The Lassa virus glycoprotein precursor GP-C is proteolytically processed by subtilase SKI-1/S1P - PubMed (original) (raw)

The Lassa virus glycoprotein precursor GP-C is proteolytically processed by subtilase SKI-1/S1P

O Lenz et al. Proc Natl Acad Sci U S A. 2001.

Abstract

The surface glycoprotein of the Lassa virus, a member of the arenaviridae family, is synthesized as a 76-kDa precursor (GP-C) that is posttranslationally cleaved into an N-terminal 44-kDa subunit and a C-terminal membrane-anchored 36-kDa subunit. Cleavage occurs at the C-terminal end of the unusual recognition motif R-R-L-L. We show here that GP-C is cleaved in the endoplasmic reticulum by the cellular subtilase SKI-1/S1P, an enzyme that has so far been observed to be involved in cholesterol metabolism. Furthermore, we present evidence that only cleaved glycoprotein is incorporated into virions and that this is necessary for the formation of infectious virus. To our knowledge, there have been no previous reports of this type of viral glycoprotein processing, one that may be an interesting target for antiviral therapy.

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Figures

Figure 1

Cleavage of Lassa virus GP-C is calcium dependent. Lassa virus glycoprotein precursor GP-C was expressed in BHK cells incubated with calcium free and normal Dulbecco's medium (GIBCO) by using pTM1-GP-C and the MVA-T7 system (31). The calcium-specific ionophore A23187 (Calbiochem; ref. 18) was added to the transfection medium in concentrations as indicated. Cells were lysed in sample buffer 6 h posttransfection, and the proteins were subjected to electrophoresis on an 11% polyacrylamide gel and electrophoretically blotted onto a nitrocellulose membrane. Cleaved GP-2 and the uncleaved precursor GP-C were identified by immunodetection by using rabbit anti-GP2 immune serum (9). GP-C* representing unglycosylated GP-C is due to overexpression in the vaccinia T7 system(9).

Figure 2

BFA does not prevent cleavage of Lassa virus GP-C. Lassa virus GP-C and FPV-HA were expressed in Vero cells by using the MVA-T7 system. (A) Four hours posttransfection cells were incubated with medium lacking methionine and cysteine for 1 h before they were labeled with [35S]methionine and [35S]cysteine for 30 min. The radioactive label was chased for 3 h in the presence of normal DMEM, the cells were solubilized in lysis buffer, and GP-C and GP-2 were immunoprecipitated with GP-2-specific rabbit-immune serum. Precipitated GP-C was treated with endoglycosidase H (New England Biolabs; lanes 2 and 5), with endoglycosidase F (New England Biolabs; lanes 3 and 6), or was left untreated (lanes 1 and 4) and subjected to SDS/PAGE and fluorography. BFA was added at a final concentration of 15 μg/ml to the medium as indicated. (B) As a control, cleavage of FPV-HA was analyzed by using mAb HA-2A11 (15).

Figure 3

SRD-12B cells cleave Lassa virus glycoprotein precursor GP-C only after transfection with recombinant SKI-1/S1P. (A) Nonconfluent cultures of SRD-12B cells and parental CHO-K1 cells were cotransfected with pCDNA3.1 encoding the human SKI-1/S1P (ref. ; lane 3) or empty vector (lanes 1 and 2) 12 h before MVA-T7-mediated expression of Lassa virus GP-C. (B) Nonconfluent SRD-12B cells were transfected with pCDNA3.1-SKI-1/S1P (lane 4) or empty vector (lane 3) 12 h before infection with Lassa virus, strain Josiah, at a moi of 1. Parental CHO-K1 cells were infected with Lassa virus in parallel (lane 1). At 48 h p.i., cells were lysed in sample buffer, and cleavage of GP-C was analyzed on Western blots.

Figure 4

SKI-1/S1P-deficient cells release noninfectious particles devoid of glycoprotein. (A) SRD-12B cells, SRD-12B cells transfected with pcDNA3.1-SKI-1/S1P, and Vero cells were infected with Lassa virus, strain Josiah. Supernatants of infected cells were analyzed by plaque titration at 0, 24, 48, 72, 96, and 144 h p.i. according to a standard protocol. (B) To detect release of noninfectious viral particles, SRD-12B cells and SRD-12B cells transfected with human SKI-1/S1P were infected with Lassa virus at a moi of 1, and virions in the supernatants were enriched and partially purified by centrifugation through a 20% sucrose cushion. These pellets (lanes 3 and 5; S) and lysates of transfected cells infected in parallel (lanes 2 and 4; C) were solubilized by treatment with sample buffer and analyzed by Western blot by using peptide sera raised against GP-2 (Upper) and against NP (Lower). (C) To study whether the NP is enclosed by a lipid envelope, supernatants of Lassa virus-infected CHO and SRD-12B cells were centrifuged as described above and treated for 30 min at 37°C with 0.1 μg/μl proteinase K (lanes 2 and 5) or with 0.1 μg/μl proteinase K and 1% Triton X-100 (lanes 3 and 6) or was left untreated (lanes 1 and 4). Samples were analyzed by using Western blots and staining with anti-NP immune serum.

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The Lassa virus glycoprotein precursor GP-C is proteolytically processed by subtilase SKI-1/S1P - PubMed (original) (raw)