Selective Degradation of mRNA by Herpes Simplex Virus 1

描述(申请人提供)：病毒粒子宿主关闭蛋白(VHS)是单纯疱疹病毒1型(HSV-1)UL41基因的产物，是宿主对感染反应的主要病毒抑制因子。VHS通过病毒粒子进入受感染的细胞，其主要功能是降解mRNA。我们对VHS的研究彻底改变了我们对蛋白质功能的理解。具体地说：(I)我们开发了一种VHS的增溶和纯化程序，使其达到均一状态。纯化的VHS是一种内切核糖核酸酶，在没有细胞或病毒蛋白的情况下，利用RNaseA的特异性切割合成的RNA。(Ii)早先的报告指出，在感染后的中后期，VHS“被病毒蛋白VP16中和”。我们发现，“中和”需要形成一个除了VP16之外还包括VP22的复合体。(3)早期报道表明VHS介导的信使核糖核酸的降解是非选择性的。我们已经鉴定和量化了3类依赖VHS的mRNAs的降解情况如下：A类由通常稳定的mRNAs组成(例如肌动蛋白、GAPDHmRNAs、正常半衰期和12h)。在受感染的细胞中，这些mRNAs在30分钟内被降解5‘到3’。B类包括感染后即刻诱导的应激反应mRNAs(如c-fos、COX-2、I：B1和IEX-1mRNAs)。这些mRNAs在其3‘UTRs中含有富含AU的元素。这些mRNAs在模拟感染细胞中的典型半衰期为30到45分钟。感染后早期，这些mRNAs去烯化，并将5‘端裂解成富含AU的元素。残留部分的半衰期为3小时。在后期(感染后6+h)，这些mRNAs的降解依赖于ICP27。A类和B类mRNAs均在多聚核糖体中降解。C类包括未降解的mRNA。这些mRNAs的产物包括Tristetraprolin和GADD452，Tristetraprolin是一种胁迫诱导蛋白，它与AU丰富的元素结合，并将mRNAs隔离在外切体中，进行3‘到5’的降解。(Iv)先前的报道表明，VHS结合了eIF4F复合体的3个组分，即eIF4H、eIF4B、eIF4AII和VP16。我们的研究表明，VHS还与Tristetraprolin、ICP27以及通过VP16与VP22发生物理相互作用。该研究提案的目标是测试A类和B类mRNAs的降解模型。该模型提出，如报道的那样，VHS降解多聚核糖体中的mRNAs，它与eIF4F的一个仍未定义的成分结合，并通过切割帽结构下游的稳定mRNAs来模拟解帽酶的功能。然后，残留的部分被隔离在P小体中并迅速降解。在B类mRNAs的情况下，VHS与Tristetraprolin结合，并将mRNA5‘切割到富AU元素。在没有结合的Tristetraprolin的情况下，残留的mRNA不能被外体迅速招募来降解3‘到5’。我们还建议区分ICP27的两个可供选择的潜在功能，使感染后期的mRNA降解。最后，鉴于VHS的多重功能，出现了每个功能在多大程度上对HSV-1的致病潜力有贡献的问题。 与公共卫生相关：细胞对病毒进入细胞作出强烈反应，单纯疱疹病毒进化出一种复杂的策略来阻止细胞干扰病毒复制。细胞反应的主要抑制因子之一是一种由病毒带入细胞的酶，它能降解因感染而产生的mRNA。这项拨款申请研究了这种酶选择性地降解因感染而产生的mrna的机制。

DESCRIPTION (provided by applicant): Virion host shutoff protein (VHS), the product of UL41 gene of herpes simplex virus 1(HSV-1) is the major viral suppressor of host responses to infection. VHS is brought into the infected cells by the virion and its primary function is to degrade mRNA. Our studies on VHS have completely revolutionized our understanding perceptions of the functions of the protein. Specifically: (i) We have developed a procedure for solubilization and purification of VHS to homogeneity. Purified VHS is an endoribonuclease that cleaves synthetic RNA with the specificity of RNase A in the absence of cellular or viral proteins. (ii) Earlier reports indicated that at mid and late times after infection VHS is "neutralized by the viral protein VP16. We showed that "neutralization" required the formation of a complex that includes VP22 in addition to VP16. (iii) Earlier reports indicated that the degradation of mRNA mediated by VHS is nonselective. We have identified and quantified the VHS- dependent degradation of 3 classes of mRNAs as follows: Class A consists of mRNAs that are normally stable (e.g. actin, GAPDH mRNAs, normal half life >12 h). In infected cells these mRNAs are degraded 5' to 3' within 30 minutes. Class B consists of stress response mRNAs (e.g. c-fos, cox-2, I:B1 and IEX-1 mRNAs) induced immediately after infection. These mRNAs contain AU-rich elements within their 3' UTRs. Typical half life of these mRNAs in mock infected cells is 30 to 45 min. Early after infection, these mRNAs are deadenylated and cleaved 5' to AU-rich elements. The residual portions linger with a half life >3 hrs. At late times (6+ h after infection) degradation of these mRNAs is dependent on ICP27. Both class A and class B mRNAs are degraded in polyribosomes. Class C includes mRNAs that are not degraded. The products of these mRNAs include tristetraprolin, a stress inducible protein that binds to AU-rich elements and sequesters the mRNAs to exosomes for 3' to 5' degradation and GADD452 a transcriptional factor. (iv) Earlier reports indicated that VHS binds 3 components of eIF4F complex, i.e. eIF4H, eIF4B and eIF4AII and VP16. Our studies have shown that VHS also interacts physically with tristetraprolin, ICP27, and through VP16 with VP22. The objective of the research proposal is to test a model of the degradation of the mRNAs in classes A and B. The model proposes that VHS degrades mRNAs in polyribosomes as reported, that it binds to a still to be defined component of eIF4F and mimics the function of the decapping enzymes by cleaving stable mRNAs downstream of the cap structure. The residual portion is then sequestered in P-bodies and rapidly degraded. In the case of class B mRNAs, VHS binds to tristetraprolin bound to AU-rich element mRNA and cleaves the mRNA 5' to the AU-rich elements. In the absence of bound tristetraprolin, the residual mRNA is not recruited promptly for degradation 3' to 5' by exosomes. We also propose to discriminate between 2 alternative potential functions of ICP27 in enabling mRNA degradation late in infection. Finally, given the multiple functions of VHS, the question arises as to the extent to which each function contributes to the pathogenic potential of HSV-1. PUBLIC HEALTH RELEVANCE: Cells respond vigorously to the entry of a virus into cells and Herpes simplex viruses evolved a complex strategy to block the cell from interfering with viral replication. One of the major suppressors of cellular responses is an enzyme brought into the cells by the virus that degrades mRNA made in response to infection. This grant application investigates the mechanisms by which this enzymes selectively degrades mRNA made in response to infection