Investigation of viral and host determinants of gammaherpesvirus pathogenesis

伽玛疱疹病毒发病机制的病毒和宿主决定因素的研究

• 批准号: 10262568
• 负责人: Laurie T Krug
• 金额: $ 155.95万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262568
• 关键词: AIDS related cancer; AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Animal Model; Animals; Area; B cell repertoire; B-Lymphocytes; Basic Science; Biological; Biopsy; Bypass; Cancer Burden; Cause of Death; Cell Culture System; Cell Culture Techniques; Cell Line; Cell Nucleus; Cells; Clinical; Clinical Trials; Clonal Expansion; Clonality; Collaborations; Complex; DNA; DNA Damage; DNA Repair; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Data Set; Development; Disease; Endothelial Cells; Enrollment; Etiology; Event; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Recombination; Genetic Risk; Genome Stability; HIV; Herpesviridae; Herpesviridae Infections; Host Defense; Human; Human Herpesvirus 4; Human Herpesvirus 8; Immune response; Immunoglobulins; Immunomodulators; Implant; Individual; Infection; Infection prevention; Interphase Cell; Intervention; Investigation; Kaposi Sarcoma; Knock-out; Knowledge; Laboratories; Life; Lytic; Malignant - descriptor; Malignant Neoplasms; Molecular; Morbidity - disease rate; Multicentric Angiofollicular Lymphoid Hyperplasia; Mus; Nucleotides; Oncogenic; Pathogenesis; Pathogenicity; Pathologic; Pathway interactions; Patients; Persons; Pharmaceutical Preparations; Process; Property; Proteins; Reporting; Research; Research Personnel; Risk; Role; STAT3 gene; Sampling; Series; Signal Transduction; Source; Structure of germinal center of lymph node; System; T-Lymphocyte; Testing; Therapeutic; Therapeutic Intervention; Thymidine; Trans-Activators; Transgenic Mice; Uracil; Viral; Viral Genome; Viral Pathogenesis; Virus; Virus Replication; Xenograft Model; antiretroviral therapy; base; cell transformation; cohort; conditional knockout; cytokine; dUTP pyrophosphatase; design; differential expression; gammaherpesvirus; infected B cell; lymph nodes; mortality; mouse model; mutant; next generation sequencing; novel; nucleotide metabolism; pathogen; patient response; pre-clinical; pre-clinical research; prevent; primary effusion lymphoma; programs; protein metabolism; reactivation from latency; receptor; recombinant virus; response; small molecule inhibitor; transcriptome sequencing; tumor; uracil-DNA glycosylase; viral DNA; virus host interaction

STAT3 activation is associated with gammaherpesvirus latency and cancer in cell culture systems, but the target genes that drive latency, proliferation and transformation are not well-defined. As a prelude to studying the effects of STAT3 activation in KSHV itself, we used transgenic mice with a B cell-specific knock-out of STAT3 to determine that STAT3 signaling is critical for the establishment of gammaherpesvirus latency in primary B cells of the host. We also determined that the viral lytic gene transactivator RTA interacts with STAT3 in a cytokine-dependent manner. Our central hypothesis is that gammaherpesviruses require STAT3 to promote B cell latency. To define the STAT3-dependent gene expression program of infected B cells, we are performing RNAseq to compare the gene expression profile in infected cells with and without STAT3. We will merge differentially expressed genes from all available gammaherpesvirus datasets to identify and prioritize common STAT3-regulated pathways and target genes. These will lead to further investigations of latency, reactivation, and transformation in KSHV and murine cell culture systems using conditional knock-out approaches. The numerous herpesvirus genes involved in nucleotide metabolism indicate that the nucleotide pool is a major restriction point in non-dividing cells. We recently reported that the enzymatic properties of the viral uracil DNA glycosylase (UNG) synergize with the viral nucleotide metabolism protein dUTPase to promote pathogenesis and counter recombination-based deletions in the viral genome. Our central hypothesis is that the viral UNG coordinates with the viral DNA polymerase to promote processivity of viral DNA synthesis and genome stability by protecting newly synthesized DNA from host UNG and DNA repair factors. We have generated multiple viral UNG mutants in the mouse system to examine their impact on viral DNA replication and genome stability and the interactions of the vUNG with other viral replication factors and host DNA repair factors. These mutants will inform the design of KSHV recombinant viruses and small molecule inhibitors of thymidine synthesis to test for the impact of uracil incorporation on reactivation from latency. The induction of a DNA damage response and block in viral DNA replication might provide a novel target to cripple KSHV reactivation and prevent AIDS malignancies. I am collaborating with other NCI investigators and clinicians in HAMB to analyze the B cell repertoire of KSHV-infected cells in HIV-associated primary effusion lymphoma and lymph node material of HIV-infected KSHV-multicentric Castleman disease patients enrolled in HAMB clinical trials. This will enable us to examine the clonality and source of the B cell transformation. Importantly, this might inform the possible etiology of these diseases which often occur in a concurrent or sequential manner in people living with HIV. Also, in collaboration with the McBride lab, we analyzed the immunoglobulin repertoire of infected and uninfected B cells from infected mice using next generation sequencing. This analysis revealed that gamamherpesvirus-infected cells undergo clonal expansion yet only a few clones were shared between the infected and uninfected germinal center B cells. There is also evidence for receptor editing and clear bias for specific IghV genes in the infected B cells. These novel data indicate the gammaherpesviruses occupy a distinct niche in the infected host and takes an active role in subverting the immunoglobulin repertoire in the B cells that it infects. This supports the existence of a critical strategy that is shared with KSHV and EBV, namely that these viruses bypass normal selection processes, placing the B cells at risk for genetic instability and for producing immunoglobulins with pathologic potential. With regard to the development of direct animal models of KS, I am collaborating with clinicians in the HIV and AIDS Malignancy Branch and in the Center for Advanced Preclinical Research (CAPR) of NCI Frederick towards the establishment of a patient-derived xenograft model. KS biopsy material from HIV infected patients in the HAMB clinical cohorts is being implanted into immunodeficient animals. Cell lines derived from this system that maintain KSHV infection and drive tumors in mice will be a tremendous advancement for the field since there is no way to maintain KSHV+ endothelial cells upon explant and there is no pre-clinical animal model of Kaposi sarcoma to screen for effective drug interventions.

STAT3 激活与细胞培养系统中的伽马疱疹病毒潜伏期和癌症相关，但驱动潜伏期、增殖和转化的靶基因尚不明确。作为研究 STAT3 激活对 KSHV 本身影响的前奏，我们使用 B 细胞特异性敲除 STAT3 的转基因小鼠来确定 STAT3 信号传导对于宿主原代 B 细胞中伽马疱疹病毒潜伏期的建立至关重要。我们还确定病毒裂解基因反式激活子 RTA 以细胞因子依赖性方式与 STAT3 相互作用。我们的中心假设是伽马疱疹病毒需要 STAT3 来促进 B 细胞潜伏期。为了定义受感染 B 细胞的 STAT3 依赖性基因表达程序，我们正在进行 RNAseq 来比较有和没有 STAT3 的受感染细胞中的基因表达谱。我们将合并所有可用的伽马疱疹病毒数据集中的差异表达基因，以识别和优先考虑常见的 STAT3 调节途径和目标基因。这些将导致使用条件敲除方法对 KSHV 和鼠细胞培养系统中的潜伏期、重新激活和转化进行进一步研究。参与核苷酸代谢的众多疱疹病毒基因表明核苷酸库是非分裂细胞中的主要限制点。我们最近报道，病毒尿嘧啶 DNA 糖基化酶 (UNG) 的酶特性与病毒核苷酸代谢蛋白 dUTPase 协同作用，促进发病机制并对抗病毒基因组中基于重组的缺失。我们的中心假设是，病毒 UNG 与病毒 DNA 聚合酶协调，通过保护新合成的 DNA 免受宿主 UNG 和 DNA 修复因子的影响，促进病毒 DNA 合成的持续性和基因组稳定性。我们在小鼠系统中产生了多种病毒 UNG 突变体，以检查它们对病毒 DNA 复制和基因组稳定性的影响以及 vUNG 与其他病毒复制因子和宿主 DNA 修复因子的相互作用。这些突变体将为 KSHV 重组病毒和胸苷合成小分子抑制剂的设计提供信息，以测试尿嘧啶掺入对潜伏期重新激活的影响。 DNA 损伤反应的诱导和病毒 DNA 复制的阻断可能为削弱 KSHV 重新激活和预防艾滋病恶性肿瘤提供新的靶点。我正在与 HAMB 的其他 NCI 研究人员和临床医生合作，分析 HIV 相关原发性渗出性淋巴瘤中 KSHV 感染细胞的 B 细胞库，以及参加 HAMB 临床试验的 HIV 感染 KSHV 多中心 Castleman 病患者的淋巴结材料。这将使我们能够检查 B 细胞转化的克隆性和来源。重要的是，这可能会揭示这些疾病的可能病因，这些疾病通常在艾滋病毒感染者中同时或相继发生。此外，我们与 McBride 实验室合作，使用下一代测序分析了受感染小鼠的受感染和未感染 B 细胞的免疫球蛋白库。该分析表明，伽马疱疹病毒感染的细胞经历克隆扩增，但感染和未感染的生发中心 B 细胞之间仅共享少数克隆。还有证据表明受感染 B 细胞中的受体编辑和特定 IghV 基因存在明显偏差。这些新数据表明，伽玛疱疹病毒在受感染宿主中占据了独特的生态位，并在破坏其感染的 B 细胞中的免疫球蛋白库方面发挥着积极作用。这支持了与 KSHV 和 EBV 共享的关键策略的存在，即这些病毒绕过正常的选择过程，使 B 细胞面临遗传不稳定和产生具有病理潜力的免疫球蛋白的风险。关于 KS 直接动物模型的开发，我正在与 HIV 和艾滋病恶性肿瘤部门以及 NCI Frederick 高级临床前研究中心 (CAPR) 的临床医生合作，建立患者来源的异种移植模型。 HAMB 临床队列中 HIV 感染患者的 KS 活检材料正在被植入免疫缺陷动物体内。从该系统衍生的维持 KSHV 感染并在小鼠中驱动肿瘤的细胞系将是该领域的巨大进步，因为没有办法在外植体上维持 KSHV+ 内皮细胞，并且没有卡波西肉瘤的临床前动物模型可供筛选。有效的药物干预。