Investigation of viral and host determinants of gammaherpesvirus pathogenesis

(1)在细胞培养系统中，STAT3激活与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STAT3激活对KSHV自身影响的前奏，我们使用B细胞特异性敲除STAT3的转基因小鼠来确定STAT3信号对于在宿主原代B细胞中建立γ疱疹病毒潜伏期至关重要。我们还确定病毒裂解基因反激活因子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细胞中STAT3依赖的基因表达程序，我们使用RNAseq来比较感染细胞中STAT3和不感染细胞的基因表达谱。到目前为止，我们已经确定了STAT3的前病毒作用，通过STAT3抑制STAT1/2和许多抗病毒干扰素刺激基因来抑制B细胞中的干扰素反应。我们将合并来自所有可用的伽玛疱疹病毒数据集的差异表达基因，以确定和优先考虑共同的stat3调控途径和靶基因。这些将导致使用条件敲除方法进一步研究KSHV和小鼠细胞培养系统中的潜伏期、再激活和转化。（2a）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的主要限制性点。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dUTPase协同作用，促进病毒基因组中基于重组的缺失的发病和对抗。我们目前正在探索病毒UNG的蛋白结合伙伴。与Kevin McBride合作，我们正在研究病毒和宿主UNG的不同生化特性。（2b）此外，我们与麦克布莱德实验室合作，利用下一代测序分析了感染小鼠感染和未感染B细胞的免疫球蛋白库。该分析表明，伽玛疱疹病毒感染的细胞进行克隆扩增，但在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细胞中存在受体编辑和特异性IghV基因的明显偏倚。这些新数据表明，伽玛疱疹病毒在被感染的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细胞处于遗传不稳定和产生具有病理潜力的免疫球蛋白的风险中。我还与HAMB的其他NCI研究人员和临床医生合作，分析hiv相关原发性积液淋巴瘤中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病患者的淋巴结物质。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，这些疾病通常在艾滋病毒感染者中并发或顺序发生。(3)我们正在研究病毒核糖核苷酸还原酶大亚基将宿主核防御因子（称为PML-NB）重新定位为轨道状结构的机制。我们假设病毒的这种重新定位中和了它们的抗病毒功能并促进了生产性感染。(4)关于艾滋病标志性肿瘤卡波西肉瘤的直接动物模型的开发，我正在与NCI Frederick的HIV和艾滋病恶性肿瘤科和高级临床前研究中心（CAPR）的临床医生合作进行更多的转化项目，以建立患者来源的异种移植模型。来自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。到目前为止，我们观察到kshv感染的内皮细胞在异种移植物中的增殖和扩增。从该系统衍生的细胞系维持KSHV感染并在小鼠体内驱动肿瘤，这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，也没有卡波西肉瘤的临床前动物模型来筛选这种艾滋病定义癌症的有效药物干预。

(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 performed RNAseq to compare the gene expression profile in infected cells with and without STAT3. Thus far we have identified a pro-viral role of STAT3 in dampening the interferon response in B cells via STAT3 inhibition of STAT1/2 and numerous antiviral interferon-stimulated genes. 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. (2a) 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. We are currently exploring protein binding partners of the viral UNG. In collaboration with Kevin McBride, we are examining the differential biochemical properties of the viral and host UNG. (2b) 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. 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. (3) We are examining the mechanism by which the viral ribonucleotide reductase large subunit relocalizes host nuclear defense factors, termed PML-NB into track-like structures. We hypothesize that this relocalization by the virus neutralizes their antiviral functions and promotes productive infection. (4) 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. Thus far, we observe KSHV-infected endothelial cell proliferation and expansion in the xenografts. 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 of this AIDS-defining cancer.