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，病毒的出现和重新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。基孔肯雅病毒(CHIKV)和委内瑞拉马脑炎病毒(VEEV)等甲型病毒属于蚊媒病毒，可引起人类病理。重新出现的CHIKV会引起长期的关节炎样症状，而VEEV感染会导致脑炎。为什么来自同一个家族的两种病毒会在不同的组织中引起病理，即关节和中枢神经系统(CNS)，到目前为止还不清楚。这种认识的差距反映在对CHIKV和VEEV的宿主因子知之甚少的事实上。在这里，我们利用我们的发现，磷脂酰丝氨酸(PS)受体(PS)受体、T细胞免疫球蛋白粘蛋白受体1(TIM-1)和THPA是CHIKV的宿主因子。我们假设(A)VEEV还劫持PS受体和Tetraspanins来感染人类细胞，(B)Tim-1和Tetraspanin与额外的蛋白质相互作用，促进CHIKV和可能的VEEV的感染，以及(C)这些宿主因子的子集有助于CHIKV和VEEV的组织趋向性。为了验证我们的假设，我们首先将使用最先进的定量蛋白质组学方法来确定CHIKV和VEEV的进入因子和受体。接下来，我们将通过RNA干扰在人类细胞中筛选PS受体和所有33个人Tetraspanins在VEEV感染中的作用。TIM-1和Tetraspanin相关蛋白将通过与高分辨率亲和富集质谱结合的邻近标记进行鉴定。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已鉴定的宿主因素对CHIKV和VEEV组织嗜性的贡献。具体地说，我们将感染皮肤器官型移植物培养物，并确定主要感染细胞类型以及已确定的宿主因子在敏感细胞和难治细胞中的表达水平。此外，我们将整合来自在线资源库的表达数据，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，我们将识别宿主因素，这些因素可能有助于CHIKV和VEEV引起的不同的组织趋向性和病理。这些宿主因子将首先在相关的细胞类型中被系统地敲除，然后在小鼠中描述对体外和活体疾病表现的贡献。综上所述，本研究计划旨在从机制上深入了解两种重要的人类病原体CHIKV和VEEV引起的感染过程。这一结果将有助于了解疾病机制，并最终可能揭示治疗基孔肯雅热和委内瑞拉马脑炎的药物靶点。

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.