Identification and characterization of alphavirus host factors determining human tissue tropism

随着全球化和气候变化促进传染病的传播，病毒的出现和再出现是一个日益严重的公共卫生问题。随着蚊子继续向新的地理区域传播，蚊子传播的感染尤其如此。甲病毒，如基孔肯雅病毒（CHIKV）和委内瑞拉马脑炎病毒（VEEV）属于蚊媒病毒，可引起人类病理。再次出现的CHIKV病毒会引起长期的关节炎样症状，而VEEV感染会导致脑炎。为什么来自同一科的两种病毒在不同组织（即关节与中枢神经系统）中引起病变，迄今尚不清楚。这种知识上的差距反映在这样一个事实上，即人们所知的CHIKV和VEEV的宿主因子很少。在这里，我们利用我们的发现，磷脂酰丝氨酸（PS）受体T细胞免疫球蛋白粘蛋白受体1 （TIM-1）和四蛋白是宿主因子的CHIKV。我们假设(a) VEEV也劫持PS受体和四跨蛋白感染人类细胞，(b) TIM-1和四跨蛋白与其他蛋白相互作用，促进CHIKV和可能的VEEV感染，以及(c)这些宿主因子的一个亚群有助于CHIKV和VEEV的组织趋向性。为了验证我们的假设，我们将首先使用最先进的无偏定量蛋白质组学方法来鉴定CHIKV和VEEV的进入因子和受体。接下来，我们将筛选PS受体和所有33种人四联蛋白在人细胞中通过RNA干扰感染VEEV中的作用。TIM-1和Tetraspanin相关蛋白将通过近距离标记结合高分辨率亲和富集质谱法进行鉴定。最后，我们将利用蛋白质组学、单细胞测序和病毒学方法研究宿主因子对CHIKV和VEEV的组织趋向性的贡献。具体来说，我们将感染皮肤器官型筏培养物，并确定主要感染的细胞类型以及易感细胞和难反应细胞中已识别的宿主因子的表达水平。此外，我们将整合来自在线存储库的表达数据，并测量相关细胞类型的蛋白质组，包括成纤维细胞和神经细胞。使用特征选择方法，我们将确定宿主因素，这些因素可能导致CHIKV和VEEV引起的不同组织倾向和病理。这些宿主因子将首先在相关细胞类型中被系统地敲除，然后在小鼠中被系统地敲除，以描述在体外和体内对疾病表现的贡献。综上所述，本研究计划旨在为两种重要的人类病原体，CHIKV和VEEV引起的感染过程提供机制见解。这些结果将有助于了解疾病机制，并可能最终揭示基孔肯雅热和委内瑞拉马脑炎治疗干预的药物靶点。

Virus emergence and re-emergence is an increasing public health problem as globalization and climate change promote the spread of infectious disease. This is particularly true for mosquito-borne infections as mosquitoes continue to spread to new geographic regions. Alphaviruses such as Chikungunya virus (CHIKV) and Venezuelan equine encephalitis virus (VEEV) belong to the group of mosquito-borne viruses, which cause pathology in humans. The re-emerging CHIKV is causing long-lasting arthritis like symptoms, while VEEV infection leads to encephalitis. Why the two viruses from the same family cause pathologies in different tissues, i.e. joints versus central nervous system (CNS), is unclear to date. This gap of knowledge is reflected by the fact that few host factors for CHIKV and VEEV are known.Here we capitalize on our findings that the phosphatidylserine (PS) receptor T cell immunoglobulin mucin receptor 1 (TIM-1) and a tetraspanin are host factors for CHIKV. We hypothesize that (a) VEEV also hijacks PS receptors and tetraspanins to infect human cells, (b) TIM-1 and the tetraspanin interact with additional proteins to promote infection with CHIKV and possibly VEEV and (c) that a subset of these host factors contribute to tissue tropism of CHIKV and VEEV.To test our hypothesis we will initially use unbiased state of the art quantitative proteomics methods to identify entry factors and receptors of CHIKV and VEEV. Next we will screen PS receptors and all 33 human tetraspanins for their role in VEEV infection by RNA interference in human cells. TIM-1 and Tetraspanin associated proteins will be identified by proximity labeling in conjunction with high-resolution affinity enrichment mass spectrometry. Finally we will investigate the contribution of the identified host factors to tissue tropism of CHIKV and VEEV using proteomics, single cell sequencing and virological methods. Specifically, we will infect skin organotypic raft cultures and determine the predominantly infected cell type as well as expression levels of identified host factors in the susceptible versus refractory cells. Additionally we will integrate expression data from online repositories and measure proteomes of relevant cell types including fibroblasts and neuronal cells. Using feature selection approaches, we will identify host factors, which likely contribute to the distinct tissue tropism and pathologies caused by CHIKV and VEEV. These host factors will be systematically knocked out first in relevant cell types and then in mice to delineate the contribution to disease manifestation in vitro and in vivo.Taken together, this research program aims at providing mechanistic insight into the infection processes caused by two important human pathogens, CHIKV and VEEV. The results will help understand disease mechanisms and may ultimately reveal drug targets for therapeutic intervention with Chikungunya fever and Venezuelan equine encephalitis.