Investigation of viral and host determinants of gammaherpesvirus pathogenesis

(1)STAT 3激活与细胞培养系统中的γ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未明确。作为研究KSHV自身中STAT 3激活作用的前奏，我们使用了具有B细胞特异性敲除STAT 3的转基因小鼠来确定STAT 3信号传导对于宿主的原代B细胞中γ疱疹病毒潜伏期的建立是至关重要的。我们还确定了病毒裂解基因反式激活因子RTA与STAT 3以精氨酸依赖的方式相互作用。我们的中心假设是γ疱疹病毒需要STAT 3来促进B细胞潜伏期。为了确定受感染的B细胞的STAT 3依赖性基因表达程序，我们正在进行RNAseq以比较有和没有STAT 3的受感染细胞中的基因表达谱。我们将从所有可用的γ疱疹病毒数据集中合并差异表达的基因，以确定和优先考虑常见的STAT 3调节途径和靶基因。这些将导致进一步调查的潜伏期，再活化，转化KSHV和鼠细胞培养系统中使用条件敲除的方法。(2)参与核苷酸代谢的许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。我们最近报道了病毒尿嘧啶DNA糖基化酶（UNG）的酶性质与病毒核苷酸代谢蛋白dUTR协同作用，以促进致病和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。我们已经在小鼠系统中产生了多个病毒UNG突变体，以研究它们对病毒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，以检测尿嘧啶掺入对潜伏期再激活的影响。诱导DNA损伤反应和阻断病毒DNA复制可能为削弱KSHV再激活和预防AIDS恶性肿瘤提供新的靶点。(2)此外，我们与McBride实验室合作，使用下一代测序分析了感染小鼠的感染和未感染B细胞的免疫球蛋白库。该分析显示，γ-疱疹病毒感染的细胞经历克隆扩增，但只有少数克隆在感染和未感染的生发中心B细胞之间共享。也有证据表明受体编辑和感染的B细胞中特异性IghV基因的明显偏倚。这些新的数据表明，γ疱疹病毒在感染宿主中占据了一个独特的生态位，并在破坏其感染的B细胞中的免疫球蛋白库方面发挥了积极作用。这支持了KSHV和EBV共有的关键策略的存在，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性和产生具有病理潜力的免疫球蛋白的风险中。(4)我正在分析HAMB临床试验中患者外周血单核细胞中的宿主免疫应答。我们正在使用流式细胞术来检查先天性和适应性免疫反应是否在HIV+和HIV-患者之间存在差异。我们还研究了在临床试验中用于治疗这些患者的化疗和免疫疗法是否会改变免疫反应。目的是确定免疫相关的临床反应的无反应者和反应者，以找到有效的治疗艾滋病相关的恶性肿瘤。我还与其他NCI研究人员和HAMB临床医生合作，分析HAMB临床试验中HIV相关原发性渗出性淋巴瘤和HIV感染KSHV多中心Castleman病患者淋巴结材料中KSHV感染细胞的B细胞库。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，这可能会告知这些疾病的可能病因，这些疾病通常以并发或顺序的方式发生在艾滋病毒感染者中。(5)关于卡波西肉瘤（艾滋病的标志性肿瘤）的直接动物模型的开发，我正在与NCI Frederick的HIV和AIDS肿瘤分支和高级临床前研究中心（CAPR）的临床医生合作，开展更多转化项目，以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活检材料正在植入免疫缺陷动物体内。源自该系统的细胞系维持KSHV感染并驱动小鼠中的肿瘤将是该领域的巨大进步，因为在外植体时没有办法维持KSHV+内皮细胞，并且没有卡波西肉瘤的临床前动物模型来筛选有效的药物干预。

(1) 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. (2) 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. (2) 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. (4) I am analyzing the host immune response in the peripheral blood mononuclear cells of patients in HAMB clinical trials. We are using flow cytometry to examine if the innate and adaptive immune response differs between HIV+ and HIV- patients. We also study if the immune response changes with the chemotherapies and immunotherapies that are used to treat these patients in the clinical trials. The goal is to identify immune correlates of the clinical response for non-responders and responders to find effective therapies for AIDS associated malignancies. I am also 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. (5) With regard to the development of direct animal models of Kaposi sarcoma, a hallmark tumor of AIDS, I am collaborating with clinicians in the HIV and AIDS Malignancy Branch and in the Center for Advanced Preclinical Research (CAPR) of NCI Frederick in more translational projects 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.