Mechanisms of cell entry of Lymphocytic Choriomeningitis Virus.

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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.