Function of gammaherpesvirus ORF75 tegument proteins

描述(申请人提供)：人类伽马疱疹病毒，爱泼斯坦-巴尔病毒(EBV)和卡波西肉瘤相关疱疹病毒(KSHV/HHV-8)，是全球与疱疹病毒相关的发病率和死亡率的主要原因。了解伽马疱疹病毒用于进行生产性复制、促进潜伏期和再激活的分子机制是合理设计临床治疗方法的关键。与病毒粒子一起进入新感染细胞的被膜蛋白在病毒生产的早期和后期都发挥着关键作用，是慢性疱疹病毒感染和相关疾病不可或缺但尚未得到充分研究的组成部分。由于EBV和KSHV严格的宿主嗜性和细胞培养中缺乏强大的病毒复制，因此很难确定被膜蛋白对EBV和KSHV的作用。在这里，我们建议使用小鼠伽马疱疹病毒68(MHV68)来检测伽马疱疹病毒被膜蛋白在病毒复制和潜伏期间的功能。令人振奋的初步结果表明，ORF75A和ORF75B被膜蛋白在病毒复制的早期和晚期具有不同的作用，这些功能削弱了ORF75B缺失病毒在感染小鼠的脾中建立潜伏期的能力。这一建议的中心假设是，MHV68的ORF75A和ORF75B被蛋白在病毒复制过程中具有分离的作用，这将损害体内的潜伏期和重新激活。这项建议描述了创新的、高影响力的实验方法，以阐明ORF75被膜蛋白在伽马疱疹病毒发病机制中的作用。在目标1中，我们将通过识别病毒复制中的缺陷来识别ORF75A和ORF75B的作用，这些缺陷是在有条件地消融进入颗粒的被膜中的蛋白质或新合成的蛋白质被消融时发生的。在目标2中，我们将使用腹腔感染途径绕过急性期经鼻接种途径的肺复制缺陷，直接检测ORF75A和ORF75B在感染小鼠B细胞和巨噬细胞建立潜伏期和从潜伏期重新激活中的作用。我们希望发现新的功能，这将为进一步的ORF75机制研究建立一个框架，并为被膜蛋白作为慢性伽马疱疹病毒感染以及KSHV和EBV相关疾病的决定因素所起的作用提供新的见解。

DESCRIPTION (provided by applicant): The human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8), are leading causes of herpesvirus-associated morbidity and mortality worldwide. An understanding of the molecular mechanisms used by gammaherpesviruses to undergo productive replication, and promote latency and reactivation is key to the rational design of clinical therapeutics. The tegument proteins that are delivered with the virion into a newly infected cell play critical roles during both early and late stages of virus production and are an integral, yet understudied component of chronic herpesvirus infection and associated disease. The role of tegument proteins are difficult to ascertain for EBV and KSHV given their strict host tropism and the lack of robust virus replication in cell culture. Here, we propose to use murine gammaherpesvirus 68 (MHV68) to disect the function of gammaherpesvirus tegument proteins during virus replication and latency. Exciting preliminary results indicate that the ORF75A and ORF75B tegument proteins have distinct roles during early and late stages of virus replication, and these functions impair the ability of the ORF75B null virus to establish latency in the spleens of infected mice. The central hypothesis of this proposal is that the ORF75A and ORF75B tegument proteins of MHV68 have separable roles during virus replication upon de novo productive infection that will impair latency and reactivation in vivo. This proposal describes innovative, high-impact experimental approaches to elucidate the contribution of ORF75 tegument proteins to gammaherpesvirus pathogenesis. In Aim 1, we will discern the role of ORF75A and ORF75B by identifying the defects in virus replication upon conditional ablation of the proteins in the tegument of incoming particles or ablation of newly synthesized protein. In Aim 2, we will use the intraperitoneal route of infection to circumvent lung replication defects in the acute phase upon the intranasal route of inoculation to directly examine the role of ORF75A and ORF75B in the establishment of latency and reactivation from latency in the B cells and macrophages of infected mice. We expect to uncover novel functions that will establish a framework for further mechanistic investigations of ORF75 and provide new insight into the role of tegument proteins as determinants of chronic gammaherpesvirus infection and KSHV- and EBV-associated diseases.