The envelope glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) is posttranslationally cleaved into two subunits. We show here that this endoproteolytic processing is not required for transport to the cell surface but is essential for LCMV GP to mediate infectivity of pseudotyped retroviral vectors. By systematic mutational analysis of the LCMV GP cleavage site, we determined that the consensus motif R-(R/K/H)-L-(A/L/S/T/F) 265 is essential for the endoproteolytic processing. In agreement with the identified consensus motif, we show that the cellular subtilase SKI-1/S1P cleaves LCMV GP.Lymphocytic choriomeningitis virus (LCMV) is the prototype of the Arenaviridae family, which includes important human pathogens causing hemorrhagic fever, such as Lassa virus, Junin virus, Machupo virus, and Guanarito virus. LCMV has been widely used as an experimental model for the study of immunology, viral persistence, and viral pathogenesis (34, 50).Virions of LCMV are composed of a nucleocapsid which is surrounded by a lipid envelope containing the envelope glycoprotein (GP). The initial steps in LCMV infection involve the interaction of GP with the cellular receptor of the target cells. Depending on the GP sequence, LCMV uses either alphadystroglycan or an alternative cellular protein as a receptor (15,41,42). After internalization of the virions within vesicles, LCMV GP mediates fusion of the viral and cellular membranes, resulting in delivery of the nucleocapsids into the cytoplasm (9,18,19).LCMV GP is initially expressed as a precursor polypeptide, GP-C, which is posttranslationally cleaved into two subunits, GP-1 and GP-2 (12). Cleavage of LCMV GP-C by a yetunidentified cellular protease occurs in the Golgi or a postGolgi compartment (47), whereas the related Lassa virus GP-C was found to be cleaved early in the secretory pathway by the subtilase SKI-1/S1P (4, 31). The amino-terminal cleavage product GP-1 is a peripheral membrane protein and is noncovalently associated with the carboxy-terminal subunit GP-2, which is an integral membrane protein (14). GP-1 presumably interacts with the cellular receptor, whereas the GP-2 subunit most likely mediates fusion of the viral envelope with the cellular membrane (8,13,26,27). The fusion peptide of GP-2 appears to be activated to a fusion-competent state by a pHdependent conformational change of the LCMV GP subunits (9,18,19).The GP of LCMV and other arenaviruses are cleaved between two uncharged amino acids (13, 30), whereas most other viral GPs are cleaved after a basic amino acid (29). In this study, we analyzed cleavage of LCMV GP in detail. We show that cleavage of LCMV GP is not required for transport to the cell surface but is required for mediating virus infectivity of retroviral pseudotypes. Furthermore, we have determined the consensus motif for cleavage of the LCMV GP, and we show that LCMV GP is cleaved in a late-Golgi or post-Golgi compartment by the cellular subtilase SKI-1/S1P. MATERIALS AND METHODS Construction of LCMV GP and Lassa virus GP expression ...