Role of HIV in KSHV oral transmission

项目总结/摘要 卡波西肉瘤（KS）仍然是全球艾滋病毒/艾滋病患者最常见的恶性肿瘤之一。 卡波西肉瘤疱疹病毒（KSHV），也称为人类疱疹病毒-8（HHV-8），是KS的致病因子。的 一旦病毒进入口腔，口腔粘膜是KSHV感染的第一目标。然而，最初的感染 KSHV对粘膜的作用过程从未被研究过，主要是由于缺乏体外模型来重现 体内病毒感染和不存在KSHV感染的动物模型。我们创造了三个， 三维（3-D）器官型培养物作为口腔粘膜模拟物，其类似于分层的口腔粘膜， KSHV感染/传播过程的组织学评估潜力。我们的研究表明，KSHV 3-D口腔粘膜模型中的感染被唾液细胞外囊泡（EV），特别是外来体， 艾滋病患者。从HIV患者的唾液或从HIV感染的T细胞的培养基中纯化的外泌体 细胞含有类似的HIV特异性货物，包括HIV TAR RNA。KSHV进入靶细胞及其感染性 在原代和永生化的人口腔上皮细胞中，HIV阳性唾液外泌体增加。此外，本发明还 在3-D器官型培养中，KSHV感染和向基底上细胞的传播被HIV-1增强。 阳性外泌体HIV阳性外泌体增加的KSHV感染性归因于HIV TAR RNA， 宿主细胞的囊泡和表皮生长因子受体（EGFR）。西妥昔单抗对EGFR的抑制作用，a EGFR的单克隆抗体，阻断由HIV阳性外泌体增强的KSHV感染。综合起来看， 这些数据使我们假设HIV阳性外泌体促进KSHV感染和传播， 口服途径是导致艾滋病毒感染者中KSHV感染发生率增加的原因。测试 根据这一假设，我们计划1）用3-D技术评估KSHV通过口腔途径的传播和感染 口腔粘膜和扁桃体组织的培养物与外周血单核细胞（PBMC）结合， 以及上皮-内皮模型。KSHV感染和通过上皮屏障传播， 将评估免疫和内皮细胞对HIV+唾液外泌体的应答; 2）描述机制 HIV阳性外泌体促进KSHV口腔传播的方法。我们将应用 单细胞RNAseq鉴定口腔粘膜和扁桃体3-D组织转录组的细胞特异性变化 KSHV感染后响应HIV+外泌体的模型;和3）阐明EGFR依赖性的 丝裂原活化蛋白激酶激活增强HIV阳性外泌体的KSHV传播。成功 拟议的研究将促进我们在分子水平上对KSHV口腔传播的理解， 开发潜在的新型治疗策略的潜在机制。

Project Summary/Abstract Kaposi sarcoma (KS) remains one of the most common malignancies in people living with HIV/AIDS worldwide. Kaposi sarcoma herpesvirus (KSHV), also called human herpesvirus-8 (HHV-8), is the causal agent for KS. The oral mucosa is the first target of KSHV infection once the virus is in the oral cavity. However, the initial infection process of the mucosa by KSHV has never been studied, mainly due to lack of an in vitro model to recapitulate the viral infection in vivo and nonexistence of an animal model for KSHV infection. We have created the 3- dimentional (3-D) organotypic culture as an oral mucosal mimic resembling a stratified oral mucosa with the potential for histological assessment of the KSHV infection/transmission process. Our studies show that KSHV infection in the 3-D oral mucosa model is enhanced by saliva extracellular vesicles (EVs), particularly exosomes, from HIV patients. Exosomes purified from the saliva of HIV patients or from culture media of HIV-infected T cells contain similar HIV-specific cargos, including HIV TAR RNA. KSHV entry into target cells and its infectivity are increased by HIV-positive saliva exosomes in primary and immortalized human oral epithelial cells. Further, KSHV infection and transmission to the suprabasal cells in the 3-D organotypic culture are enhanced by HIV- positive exosomes. Increased KSHV infectivity by HIV-positive exosomes is attributed to the HIV TAR RNA within the vesicles and epidermal growth factor receptor (EGFR) of host cells. Inhibition of EGFR by Cetuximab, a monoclonal antibody to EGFR, blocks KSHV infection enhanced by HIV-positive exosomes. Taken together, these data lead us to hypothesize that HIV-positive exosomes promote KSHV infection and transmission through the oral route and are responsible for increased incidence of KSHV infection in people living with HIV. To test this hypothesis, we plan to 1) assess KSHV transmission and infection through the oral route with the 3-D cultures of oral mucosal and tonsil tissues incorporated with peripheral blood mononuclear cells (PBMCs) as well as the epithelium-endothelium model. KSHV infection and transmission through the epithelial barrier to immune and endothelial cells in response to HIV+ saliva exosomes will be assessed; 2) delineate the mechanism by which HIV-positive exosomes promote KSHV oral transmission using the 3-D tissue models. We will apply single cell RNAseq to identify cell-specific changes in transcriptome of the oral mucosal and tonsil 3-D tissue models following KSHV infection in response to HIV+ exosomes; and 3) elucidate the role of EGFR-dependent mitogen-activated protein kinase activation in enhanced KSHV transmission by HIV-positive exosomes. Success of the proposed research will advance our understanding of KSHV oral transmission at the molecular level and the underlying mechanisms for developing potential novel therapeutic strategies.