Processing of the Ebola virus glycoprotein by the proprotein convertase furin - PubMed (original) (raw)
Processing of the Ebola virus glycoprotein by the proprotein convertase furin
V E Volchkov et al. Proc Natl Acad Sci U S A. 1998.
Abstract
In the present study, we have investigated processing and maturation of the envelope glycoprotein (GP) of Ebola virus. When GP expressed from vaccinia virus vectors was analyzed by pulse-chase experiments, the mature form and two different precursors were identified. First, the endoplasmic reticulum form preGPer, full-length GP with oligomannosidic N-glycans, was detected. preGPer (110 kDa) was replaced by the Golgi-specific form preGP (160 kDa), full-length GP containing mature carbohydrates. preGP was finally converted by proteolysis into mature GP1,2, which consisted of two disulfide-linked cleavage products, the amino-terminal 140-kDa fragment GP1, and the carboxyl-terminal 26-kDa fragment GP2. GP1,2 was also identified in Ebola virions. Studies employing site-directed mutagenesis revealed that GP was cleaved at a multibasic amino acid motif located at positions 497 to 501 of the ORF. Cleavage was blocked by a peptidyl chloromethylketone containing such a motif. GP is cleaved by the proprotein convertase furin. This was indicated by the observation that cleavage did not occur when GP was expressed in furin-defective LoVo cells but that it was restored in these cells by vector-expressed furin. The Reston subtype, which differs from all other Ebola viruses by its low human pathogenicity, has a reduced cleavability due to a mutation at the cleavage site. As a result of these observations, it should now be considered that proteolytic processing of GP may be an important determinant for the pathogenicity of Ebola virus.
Figures
Figure 1
Synthesis, processing, and transport of EBOV GP. Pulse–chase analyses of EBOV GP were done in RK13 cells infected with vSCGP8 (A) or in HeLa cells infected with vTF7-3 and transfected with pGEM-GPLT (B). At 6 h postinfection, cells were pulse-labeled for 20 min (lane 0) and chased for the indicated time intervals. Lysed cells were immunoprecipitated with goat anti-EBOV Igs. Precipitated proteins were separated on 15% (A, C) or 8% (B) polyacrylamide gels, and proteins were visualized by fluorography. (D) Endoglycosidase digestion of the tail elongation mutant GPLT expressed in HeLa cells. Cells were infected with vTF7-3 and subsequently transfected with pGEM-mGP8 expressing wild-type GP (GP) or pGEM-GPLT expressing GPLT. At 18 h posttransfection, cells were lysed and subjected to endoglycosidase treatments. Digested samples were separated by SDS/PAGE and blotted onto a poly(vinylidene difluoride) membrane. Detection of GP-specific proteins was performed with goat anti-EBOV Igs. The positions of the precursors (preGPer and preGP) and the proteolytically cleaved subunit GP1 before and after endoglycosidase treatment are indicated.
Figure 2
Virion GP of EBOV consists of two cleavage subunits. EBOV GP metabolically labeled with [3H]glucosamine or with [35S]methionine-cysteine was immunoprecipitated from purified virions grown in Vero E6 cells and analyzed by 15% SDS/PAGE under reducing or nonreducing conditions. The positions of the mature GP (GP1,2), and the two cleavage subunits GP1 and GP2, are indicated.
Figure 3
Intracellular processing of the cleavage site mutants Z/F1 and Z/F2. HeLa cells were infected with vTF7-3 and transfected with the plasmids pGEM-mGP8 (wild-type GP, WT), pGEM-Z/F1 (mutant Z/F1), and pGEM-Z/F2 (mutant Z/F2). At 6 h postinfection, cells were pulse-labeled for 20 min and chased for 240 min. Immunoprecipitated proteins were separated under reducing conditions on 8% (Upper gel) or 15% (Lower gel) polyacrylamide gels. The positions of the noncleaved precursor (preGP) of both mutants and the cleavage subunits GP1 and GP2 are indicated. The sequences at the cleavage sites are shown at Top.
Figure 4
Effect of decanoylated R-V-K-R chloromethylketone on cleavage of EBOV GP. RK13 cells were infected with vSCGP8. At 6 h postinfection, cells were pulse-labeled for 20 min and chased for various times in the absence (0 μM) or presence (25 μM, 80 μM) of inhibitor. Immunoprecipitated samples were separated by PAGE under reducing conditions on 15% gels (Top) and 8% gels (Middle) and under nonreducing conditions on 8% gels (Bottom). The positions of the precursors (preGPer and preGP) as well as the cleavage subunits GP1 and GP2 are indicated. Under nonreducing conditions, high _M_r forms of both precursors are seen (*preGP and *preGPer).
Figure 5
Synthesis and processing of EBOV GP in LoVo cells with or without coexpression of furin. LoVo cells were either infected with vSCGP8 alone (moi of 10 pfu/cell) or coinfected with vSCGP8 and VVHfur (each with a moi of 10 pfu/cell). Infected cells were pulse-labeled for 20 min at 6 h postinfection and chased for different time intervals as indicated. Immunoprecipitated samples were separated on 8% (Upper) and 15% (Lower) polyacrylamide gels under reducing (A) and an 8% polyacrylamide gel under nonreducing (B) conditions. The positions of the precursors (preGPer and preGP) as well as the cleavage subunits GP1 and GP2 are indicated. Under nonreducing conditions, the uncleaved precursor showed an _M_r higher than 220 kDa (*preGP).
Figure 6
Processing of wild-type and cleavage site mutants of EBOV subtype Reston. HeLa cells were infected with vTF7-3 and transfected with the plasmids pGEM-PR8 (wild-type Reston GP, WT), pGEM-R/K (mutant R/K), and pGEM-R/R (mutant R/R). At 6 h postinfection, cells were pulse-labeled for 20 min and chased for 240 min. Immunoprecipitated proteins were separated under reducing conditions on an 8% polyacrylamide gel. The positions of the noncleaved precursors (preGPer and preGP) and the cleavage subunit GP1 are indicated. The sequences at the cleavage sites are shown at Top.
Figure 7
Scheme of EBOV GP, cleavage sites of different subtypes, and a model for the structure of the mature GP monomer. Signal peptide sequence, the carboxyl-terminal transmembrane domain, and the putative fusion domain are indicated by gray boxes. The altered amino acid in the cleavage site of EBOV subtype Reston is marked by a box. Asterisk, potential _N_-glycosylation site; C, cysteine residue; GP1, larger cleavage product of GP; GP2, smaller cleavage product of GP; S-S, disulfide bridge.
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