Endoproteolytic processing of the lymphocytic choriomeningitis virus glycoprotein by the subtilase SKI-1/S1P

Abstract

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.

FIG. 1.

Endoproteolytic processing of wild-type and mutant LCMV GPs. 293T cells were transfected with pHCMV expression plasmids encoding C-terminally HA-tagged wild-type and mutant LCMV GPs, as indicated in Table 1, or untagged LCMV GP (control). At 48 h after transfection, proteins from lysed cells were analyzed by immunoblotting with the anti-HA tag-specific antibody 3F10. GP-C, precursor GP; GP-2, C-terminal cleavage product of GP-C containing the HA tag.

FIG. 2.

Cell surface expression of wild-type and mutant LCMV GPs. (A) HeLa cells were mock treated (control) or transfected with pHCMV expression plasmids encoding wild-type and mutant LCMV GPs, as indicated in Table 1. At 48 h after transfection, cells were analyzed by immunostaining. Cell surface LCMV GP was analyzed by immunostaining with the anti-LCMV GP-directed monoclonal antibody KL25. (B) 293T cells were mock treated (shaded) or transfected with pHCMV expression plasmids for LCMV GP (open). At 48 h after transfection, LCMV GP was detected by flow cytometry with antibody KL25.

FIG. 3.

Quantification of cell surface expression of wild-type and mutant LCMV GPs and pseudotyped retroviruses. Cell surface expression and pseudotyping efficiency of LCMV GP are shown in relation to those of wild-type LCMV GP, which were set 100%. Cell surface expression was quantified by flow cytometry with the KL25 antibody. Pseudotyping efficiency was determined by titration of virus from transfected retroviral packaging cell supernatants. Shown are the means and standard deviations from three independent transfections.

FIG. 4.

SKI-1/S1P cleaves LCMV GP-C. SKI-1/S1P-deficient SRD-12B cells and the parental CHO-K1 cells were transfected with expression plasmids encoding C-terminally HA-tagged LCMV GP and SKI-1/S1P, as indicated. At 48 h after transfection, proteins from lysed cells were analyzed by immunoblotting with an anti-HA tag-specific antibody. GP-C, precursor GP; GP-C*, unglycosylated GP-C most likely due to overexpression (30, 31); GP-2, C-terminal cleavage product of GP-C containing the HA tag.

FIG. 5.

Effects of brefeldin A and monensin on the cleavage of GP-C of LCMV and Lassa virus. Vero cells transfected with a pCAGGS plasmid encoding either Lassa virus GP (lanes 2 and 3), LCMV GP (lanes 5, 6, and 7), or empty vector (lanes 1 and 4) were labeled with [³⁵S]methionine/[³⁵S]cysteine at 24 h after transfection for 30 min, followed by a 3-h chase. GP-C and GP-2 were immunoprecipitated by using a tetra-His antibody and analyzed by SDS-PAGE and fluorography.

FIG. 6.

Amino acid sequences at cleavage site of LCMV GP and GPs from other arenaviruses. The alignment shows the GP sequences between amino acid positions 258 and 268 (numbering of LCMV GP) of the Old World arenaviruses LCMV (1, 6, 36, 39, 44), Lassa virus (3), and Mopeia virus (46), as well as the New World arenaviruses Pichinde virus (2), Oliveros virus (10), Guanarito virus (43), Junin virus (25), Machupo virus (43), and Tacaribe virus (24). The arrow indicates the cleavage site, which has been confirmed for Lassa virus GP-C (30), LCMV GP-C (ARM53b), Pichinde virus GP-C, and Tacaribe virus GP-C (13). Amino acids which are conserved in all sequences are shaded in black, and those which are present in the majority of sequences are shaded in gray. GP-1, C-terminal sequence of GP-1; GP-2, N-terminal sequence of GP-2.