The glycoproteins (GP) of enveloped viruses facilitate entry into the host cell by interacting with specific cellular receptors. Despite extensive study, a cellular receptor for the deadly filoviruses Ebolavirus and Marburgvirus has yet to be identified and characterized. Here, we show that T-cell Ig and mucin domain 1 (TIM-1) binds to the receptor binding domain of the Zaire Ebola virus (EBOV) glycoprotein, and ectopic TIM-1 expression in poorly permissive cells enhances EBOV infection by 10-to 30-fold. Conversely, reduction of cell-surface expression of TIM-1 by RNAi decreased infection of highly permissive Vero cells. TIM-1 expression within the human body is broader than previously appreciated, with expression on mucosal epithelia from the trachea, cornea, and conjunctiva-tissues believed to be important during in vivo transmission of filoviruses. Recognition that TIM-1 serves as a receptor for filoviruses on these mucosal epithelial surfaces provides a mechanistic understanding of routes of entry into the human body via inhalation of aerosol particles or hand-to-eye contact. ARD5, a monoclonal antibody against the IgV domain of TIM-1, blocked EBOV binding and infection, suggesting that antibodies or small molecules directed against this cellular receptor may provide effective filovirus antivirals. viral entry | viral receptor | virion internalization T he Filoviridae family of viruses is composed of two genera, Ebolavirus and Marburgvirus, which cause hemorrhagic fever in humans and nonhuman primates. Infection with some strains of filoviruses causes fatality in 50-90% of human cases (1). The viral glycoprotein (GP) of Ebolavirus, which consists of surfaceexposed subunit GP1 attached to membrane-bound subunit GP2 by a disulfide bond (2), mediates binding to, penetration of, and fusion with host-cell membranes (3, 4). Pseudovirions bearing Ebolavirus GP transduce a broad range of cells through interactions that require the GP1 receptor-binding domain (RBD) (5-8). Upon internalization into low-pH endosomes, the filovirus GP1 is proteolyzed by cathepsins B and L, leading to GP2-dependent fusion of the viral and host membranes (9-12). Several proteins enhance filovirus entry in host cells, including the C-type lectins L-SIGN, DC-SIGN, and hMGL, as well as RhoB/C, integrin α5β1, folate receptor-α, and the tyrosine kinase receptor Axl (13-26); however, because none of these molecules has been shown to interact with the RBD of the filovirus GP1, it is unlikely that any of these proteins serve as a receptor for this family of viruses. Thus, we used gene correlation analysis to search for additional potential receptors. Here, we identify T-cell Ig and mucin domain 1 (TIM-1), which interacts with Zaire ebolavirus (EBOV) GP and enhances EBOV infection by 10-to 30-fold upon expression, providing strong evidence that TIM-1 serves as a receptor for EBOV. As we found that TIM-1 is expressed on a number of mucosal epithelial surfaces, we propose that TIM-1/ EBOV interactions may serve as a conduit for filovirus entry into ...

In a bioinformatics-based screen for cellular genes that enhance Zaire ebolavirus (ZEBOV) transduction, AXL mRNA expression strongly correlated with ZEBOV infection. A series of cell lines and primary cells were identified that require Axl for optimal ZEBOV entry. Using one of these cell lines, we identified ZEBOV entry events that are Axl-dependent. Interactions between ZEBOV-GP and the Axl ectodomain were not detected in immunoprecipitations and reduction of surface expressed Axl by RNAi did not alter ZEBOV-GP binding, providing evidence that Axl does not serve as a receptor for the virus. However, RNAi knock down of Axl reduced ZEBOV pseudovirion internalization and α-Axl antisera inhibited pseudovirion fusion with cellular membranes. Consistent with the importance of Axl for ZEBOV transduction, Axl transiently co-localized on the surface of cells with ZEBOV virus particles and was internalized during virion transduction. In total, these findings indicate that endosomal uptake of filoviruses is facilitated by Axl.

To explore mechanisms of entry for Ebola virus (EBOV) glycoprotein (GP) pseudotyped virions, we used comparative gene analysis to identify genes whose expression correlated with viral transduction. Candidate genes were identified by using EBOV GP pseudotyped virions to transduce human tumor cell lines that had previously been characterized by cDNA microarray. Transduction profiles for each of these cell lines were generated, and a significant positive correlation was observed between RhoC expression and permissivity for EBOV vector transduction. This correlation was not specific for EBOV vector alone as RhoC also correlated highly with transduction of vesicular stomatitis virus GP (VSVG) pseudotyped vector. Levels of RhoC protein in EBOV and VSV permissive and nonpermissive cells were consistent with the cDNA gene array findings. Additionally, vector transduction was elevated in cells that expressed high levels of endogenous RhoC but not RhoA. RhoB and RhoC overexpression significantly increased EBOV GP and VSVG pseudotyped vector transduction but had minimal effect on human immunodeficiency virus (HIV) GP pseudotyped HIV or adeno-associated virus 2 vector entry, indicating that not all virus uptake was enhanced by expression of these molecules. RhoB and RhoC overexpression also significantly enhanced VSV infection. Similarly, overexpression of RhoC led to a significant increase in fusion of EBOV virus-like particles. Finally, ectopic expression of RhoC resulted in increased nonspecific endocytosis of fluorescent dextran and in formation of increased actin stress fibers compared to RhoA-transfected cells, suggesting that RhoC is enhancing macropinocytosis. In total, our studies implicate RhoB and RhoC in enhanced productive entry of some pseudovirions and suggest the involvement of actin-mediated macropinocytosis as a mechanism of uptake of EBOV GP and VSVG pseudotyped viral particles.Enveloped viruses enter cells by a variety of different pathways. Productive internalization of enveloped viruses with targeted cells is mediated through interactions of the viral glycoprotein(s) (GPs) with moieties on the surface of the cell. In general, enveloped viral entry occurs through viral adherence to the cell surface, interaction with a specific plasma membrane-associated receptor that results in a series of GP conformational changes leading to fusion of viral and cellular membranes, and delivery of the viral core particle into the cytoplasm. Fusion of the two membranes can occur at the plasma membrane or by uptake of the intact virions into endosomes with subsequent membrane fusion between the viral membrane and the lipid bilayer of the endocytic vesicle. Human immunodeficiency virus (HIV) is an example of a virus that fuses directly to the plasma membrane (5), whereas influenza virus must be internalized into acidified vesicles where the appropriate GP conformational changes can occur, mediating membrane fusion (21). Most enveloped viruses that enter through vesicles utilize a low-pH environment to mediate the necessary...