Function of gammaherpesvirus ORF75 tegument proteins

描述（由申请方提供）：人γ疱疹病毒、EB病毒（EBV）和卡波西肉瘤相关疱疹病毒（KSHV/HHV-8）是全球疱疹病毒相关发病率和死亡率的主要原因。理解γ疱疹病毒进行生产性复制、促进潜伏和再激活的分子机制是合理设计临床治疗方法的关键。与病毒体一起递送到新感染的细胞中的被膜蛋白在病毒产生的早期和晚期都起着关键作用，并且是慢性疱疹病毒感染和相关疾病的不可或缺但尚未充分研究的组成部分。由于EBV和KSHV具有严格的宿主嗜性，且在细胞培养中缺乏稳健的病毒复制，因此很难确定被膜蛋白的作用。在这里，我们建议使用小鼠γ疱疹病毒68（MHV 68）来解剖γ疱疹病毒被膜蛋白在病毒复制和潜伏期的功能。令人兴奋的初步结果表明，ORF 75 A和ORF 75 B外皮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ORF 75 B无效病毒在感染小鼠脾脏中建立潜伏期的能力。该提议的中心假设是MHV 68的ORF 75 A和ORF 75 B被膜蛋白在从头产生性感染后的病毒复制期间具有可分离的作用，这将损害体内的潜伏期和再活化。该提案描述了创新的，高影响力的实验方法来阐明ORF 75被膜蛋白对γ疱疹病毒发病机制的贡献。在目的1中，我们将通过鉴定在条件性消融进入颗粒的被膜中的蛋白质或消融新合成的蛋白质时病毒复制中的缺陷来辨别ORF 75 A和ORF 75 B的作用。在目的2中，我们将使用腹腔内感染途径来规避鼻内接种途径后急性期的肺复制缺陷，以直接检查ORF 75 A和ORF 75 B在感染小鼠的B细胞和巨噬细胞中建立潜伏期和从潜伏期再激活中的作用。我们希望发现新的功能，将建立一个框架，为进一步的机制调查的ORF 75和提供新的见解被蛋白的作用，作为决定因素的慢性γ疱疹病毒感染和KSHV和EBV相关疾病。 公共卫生相关性：EB病毒和卡波西肉瘤相关疱疹病毒与免疫功能正常和免疫功能低下患者的许多癌症、肿瘤和疾病有很强的病因学联系，因此是世界范围内的重要病原体。鼠γ疱疹病毒-68模型系统提供了在从头感染培养和研究整个动物中疾病发展时检查生产性复制的多个方面的机会，使得能够快速验证细胞培养中鉴定的病毒-宿主相互作用的生物学相关性。这些研究的结果将阐明病毒被膜蛋白ORF 75 A和ORF 75 B在小鼠γ疱疹病毒发病机制中的关键作用，并可能对病毒相关疾病的预防和治疗产生翻译影响。!

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. PUBLIC HEALTH RELEVANCE: Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus have strong etiological links to numerous cancers, neoplasms, and disease in immunocompetent and immunocompromised patients, and as such, are significant pathogens worldwide. The murine gammaherpesvirus-68 model system provides the opportunity to examine multiple aspects of productive replication upon de novo infection culture and study disease development in the whole animal, enabling rapid validation of the biological relevance of virus-host interactions identified in cell culture. The information from these studies wil elucidate the key role that the viral tegument proteins ORF75A and ORF75B play in murine gammaherpesvirus pathogenesis and may lead to a translational impact on the prevention and treatment of virus-associated diseases. !