Mechanisms of cell entry of Lymphocytic Choriomeningitis Virus.

项目摘要 阿雷诺病毒是与特定啮齿动物宿主密切相关的人畜共患负义RNA病毒(1)。一些人 然而，这些病毒中，已知会导致人类疾病，如拉沙氏菌、淋巴细胞性脉络膜脑膜炎 病毒(LCMV)、Lujo、Machupo和Junín病毒(6)。属于东半球(OW)的原型阿雷诺病毒 分支，淋巴细胞性脉络膜脑膜炎病毒(LCMV)，感染家鼠(Mus Musculus)，因此 全球分布(7、8、9、10)。虽然在人类中LCMV感染通常是无症状的或与 如果病情轻微，它还会导致流产、先天出生缺陷，并可能导致 移植受者(11、12、13、14、15)。此外，小鼠的巨细胞病毒感染已被证明是一种例外 研究病毒-宿主相互作用的有效模型系统，这导致了该领域的开创性发现 微生物学和免疫学(16、17、18)。虫媒病毒糖蛋白复合体(GPC)介导病毒 进入细胞，最终形成膜融合。第一个被发现的阿拉伯病毒受体是阿尔法- 针对东半球病毒LASV和LCMV以及新大陆C分支病毒的营养不良聚糖(α-DG) 奥利维罗和拉丁裔(19，25)。我们和其他人之前的工作表明，LASV进入要复杂得多 比之前预想的要好。通过使用单倍体遗传筛选，该病毒被发现依赖于 溶酶体相关膜蛋白1(LAMP1)，不依赖于α-DG(28，29)。拉萨病毒 在中性pH条件下，在细胞表面附着于糖基化的α-DG，并在细胞内摄取和转运到 内体和溶酶体细胞隔间，GPC经历酸性pH诱导的转换以接合 溶酶体膜蛋白LAMP1与驱动膜融合(29)。到目前为止，入境流程LCMV仍然 不完全理解。据报道，LCMV在没有已公布的受体的情况下可以有效地感染。 α-DG，感染也不依赖于LAMP1，这表明关键进入因子(S)仍然未知(20，21，29)。 为了确定LCMV进入的未知因素，我们进行了初步的全基因组CRISPR-Cas9筛查 并确定CD164，一种溶酶体蛋白，是最重要的打击。这一初步数据使我们提出了假设 LCMV gp通过胞内转运至溶酶体室后与CD164结合，CD164 然后促进酸性pH依赖的膜融合。在这里，我们建议使用遗传学、细胞生物学、 生化和结构研究，以确定CD164在LCMV进入过程中的作用。第一个目标是确定 利用CRISPR-Cas9全基因组功能丧失基因进入LCMV细胞所需的宿主基因 筛选，然后通过个体基因敲除和慢病毒地址验证显著的命中。 第二个目的是探索CD164通过确定结构域促进LCMV进入的机制 在CD164及其通过遗传操作细胞所必需的糖基化，以及生化和 研究LCMV gp与CD164相互作用的结构方法。

Project Summary Arenaviruses are zoonotic negative-sense RNA viruses closely associated with specific rodent hosts (1). Some of these viruses, however, are known to cause human disease such as Lassa, Lymphocytic Choriomeningitis Virus (LCMV), LuJo, Machupo, and Junín virus (6). The prototypic arenavirus that belongs to the Old World (OW) clade, Lymphocytic Choriomeningitis Virus (LCMV), infects house mice (Mus musculus) and consequently has a worldwide distribution (7, 8, 9, 10). While LCMV infection in humans is typically asymptomatic or associated with mild illness, it can also cause abortions, congenital birth defects, and has been implicated in mortality of transplant recipients (11, 12, 13, 14, 15). Moreover, LCMV infection of mice has proven to be an exceptionally effective model system for the study of virus-host interactions, which has led to seminal discoveries in the fields of microbiology and immunology (16, 17, 18). The glycoprotein complex (GPC) of arenaviruses mediates virus entry into cells culminating in membrane fusion. The first arenavirus receptor to be discovered was alpha- dystroglycan (α-DG) for the Old World viruses LASV and LCMV, and the Clade C New World arenaviruses Olivero and Latino (19, 25). Previous work by us and others revealed that LASV entry was more complicated than previously appreciated. Through the use of haploid genetic screens, the virus was found to rely on Lysosomal Associated Membrane Protein 1 (LAMP1), in an α-DG-independent manner (28, 29). Lassa virus attaches to glycosylated α-DG at neutral pH at the cell surface, and upon endocytic uptake and trafficking to endosomal and lysosomal cell compartments, the GPC undergoes an acid pH-induced transition to engage lysosomal membrane protein LAMP1 and drive membrane fusion (29). To date, the entry process LCMV remains incompletely understood. LCMV has been reported to infect efficiently in the absence of its published receptor, α-DG, nor is infection reliant upon LAMP1, suggesting that critical entry factor(s) remain unknown (20, 21, 29). To identity unknown factors involved in LCMV entry, we did a preliminary genome-wide CRISPR-Cas9 screen and identified CD164, a lysosomal protein, as the most significant hit. This preliminary data led us to hypothesize that upon endocytic transport of LCMV to lysosomal compartments, LCMV GP engages CD164, which then facilitates acid pH-dependent membrane fusion. Here, we propose to use genetic, cell biological, biochemical, and structural studies to characterize the role of CD164 in LCMV entry. The first aim is to determine the host genes required for cell entry of LCMV through the use of a CRISPR-Cas9 genome-wide loss-of-function screen, followed by validation of significant hits through individual genetic knockouts and lentiviral addbacks. The second aim is to probe the mechanism by which CD164 facilitates entry of LCMV by determining the domain within CD164 and its necessary glycosylation through genetic manipulation of cells, as well as biochemical and structural approaches to characterize the interaction between LCMV GP and CD164.