Defining mechanisms of KSHV pathogenesis using MHV68-KSHV chimeric viruses

项目总结 伽马疱疹病毒(GHV)是一种dna肿瘤病毒，可终生、慢性感染宿主。 淋巴细胞。调控宿主细胞生理和抗病毒作用的病毒基因产物的表达 免疫反应使受感染的宿主面临患多种癌症的风险。对于爱滋病患者来说，感染 卡波西肉瘤相关疱疹病毒(KSHV)是导致发病和死亡的主要原因。然而，KSHV 不容易感染小鼠，这使得试图定义KSHV建立长期- 长期感染和疾病。为了克服这一障碍，我们使用了嵌合病毒方法，在这种方法中，KSHV 基因被转移到密切相关的病毒，小鼠伽马疱疹病毒68(MHV68)，这是一种天然的 容易感染实验室老鼠的啮齿动物病原体。我们使用这种MHV68-KSHV嵌合病毒方法 评估KSHV潜伏期相关核抗原(KLANA)在病毒生产性复制、潜伏期 建立和维护。我们发现，kLANA足以取代MHV68 LANA(MLANA) 病毒潜伏期。然而，kLANA，而不是mLANA，通过抑制MHV68的活性来抑制MHV68的裂解复制 裂解反式激活因子RTA的启动子。我们的工作表明，嵌合病毒为 解剖KSHV蛋白在活体宿主中的功能，以及该系统可以定义进化获得的 一种病毒癌蛋白的特征。本提案中描述的实验将进一步定义KSHV LANA的功能 在病毒致病机制和进化上与mLANA的分歧。我们将使用被抑制的裂解复制 表达KLANA的嵌合病毒确定裂解复制的幅度如何影响感染免疫 以及病毒的致癌潜力。最后，我们将扩展我们的嵌合病毒研究与LANA，吻合 将它们与MHV68-KSHV嵌合病毒进行v-Cyclin和v-GPCR，以开发下一代嵌合体 其中多个MHV68基因被其对应的KSHV基因替换。通过这项工作，我们将提高 用小动物模型研究KSHV癌蛋白在病毒感染和疾病中的分子机制 并提供临床前测试平台，以促进针对LANA和其他病毒的疗法的开发 治疗或预防KSHV相关癌症的蛋白质。

PROJECT SUMMARY Gammaherpesviruses (GHVs) are DNA tumor viruses that establish lifelong, chronic infections of host lymphocytes. The expression of viral gene products that manipulate host cell physiology and thwart antiviral immune responses places the infected host at risk for numerous cancers. For individuals with AIDS, infection by Kaposi sarcoma-associated herpesvirus (KSHV) is a major cause of morbidity and mortality. However, KSHV does not readily infect mice, which complicates attempts to define mechanisms by which KSHV establishes long- term infections and disease. To overcome this barrier, we have used a chimeric virus approach in which KSHV genes are transferred into the closely related virus, murine gammaherpesvirus 68 (MHV68), which is a natural rodent pathogen that readily infects laboratory mice. We used this MHV68-KSHV chimeric virus approach to evaluate the KSHV latency-associated nuclear antigen (kLANA) during productive viral replication, latency establishment, and maintenance. We found that kLANA was sufficient to replace MHV68 LANA (mLANA) for viral latency. However, kLANA, but not mLANA, suppressed MHV68 lytic replication by inhibiting the activity of the promoter for lytic transactivator RTA. Our work demonstrates that chimeric viruses offer opportunities for dissecting functions of KSHV proteins in a living host, and that this system can define evolutionarily acquired features of a viral oncoprotein. Experiments described in this proposal will further define functions of KSHV LANA in viral pathogenesis and evolutionary divergence from mLANA. We will use the repressed lytic replication of the kLANA-expressing chimeric virus to define how the amplitude of lytic replication influences immunity to infection and oncogenic potential of the virus. Finally, we will extend our chimeric virus studies with LANA, dovetailing them with MHV68-KSHV chimeric viruses for v-Cyclin and v-GPCR, to develop the next generation of chimeras in which multiple MHV68 genes are replaced with their KSHV counterparts. Through this work we will improve small animal models to enable molecular mechanistic studies of KSHV oncoproteins in viral infection and disease and provide a preclinical testing platform to foster the development of therapies that target LANA and other viral proteins to treat or prevent KSHV-related cancers.