Investigation of viral and host determinants of gammaherpesvirus pathogenesis

(1)STAT 3激活与细胞内γ疱疹病毒潜伏期和癌症相关 培养系统，但驱动潜伏期，增殖和转化的靶基因是 不明确。作为研究KSHV自身中STAT 3激活作用的前奏，我们 使用B细胞特异性敲除STAT 3的转基因小鼠来确定STAT 3 信号传导对于在免疫缺陷病毒的原代B细胞中建立γ疱疹病毒潜伏期是至关重要的。 主持人我们还确定了病毒裂解基因反式激活因子RTA与STAT 3相互作用， 以依赖尼古丁的方式。我们的中心假设是γ疱疹病毒需要 STAT 3促进B细胞潜伏期。为了确定STAT 3依赖的基因表达程序， 感染的B细胞中，我们正在进行RNAseq以比较感染的B细胞中的基因表达谱。 有和没有STAT 3的细胞到目前为止，我们已经确定了STAT 3在细胞凋亡中的前病毒作用。 通过STAT 3抑制STAT 1/2和许多其它途径抑制B细胞中的干扰素应答， 抗病毒干扰素刺激基因。我们将合并所有基因的差异表达基因， 可用的γ疱疹病毒数据集，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延迟，重新激活， 使用条件敲除方法在KSHV和鼠细胞培养系统中进行转化。 (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中的主要限制点。我们最近报道说， 病毒尿嘧啶DNA糖基化酶（UNG）的酶性质与病毒DNA糖基化酶协同作用， 核苷酸代谢蛋白dUTR以促进致病和对抗重组为主 病毒基因组的缺失。我们目前正在探索蛋白质结合伙伴的 病毒性UNG。与凯文·麦克布莱德合作，我们正在研究 病毒和宿主UNG的特性。(2b)此外，与麦克布莱德实验室合作，我们 分析了来自感染小鼠的感染和未感染B细胞的免疫球蛋白库 使用下一代测序。这项分析显示，感染了γ-疱疹病毒的细胞 进行克隆扩增，但只有少数克隆在感染和未感染之间共享 生发中心B细胞。也有证据表明受体编辑和明显的偏见， 感染的B细胞中的特异性IghV基因。这些新数据表明γ疱疹病毒 在受感染的宿主中占据独特的生态位，并在破坏宿主的 它感染的B细胞中的免疫球蛋白库。这证明了存在一个 KSHV和EBV共享的关键策略，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和产生 免疫球蛋白与病理潜力。我还与其他NCI合作， HAMB的研究人员和临床医生分析KSHV感染细胞的B细胞库 在HIV相关原发性渗出性淋巴瘤和HIV感染者淋巴结物质中 参加HAMB临床试验的KSHV-多中心Castleman病患者。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 可能告知这些疾病的可能病因，这些疾病通常同时发生， 艾滋病病毒感染者的生活方式。(3)我们正在研究 病毒核糖核苷酸还原酶大亚基重新定位宿主核防御因子，称为 PML-NB进入轨道状结构。我们假设病毒的这种重新定位 中和它们的抗病毒功能并促进生产性感染。(4)关于 卡波西肉瘤是艾滋病的标志性肿瘤， 与艾滋病毒和艾滋病咨询分支和中心的临床医生合作， NCI Frederick的高级临床前研究（CAPR）在更多的转化项目中， 建立患者来源的异种移植模型。来自HIV感染者的KS活检材料 将HAMB临床组群中的患者植入免疫缺陷动物中。因此 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增殖和扩增。 来自该系统的细胞系维持KSHV感染并驱动小鼠肿瘤， 这是该领域的巨大进步，因为没有办法维持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 有效的药物干预这种艾滋病定义的癌症。

(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. 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.