Investigation of viral and host determinants of gammaherpesvirus pathogenesis

在细胞培养系统中，STAT3的激活与伽马疱疹病毒潜伏期和癌症有关，但驱动潜伏期、增殖和转化的靶基因还没有很好的定义。作为研究STAT3激活在KSHV中的作用的前奏，我们使用了B细胞特异性STAT3基因敲除的转基因小鼠来确定STAT3信号在宿主原代B细胞中建立伽马疱疹病毒潜伏期的关键。我们还确定了病毒裂解基因反式激活因子RTA以细胞因子依赖的方式与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。为了确定感染B细胞的STAT3依赖的基因表达程序，我们正在使用RNAseq来比较感染细胞中有和没有STAT3的基因表达谱。我们将从所有可用的伽马疱疹病毒数据集中合并差异表达的基因，以识别和优先考虑STAT3调节的共同途径和靶基因。这将导致使用条件基因敲除方法进一步研究KSHV和小鼠细胞培养系统中的潜伏期、重新激活和转化。参与核苷酸代谢的众多疱疹病毒基因表明，核苷酸池是未分裂细胞的主要限制点。我们最近报道，病毒尿嘧啶DNA糖基酶(UNG)的酶性质与病毒核苷酸代谢蛋白dUTPase协同作用，促进病毒基因组中基于重组的缺失，促进发病。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合成的过程和基因组的稳定性。我们在小鼠系统中产生了多个病毒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。这些突变体将为设计KSHV重组病毒和胸苷合成的小分子抑制剂提供信息，以测试尿嘧啶掺入对潜伏期重新激活的影响。诱导DNA损伤反应和阻断病毒DNA复制可能为削弱KSHV的重新激活和预防艾滋病提供一个新的靶点。我正在与HAMB的其他NCI研究人员和临床医生合作，分析参加HAMB临床试验的HIV感染的原发渗出性淋巴瘤患者的KSHV感染细胞的B细胞库和HIV感染的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，这可能为这些疾病的可能病因提供信息，这些疾病经常以并发或顺序的方式在艾滋病毒携带者中发生。此外，在与麦克布莱德实验室的合作中，我们使用下一代测序技术分析了感染小鼠和未感染小鼠的B细胞的免疫球蛋白谱。这一分析表明，感染了伽马疱疹病毒的细胞经历了克隆性扩增，但感染和未感染的生发中心B细胞之间只有少数克隆共享。也有证据表明，在受感染的B细胞中，受体编辑和特定的IghV基因存在明显的偏见。这些新的数据表明，伽马疱疹病毒在受感染的宿主中占据了一个独特的生态位，并在颠覆其感染的B细胞中的免疫球蛋白谱系方面发挥了积极作用。这支持了与KSHV和EBV共同的关键策略的存在，即这些病毒绕过正常的选择过程，将B细胞置于遗传不稳定的风险中，并产生具有病理潜力的免疫球蛋白。关于KS的直接动物模型的开发，我正在与HIV/AIDS恶性病科和NCI Frederick的高级临床前研究中心(CAPR)的临床医生合作，以建立患者来源的异种移植模型。来自HAMB临床队列中艾滋病毒感染者的KS活检材料正被植入免疫缺陷动物体内。从该系统获得的维持KSHV感染和驱动小鼠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，也没有临床前的Kaposi肉瘤动物模型来筛选有效的药物干预。

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.