs的降解。实质上，UL41蛋白介导的信使核糖核酸的降解不是无差别的，而是高度选择性的。这项应用有4个目的，即(1)鉴定TTP mRNA中的序列，使野生型病毒感染细胞中的mRNA具有选择性稳定性；(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与未感染细胞或UL41突变病毒感染细胞相比，含有富含A-U元件的mRNAs降解速度差异的基础，以及(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.