Three-dimensional human epithelial cultures as a model for evaluation of flavivirus-host interactions driving infection in the skin

项目总结 寨卡病毒(ZIKV)、登革热病毒(DENV)和西尼罗河病毒(WNV)都会导致严重的人类疾病 它们广泛的蚊媒传播。虽然这些黄病毒共享一个共同的主要站点 感染在真皮，它们扩散到不同的继发性感染部位，临床上差异很大。 结果。我们认为，ZIKV、DENV和WNV之间早期病毒-宿主相互作用的差异是 对疾病的这些差异负有责任；然而，我们对分子的了解存在差距 负责的机制。因此，在初始感染部位发生的不同的病毒-宿主相互作用的阐明 了解这些病毒如何在皮肤和其他地方产生生产性感染的关键。建议数 研究将建立器官型上皮细胞培养作为一种遗传易驯化和免疫能力强的 黄病毒感染的三维人体皮肤模型，用于病毒与宿主的相互作用研究。我们将在以下方面进行扩展 目前通常用于研究DNA病毒的模型，通常仅由人成纤维细胞和 角质形成细胞，加上人类皮肤常驻树突状细胞和巨噬细胞，以准确地重建 皮肤中的自然黄病毒感染。此外，我们还将纳入蚊子唾液，其中包括 免疫调节和抗炎蛋白，进入病毒接种进一步概括分子 在黄病毒感染的主要部位发生的事件。我们将确定个人感染情况 ZIKV、DENV和WNV，并评估每种病毒的细胞嗜性和宿主反应。一旦成立，我们 将利用这种3D人体皮肤模型来评估宿主核糖核酸酶L(核糖核酸酶L)蛋白在 ZIKV在皮肤中的感染，这将验证该系统用于黄病毒与宿主的相互作用研究。虽然抗病毒药物 核糖核酸酶L的活性被很好地描述，我们最近发现了前病毒核糖核酸酶L在寨卡病毒感染过程中的活性。 相反，我们观察到典型的抗病毒核糖核酸酶L活性在DENV和WNV感染期间。这些研究 在2D单层培养系统中进行永生化细胞系，因此我们将使用我们的3D皮肤 含有原代细胞的模型，以检验核糖核酸酶L在ZIKV细胞趋向性和 以更相关的系统在皮肤中传播。我们将使用CRISPR-CAS9基因编辑删除核糖核酸酶L 不同的皮肤细胞组成上皮细胞培养，继而产生核糖核酸酶L缺失的皮肤培养 用于感染ZIKV、DENV或WNV。我们将评估核糖核酸酶L缺失对人乳头瘤病毒感染和传播的影响 皮肤中不同的黄病毒，以及宿主的反应如何受到影响。拟议的研究将 描述核糖核酸酶L在皮肤感染过程中被寨卡病毒改变用途的重要宿主因子的作用。 此外，该项目将建立器官型上皮细胞培养，作为一种令人兴奋的新人类模型 到基因编辑，这将促进对虫媒病毒-宿主相互作用和宿主反应的研究 皮肤的主要感染部位。

PROJECT SUMMARY Zika virus (ZIKV), dengue virus (DENV), and West Nile virus (WNV) all cause severe human disease as a result of their widespread mosquito-borne transmission. While these flaviviruses share a common primary site of infection in the dermis, they disseminate to varying secondary sites of infection with highly divergent clinical outcomes. We propose that discrepancies in early virus-host interactions among ZIKV, DENV, and WNV are responsible for these differences in disease; however, there is a gap in our knowledge of the molecular mechanisms responsible. Elucidation of distinct virus-host interactions occurring at the initial infection site is thus key to understanding how these viruses establish productive infections in the skin and elsewhere. The proposed research will establish organotypic epithelial cultures as a genetically tractable and immunocompetent 3D human skin model of flavivirus infection, to be used for virus-host interaction studies. We will expand on the current model commonly used to study DNA viruses, typically comprised solely of human fibroblasts and keratinocytes, with the addition of human skin-resident dendritic cells and macrophages, to accurately recreate natural flavivirus infection in the skin. Additionally, we will incorporate mosquito saliva, which includes immunomodulatory and anti-inflammatory proteins, into the virus inoculum to further recapitulate the molecular events occurring at the primary site of flavivirus infection. We will determine individual infection conditions for ZIKV, DENV, and WNV, and evaluate cell tropism and host responses with each virus. Once established, we will employ this 3D human skin model to evaluate the role of the host ribonuclease L (RNase L) protein during ZIKV infection in the skin, which will validate this system for flavivirus-host interaction studies. While antiviral RNase L activity is well described, we have recently discovered proviral RNase L activity during ZIKV infection. In contrast, we observed canonical antiviral RNase L activity during DENV and WNV infections. These studies were performed in 2D monolayer culture systems with immortalized cell lines, therefore we will use our 3D skin model containing primary cells to test the hypothesis that RNase L plays a role in ZIKV cell tropism and spread in the skin in a more relevant system. We will use CRISPR-Cas9 gene editing to delete RNase L from the different skin cells comprising epithelial cultures, and subsequently generate RNase L-deficient skin cultures for infections with ZIKV, DENV, or WNV. We will assess effects of RNase L deletion on infection and spread of the different flaviviruses in the skin, as well as how host responses are impacted. The proposed studies will characterize the role of RNase L as an important host factor repurposed by ZIKV during infection in the skin. Furthermore, this project will establish organotypic epithelial cultures as an exciting new human model amenable to gene editing which will facilitate inquiries of arbovirus-host interactions and host responses at the common primary infection site of the skin.