Modulation of histone deacetylase activity by a gammaherpesviral kinase

描述（由申请人提供）：伽玛疱疹病毒是普遍存在的病原体，在其宿主中建立终身感染。人类伽玛疱疹病毒eb病毒（EBV）和卡波西肉瘤相关疱疹病毒（KSHV）与多种疾病有关，包括几种癌症。命名为小鼠γ疱疹病毒68 （MHV68）的小鼠γ疱疹病毒作为一个模型系统，并提供了使用强大的小鼠遗传学来了解病毒与宿主的关系。所有的伽玛疱疹病毒都编码一种病毒蛋白激酶，该蛋白激酶磷酸化并与多种底物相互作用，尽管许多这些底物相互作用的生理相关性仍有待证实。作为核DNA病毒，疱疹病毒与细胞核内包裹DNA的细胞组蛋白相互作用形成染色质。组蛋白乙酰化是一种常见的翻译后修饰，可促进基因表达的增强。组蛋白去乙酰化酶（hdac）催化从组蛋白中去除乙酰基团，并被经典地认为是抑制转录。由于其抑制性质，hdac经常被认为是抗病毒的，并且一些病毒已经进化出灭活、破坏、重新定位或修饰hdac以促进感染的机制。例如，单纯疱疹病毒-1 （HSV-1）通过两种不同的病毒蛋白（ICP02和Us3激酶）进化出拮抗hdac的机制；我们发现orf36， MHV68激酶，在感染期间与hdac相互作用并调节hdac。我们假设这种调节促进了病毒感染的几个阶段，包括病毒原表达、DNA复制和感染性病毒粒子的产生。为了验证这一假设，我们将使用具有条件HDAC等位基因的细胞和小鼠。在体外细胞中切除hdac后，我们将通过qRT-PCR检测病毒基因表达和DNA复制，并通过斑块检测检测感染性病毒粒子的产生。在体内HDAC切除后，我们将通过斑块测定法测量病毒滴度峰值，并通过限制性稀释法测定病毒潜伏期参数。体外和体内技术的结合将包括对作用的定向而广泛的表征

DESCRIPTION (provided by applicant): Gammaherpesviruses are pervasive pathogens that establish lifelong infection in their hosts. The human gammaherpesviruses Epstein-Barr Virus (EBV) and Kaposi's Sarcoma Associated Herpesvirus (KSHV) are associated with diverse diseases, including several cancers. The murine gammaherpesvirus named mouse gammaherpesvirus 68 (MHV68) serves as a model system and provides for the use of powerful mouse genetics to understand the virus-host relationship. All gammaherpesviruses encode a viral protein kinase that phosphorylates and interacts with a multitude of substrates, though the physiological relevance of many of these substrate interactions remains to be verified. As nuclear DNA viruses, herpesviruses interact with cellular histone proteins that enwrap DNA in the nucleus to form chromatin. Histone acetylation is a common post-translational modification that promotes enhanced gene expression. Histone deacetylases (HDACs) catalyze the removal of acetyl moieties from histones and are classically considered to repress transcription. Due to their repressive nature, HDACs are frequently characterized as antiviral, and several viruses have evolved mechanisms to inactivate, disrupt, relocalize, or modify HDACs to facilitate infection. For example, herpes simplex virus-1 (HSV-1) has evolved mechanisms to antagonize HDACs through two distinct viral proteins: ICP02 and the Us3 kinase3;4. We have discovered that orf36, the MHV68 kinase, interacts with and modulates HDACs during infection. We hypothesize that this modulation facilitates viral infection at several stages, including viral gen expression, DNA replication, and infectious virion production. To test this hypothesis, we will use cells and mice with conditional HDAC alleles. After excision of HDACs in cells in vitro, we will measure viral gene expression and DNA replication via qRT-PCR and infectious virion production via plaque assay. After HDAC excision in vivo, we will measure peak viral titers via plaque assay and parameters of viral latency with limiting dilution assays. The combination of in vitro and in vivo techniques will encompass a directed, yet extensive, characterization of the role of HDACs in MHV68 infection. Herpesviral kinases are critical regulators of infection and represent a tractable target for therapeutics. Additionally, histone deacetylases have emerged as important therapeutic targets in cancer treatments. Thus, understanding the interactions between herpesviral kinases, chromatin, and chromatin-modifying enzymes is critical to the development of rational therapeutics. Several orf36 functions are conserved in other gammaherpesviral kinases; thus, the results of our studies are expected to translate to human pathogens.