Cell Host inits absence, GP-containing virus particles form but are noninfectious, due in part to their inability to attach to host cells. Thus, we have identifi ed a class of pathogen-derived ERGIC-53 ligands, a lectin-independent basis for their association with ERGIC-53, and a role for ERGIC-53 in the pro- pagation of several highly pathogenic RNA virus families. INTRODUCTION Arenaviruses and hantaviruses are rodent-borne, negative- sense RNA viruses that cause signifi cant morbidity and mortal- ity in humans (Buchmeier et al., 2007; Schmaljohn and Nichol, 2007). Most pathogenic arenaviruses are associated with severe hemorrhagic fever syndromes in humans. Examples include the New World arenaviruses Junin virus (JUNV), Machupo virus (MACV), and Guanarito virus (GTOV), which are the etiologic agents of Argentine, Bolivian, and Venezuelan hemorrhagic fevers, respectively, as well as Lassa virus (LASV), an Old World arenavirus that causes Lassa fever along the coast of West Africa (Buchmeier et al., 2007). Additionally, lymphocytic choriomeningitis virus (LCMV) can cause aseptic meningitis in immunocompetent individuals and is a potent teratogen (Buchmeier et al., 2007). LCMV and Dandenong virus (DANV), an LCMV-like virus, are also responsible for a nearly uniform lethality in immunosuppressed recipients of virus- infected tissues (Fischer et al., 2006; Palacios et al., 2008). Han- taviruses cause two human illnesses: hemorrhagic fever with renal syndrome in the Old World and hantavirus cardiopulmo- nary syndrome (HCPS) in the New World (Schmaljohn and Nichol, 2007). Sin Nombre virus (SNV) and Andes virus (ANDV) are the primary etiologic agents of HCPS in North and South America, respectively, and are associated with a fatality rate of 35%39% (da Rosa Elkhoury et al., 2012; MacNeil et al., 2011). US Food and Drug Administration (FDA)-approved vac- cines or effective antivirals do not currently exist for the preven- tion and/or therapeutic treatment of arenavirus or hantavirus disease. Arenaviruses and hantaviruses each encode an envelope glycoprotein (GP) that decorates the surface of the virion and functions to mediate attachment and entry of virions into permis- sive host cells. Each GP is encoded as a precursor (GPC) that is proteolytically processed into mature subunits. The arenavirus GPC is posttranslationally modifi ed to yield a stable signal pep- tide (SSP) as well as GP1 and GP2 subunits (Lenz et al., 2001), whereas the hantavirus GPC is cotranslationally processed 522Cell Host G1-G2 for hantaviruses) that facilitates receptor binding and entry (Buchmeier et al., 2007; Schmaljohn and Nichol, 2007). Relatively little is known regarding interactions that arenavirus or hantavirus GPs have with host proteins or the importance of such interactions for viral replication and disease pathogenesis. Herein, we utilized a proteomics approach to comprehensively identify human proteins that interact with GPs encoded by a prototypic arenavirus or hantavirus. We show that the endoplasmic reticulum (ER)-Golgi intermediate compartment 53 kDa protein (ERGIC-53), an intracellular cargo receptor that facilitates the anterograde transport of a limited number of glycoprotein ligands in the early exocytic pathway (Appenzeller et al., 1999), has a conserved interaction with GPs encoded by multiple families of RNA viruses and is essential for the forma- tion of infectious arenavirus, coronavirus, and fi lovirus particles in a GP-specifi c manner. Our results suggest that loss of ERGIC-53oritsfunctionalityleadstotheformationof GP-containing virions that are defective in their ability to attach to permissive host cells. RESULTS Identifi cation of Cellular Proteins that Associate with Arenavirus and Hantavirus GPs and Choice of ERGIC-53 To identify human proteins that associate with arenavirus and hantavirus GPs, we used an approach that featured affi nity purifi cation (AP) of biotinylated viral proteins (LCMV GP to represent arenaviruses or ANDV GP for hantaviruses) in com- plex with host proteins, followed by mass spectrometry to iden- tify host protein partners as described in Figure S1A and the Supplemental Experimental Procedures (available online). We identifi ed a number of host proteins that associated with LCMV GP (n = 309), ANDV GP (n = 134), or both GPs (n = 51) (Figures 1A1C, S1B, and S1C; Tables S1AS1C). As shown in Figure S1D and Table S1D, host proteins that associated with both GPs were enriched for processes involving the ER, protein folding, and vesicular transport. The LCMV GP- only partners were enriched for processes that included the ER, the proteasome, and nuclear import, while the ANDV GP- only partners were enriched for protein translation and ribo- some biogenesis. We were particularly interested in the subset of proteins that interacted with both GPs, as they could serve as broad-spectrum antiviral targets. Of these, we chose ERGIC-53 for further study based upon several criteria. First, the ERGIC