The functions of the US3 protein kinase of herpes simplex virus

描述（由申请人提供）：本申请是研究单纯疱疹病毒（HSV）阻止宿主细胞自杀（凋亡）以阻止病毒复制的机制的延续和替代。在发现US3激酶阻断由缺陷病毒、活化的促凋亡基因或外源性药物（如山梨糖醇）诱导的细胞凋亡之后，发现(a) HSV编码US3的两个异构体US3和US3.5，只有全长US3激酶，而不是在蛋氨酸77处启动的US3.5激酶阻断细胞凋亡，(b) US3比US3.5更有效地使受感染细胞释放衣壳。(c) US3和US3.5磷酸化组蛋白去乙酰化酶1和2 (HDAC-1/2)，并使通过共转导（辅助功能）引入细胞的基因得以表达。在缺乏激酶的情况下，病毒在小鼠中完全无毒。目前的应用程序提出了一个更广泛的研究这种独特的调控病毒激酶。AIM 1的目的是确定US3蛋白激酶翻译后修饰的功能。US3蛋白通过自磷酸化、UL13病毒激酶和细胞激酶进行广泛的翻译后修饰。此外，有证据表明，它聚集形成更高阶的结构。这个目的的目的是阐明翻译后修饰在US3激酶的各种功能中的作用，通过适当的定位和突变位点。AIM 2的目标是绘制编码US3蛋白激酶关键功能的结构域，并确定每种功能的相互作用蛋白。我们发现抗凋亡功能和核包膜的修饰使衣壳从细胞核中释放在N端结构域，而激酶活性，HDAC-1/2的磷酸化和功能设计使病毒基因表达在c端与残基182相关。这些研究的目的是绘制负责抗凋亡活性和衣壳从细胞核出口的结构域，并识别和研究与这些结构域相互作用的病毒或细胞蛋白的作用。AIM 3的目的是验证US3蛋白激酶的辅助功能是由于HDAC1/2的磷酸化，而不是由于一些未知蛋白的磷酸化。AIM 4的目的是确定HSV-1 US3激酶的各种功能在HSV-1感染的神经元转运和发病机制中的作用。US3突变体是完全无毒的，并且在神经元运输中受损。我们想要确定上述US3蛋白激酶的各种功能对HSV在实验动物系统中引起发病率和死亡率的能力的贡献。本质上，US3激酶被证明是关键酶，它不仅阻止宿主细胞阻止病毒复制的尝试，而且在病毒的神经侵入和复制中也起着关键作用。病毒征服人类细胞的机制是当今病毒学的主要目标。公共卫生相关性：US3蛋白激酶是一种重要的蛋白，具有两套功能：它阻断宿主对感染的关键反应（阻断细胞凋亡，阻断宿主蛋白对DNA的沉默，并使干扰素3受体失活），并修饰病毒和细胞蛋白，以实现病毒的有效复制和传播。这些研究将阐明US3发挥这些功能的机制，有助于我们理解细胞凋亡和DNA沉默，并查明单纯疱疹病毒1的致病性。

DESCRIPTION (provided by applicant): This application is a continuation and replacement of a grant to study the mechanisms by which herpes simplex viruses (HSV) thwart attempts of the host cell to commit suicide (apoptosis) to block viral replication. The discovery that the US3 kinase blocks apoptosis induced by defective viruses, activated pro-apoptotic genes, or exogenous agents (e.g. sorbitol) was followed by the findings that (a) HSV encodes two isomers of US3 designated US3 and US3.5 that only the full- length US3 kinases, and not the US3.5 kinase initiating at methionine 77 blocks apoptosis, (b) US3 is more efficient than US3.5 in enabling the release of capsids from infected cells, (c) both US3 and US3.5 phosphorylate histone deacetylases 1 and 2 (HDAC-1/2) and enable the expression of genes introduced into cells by co-transduction (helper function). In the absence of the kinase, the virus is totally avirulent in mice. The present application proposes a broader investigation of this unique regulatory viral kinase. The objective of AIM 1 is to define the functions of post-translational modifications of the US3 protein kinase. The US3 protein is extensively posttranslationally modified by auto-phosphorylation, by UL13 viral kinase and by cellular kinases. In addition there is evidence that it aggregates to form higher order structures. The objective of this aim is to elucidate the role of posttranslational modifications in the various functions of the US3 kinase by appropriate mapping and mutagenesis of the sites. The objective of AIM 2 is to map the domains encoding key functions of the US3 protein kinase and identify the interacting proteins for each of the functions. We found that the anti-apoptotic functions and the modification of the nuclear envelope to enable release of capsids from nuclei map in the N- terminal domain whereas the kinase activity, the phosphorylation of HDAC-1/2 and functions designed to enable viral gene expression map C-terminal with respect to residue 182. The objective of these studies is to map the domains responsible for anti-apoptotic activity and egress of capsids from nuclei and to identify and investigate the role of viral or cellular proteins that interact with these domains. The objective of AIM 3 is to verify that the helper function of the US3 protein kinase is due to the phosphorylation of HDAC1/2 and not to the phosphorylation of some unknown proteins. The objective of AIM 4 is to define the role of the various functions of the HSV-1 US3 kinase in neuronal transport and pathogenesis of HSV-1 infections. The US3 mutants are totally avirulent and are impaired in neuronal transport. We want to determine the contributions of the various functions of the US3 protein kinase enumerated above to the ability of HSV to cause morbidity and mortality in experimental animals systems. In essence, the US3 kinases turn out to be key enzymes that not only block the attempts of the host cell to thwart viral replication but they also play a key role in neuroinvasiveness and replication of the virus. The mechanisms by which viruses conquer human cells are key objectives of virology today. PUBLIC HEALTH RELEVANCE: The US3 protein kinase is an essential protein that performs two sets of function: it blocks key host responses to infection (blocks apoptosis, blocks silencing of DNA by host proteins, and inactivates interferon 3 receptor) and modifies viral and cellular proteins for efficient replication and spread of virus. The proposed studies will elucidate the mechanisms by which US3 performs these functions, contribute to our understanding of apoptosis and silencing of DNA, and pinpoint the functions that account for the pathogenicity of herpes simplex virus 1.