Defining mechanisms of KSHV pathogenesis using MHV68-KSHV chimeric viruses

使用 MHV68-KSHV 嵌合病毒定义 KSHV 发病机制

• 批准号: 10243300
• 负责人: James Craig Forrest
• 金额: $ 52.58万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-14 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10243300
• 关键词: Acquired Immunodeficiency Syndrome; Animal Model; Antiviral Agents; Biological Models; Cell Lineage; Cell physiology; Cells; Chimera organism; Chronic; Cytometry; DNA Tumor Viruses; DNA-Binding Proteins; Development; Disease; Episome; Evaluation; Fostering; G-Protein-Coupled Receptors; Gene Transfer; Genes; Genetic Transcription; Genome; Goals; HIV; Herpesviridae Infections; Human; Human Herpesvirus 8; Immune; Immune response; Immunity; Individual; Infection; Inflammation; Inflammatory; Kaposi Sarcoma; Knock-in; Laboratory mice; Lymphocyte; Lytic; Maintenance; Malignant Neoplasms; Methods; Modeling; Molecular; Morbidity - disease rate; Multicentric Angiofollicular Lymphoid Hyperplasia; Mus; Oncogenic; Oncoproteins; Organism; Outcome; Pathogenesis; Pathogenicity; Persons; Preclinical Testing; Prevention; Process; Production; Proteins; Risk; Rodent; Role; Signal Pathway; System; Time; Trans-Activators; Transcriptional Regulation; Transgenic Mice; Viral; Viral Genes; Viral Pathogenesis; Viral Proteins; Virus; Virus Diseases; Virus Latency; Virus Replication; Work; adaptive immune response; adaptive immunity; antiretroviral therapy; cancer risk; cell type; chronic infection; experimental study; gammaherpesvirus; gene replacement; humanized mouse; improved; in vivo; infected B cell; latency-associated nuclear antigen; lytic replication; mortality; next generation; novel strategies; pathogen; prevent; primary effusion lymphoma; promoter; protein function; response; single-cell RNA sequencing; targeted treatment; therapy development; tissue culture; tool; viral cyclin

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.

项目摘要 γ疱疹病毒（GHV）是DNA肿瘤病毒，其建立宿主的终身、慢性感染 淋巴细胞操纵宿主细胞生理学和阻碍抗病毒的病毒基因产物的表达 免疫反应使受感染的宿主处于多种癌症的风险中。对于艾滋病患者， 卡波西肉瘤相关疱疹病毒（KSHV）是发病率和死亡率的主要原因。然而，KSHV 不容易感染小鼠，这使得试图确定KSHV建立长- 术语感染和疾病。为了克服这一障碍，我们使用了嵌合病毒的方法，其中KSHV 基因转移到密切相关的病毒，鼠γ疱疹病毒68（MHV 68），这是一种天然的病毒。 容易感染实验室老鼠的啮齿动物病原体。我们使用这种MHV 68-KSHV嵌合病毒方法， 在生产性病毒复制期间评价KSHV潜伏相关核抗原（kLANA），潜伏期 建立和维护。我们发现k拉娜足以取代MHV 68拉娜（m拉娜）， 病毒潜伏期然而，kLANA，而不是mLANA，抑制MHV 68裂解复制通过抑制的活性， 裂解性反式激活因子RTA的启动子。我们的工作表明，嵌合病毒提供了机会， 解剖KSHV蛋白在活宿主中的功能，并且该系统可以定义进化获得的 病毒癌蛋白的特征。本提案中描述的实验将进一步定义KSHV拉娜的功能 在病毒发病机制和进化分歧从mLANA。我们将使用受抑制的裂解性复制， 表达kLANA的嵌合病毒，以确定裂解复制的幅度如何影响对感染的免疫力 以及病毒的致癌潜力。最后，我们将用拉娜扩展我们的嵌合病毒研究， 将它们与用于v-Cyclin和v-GPCR的MHV 68-KSHV嵌合病毒一起，以开发下一代嵌合体 其中多个MHV 68基因被它们的KSHV对应物替换。通过这项工作，我们将提高 小动物模型使KSHV癌蛋白在病毒感染和疾病中的分子机制研究成为可能 并提供一个临床前测试平台，以促进靶向拉娜和其他病毒的治疗方法的开发， 治疗或预防KSHV相关癌症的蛋白质。