2A), and transfection of an exogenous DAG1-expressing plasmid did not significantly increase either DG expression or LASV-VSV transduction (data not shown)

2A), and transfection of an exogenous DAG1-expressing plasmid did not significantly increase either DG expression or LASV-VSV transduction (data not shown). from murine leukemia virus envelope (Env) for the mucin-like domain served as a competent receptor. These studies provide evidence that, in the absence of a functional DG, TIM-1 mediates the entry of LASV pseudoviral particles through interactions of virions with the IgV PtdSer-binding pocket of TIM-1. IMPORTANCE PtdSer receptors, such as TIM-1, are emerging as critical entry factors for many enveloped viruses. Most recently, hepatitis C virus and Zika virus have been added to a growing list. PtdSer receptors engage with enveloped viruses through the binding of PtdSer embedded in the viral envelope, defining them as GP-independent receptors. This GP-independent entry mechanism should effectively mediate the entry of all enveloped viruses, yet LASV GP-pseudotyped viruses were previously found to be unresponsive to PtdSer receptor enhancement in HEK 293T cells. Here, we demonstrate that LASV pseudovirions can utilize the PtdSer receptor TIM-1 but only in the absence of appropriately glycosylated -dystroglycan (DG), the high-affinity cell surface receptor for LASV. Our studies shed light on LASV receptor utilization and explain why previous studies performed with -DG-expressing cells did not find that LASV pseudovirions utilize PtdSer receptors for virus uptake. remains Furosemide unclear, as Sullivan et al. demonstrated that Axl knockout (Axl-KO) mice are readily susceptible to LCMV (48). A number of the studies indicating that Axl does not mediate LASV pseudovirion entry were performed with cells that expressed wild-type (WT) DG. Hence, the use of alternative receptors by LASV may occur only when functional DG is not present. Consistent with this, Fedeli et al. recently demonstrated that Axl serves as a LASV receptor in cells where functional DG is either absent or present at low levels (49). In this study, we found that that PtdSer receptor TIM-1 mediates the entry of either LCMV or vesicular stomatitis virus (VSV) pseudovirions bearing LASV GP but only when DG either is not expressed or does not contain the necessary LARGE-dependent alterations of the O-linked glycans. This is consistent with findings that the high-affinity interactions of LASV GP and DG prevail over lower-affinity PtdSer/PtdSer receptor Furosemide interactions (49). Furthermore, we found that the TAM receptor Axl Gdf11 was unable to serve as a receptor for LASV pseudovirions in HEK 293T and Vero cells, irrespective of the status of DG in these cells. RESULTS LASV entry is TIM-1 dependent in Vero cells. Multiple lines of evidence indicate that DG is not the only receptor available to Old World arenaviruses (45, 49,C51), although when appropriately glycosylated, DG binds to LASV GP with high affinity Furosemide and mediates Old World arenavirus entry (21, 22). Although DG is widely expressed throughout the body, some cell types do not glycosylate DG in a way that is compatible with LASV GPC engagement and laminin binding (22). As Vero cells are readily permissive to LASV but are not sensitive to laminin-mediated competition (22), we Furosemide assessed the ability of mAb IIH6 to bind to Vero cells. IIH6 has been previously shown to distinguish if DG is glycosylated in a LASV GPC-compatible manner (22, 52). Surface staining of cells with IIH6 demonstrated that suitably glycosylated DG was detected on HEK 293T cells, but not Vero cells, yet both cell types had readily detectable dystroglycan on their surface (Fig. 1A). These findings are consistent with previous studies proposing that DG is not used by LASV for entry into Vero cells (22, 45). Open in a separate window FIG 1 LASV pseudovirion Furosemide entry is TIM-1 dependent in Vero cells. (A) Cell surface detection of endogenous DG expression on Vero or HEK 293T cells. Live cells were stained with polyclonal DG antisera (top) or the mannosylation-dependent anti-DG mAb IIH6 (bottom). Filled histograms represent cells stained with isotype control antisera or mAb; unfilled.