Crimean-Congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase SKI-1

Abstract

Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne member of the genus Nairovirus, family Bunyaviridae. The mature virus glycoproteins, Gn and Gc (previously referred to as G2 and G1), are generated by proteolytic cleavage from precursor proteins. The amino termini of Gn and Gc are immediately preceded by tetrapeptides RRLL and RKPL, respectively, leading to the hypothesis that SKI-1 or related proteases may be involved (A. J. Sanchez, M. J. Vincent, and S. T. Nichol, J. Virol. 76:7263-7275, 2002). In vitro peptide cleavage data show that an RRLL peptide representing the Gn processing site is efficiently cleaved by SKI-1 protease, whereas an RKPL peptide representing the Gc processing site is cleaved at negligible levels. The efficient cleavage of RRLL peptide is consistent with the known recognition sequences of SKI-1, including the sequence determinants involved in the cleavage of the Lassa virus (family Arenaviridae) glycoprotein precursor. These in vitro findings were confirmed by expression of wild-type or mutant CCHF virus glycoproteins in CHO cells engineered to express functional or nonfunctional SKI-1. Gn processing was found to be dependent on functional SKI-1, whereas Gc processing was not. Gn processing occurred in the endoplasmic reticulum-cis Golgi compartments and was dependent on an R at the -4 position within the RRLL recognition motif, consistent with the known cleavage properties of SKI-1. Comparison of SKI-1 cleavage efficiency between peptides representing Lassa virus GP2 and CCHF virus Gn cleavage sites suggests that amino acids flanking the RRLL may modulate the efficiency. The apparent lack of SKI-1 cleavage at the CCHF virus Gc RKPL site indicates that related proteases, other than SKI-1, are likely to be involved in the processing at this site and identical or similar sites utilized in several New World arenaviruses.

FIG. 1.

Peptides containing RRLL sequences are efficiently processed by hSKI-1 in vitro. (A and B) RP-HPLC chromatogram of the crude digest following 1 h of incubation at 37°C of 20 μg of Q-CCHFV-A [Abz-S-S-G-S-R-R-L-L↓S-E-E-S-Y(NO₂)-Ala-NH₂] with recombinant hSKI-1 (10 μl) in 25 mM Tris-25 mM MES-2.5 mM CaCl₂, pH 7.4. The elution of the peaks was monitored in line by UV absorbance (absorbance unit full scale [AUFS] at a 214-nm wavelength) (A) as well as fluorescence (λ_ex, 320 nm; λ_em, 420 nm) (B) detectors. The peaks eluting at retention times (R_t) 41.45, 39.5, and 33.5 min were characterized by MALDI-TOF (MS) as the undigested peptide [m/z = 1,704.1 (M+H)⁺], the highly fluorescent N-terminal (NT) Abz-S-S-G-S-R-R-L-L-OH [m/z = 994.8 (M+H)⁺], and the nonfluorescent C-terminal (CT) S-E-E-S-Y(NO₂)-Ala-NH₂ [m/z = 729.0 (M+H)⁺]. Note that both NT fragment and the undigested peptides are detectable by UV absorbance (A) as well as by fluorescence measurement (B) while the CT fragment is detectable by UV absorbance alone. Notice the loss of one or multiple 16 mass units (mu) (either NH₂ or OH groups) or the addition of 22 mu (Na) in the observed mass spectra.

FIG. 2.

Peptides containing RKPL sequences are poor substrates for hSKI-1. RP-HPLC chromatogram of the crude digest following 24 h of incubation at 37°C of 20 μg of desAbzQ-CCHFV-B [A-L-V-L-R-K-P-L↓F-L-D-S-Y(NO₂)-Ala-NH₂] with excess recombinant hSKI-1 (50 μl) in 25 mM Tris-25 mM MES-2.5 mM CaCl₂, pH 7.4. The elution of the peaks was monitored in line by UV (214-nm wavelength) absorbance. The peaks eluting at retention times 48.03, 42.09, and 41.5 min were characterized by MALDI-TOF (MS) as the undigested peptide [m/z = 1,651.2 (M+H)⁺], the N-terminal (NT) A-L-V-L-R-K-P-L-OH [m/z = 909.9 (M+H)⁺], and the C-terminal (CT) F-L-D-S-Y(NO₂)-Ala-NH₂ [m/z = 759.7 (M+H)⁺]. The three minor peaks eluting at 45.4, 46.3, and 46.7 min were identified by MS as A-L-V-L-R-K-P-L-F-OH (m/z = 1,057.2), A-L-V-L-R-K-P-L-F-L-D-OH (m/z = 1,286.1), and A-L-V-L-R-K-P-L-F-L-OH (m/z = 1,169.5).

FIG. 3.

Comparison of the in vitro processing kinetics of peptides containing RRLL and RKPL sequences. desAbz Q-CCHFV-A (A) or desAbz Q-CCHFV-B (B) was incubated with SKI-1 (10 μl) for 5 min and 18 h in 25 mM Tris-25 mM MES-2.5 mM CaCl₂, pH 7.4. Note the near disappearance of the peak at an m/z value of 1,704 for the 18-h digest of Q-CCHFV-A peptide, whereas the desAbz Q-CCHFV-B exhibited only a minor cleavage even after 18 h of digestion.

FIG. 4.

Processing of Gn is abolished in cells which are deficient in SKI-1 expression. Plasmid DNA encoding CCHF virus glycoproteins was transfected into SKI-1(+) and SKI-1(−) cells and was labeled with [³⁵S]cysteine. Precleared cell lysates were immunoprecipitated by using HMAF specific to CCHF virus proteins. Proteins were resolved in 10% NuPAGE gels, and proteins were visualized by autoradiography. (A) Expression of CCHF virus glycoproteins in SKI-1(+) and SKI-1(−) cells. Cells were labeled for 30 min (lanes 1 and 4) and were chased for 2 h (lanes 2 and 5) and 3 h (lanes 3 and 6). The numbers at the right-hand side denote molecular size markers. PreGn refers to the 140-kDa Gn precursor, and Gn refers to the mature Gn protein. PreGc and Gc correspond to the precursors of Gc and mature Gc, respectively. (B) Processing of the glycoproteins in the presence of BFA. SW13 cells were transfected with plasmid expressing the wt protein, and the treatments are indicated on the top. Proteins were resolved by using 3 to 8% gradient NuPAGE. P160 is likely to be a higher-molecular-weight protein secreted into the medium. The intracellular accumulation is due to BFA treatment.

FIG. 5.

Mutation of critical R at the P-4 position in the context of RRLL at the SKI-1 recognition site abolishes Gn processing. SKI-1(+) cells were transfected with plasmid encoding CCHF virus glycoprotein with a mutation at the potential processing site (516R of the RRLL tetrapeptide to I or 804R of RKLL tetrapeptide to I). Transfected cells were labeled with [³⁵S]cysteine for 30 min and were chased for 2 and 3 h. Proteins were immunoprecipitated by using CCHF virus-specific antibodies and were resolved in 10% NuPAGE. The numbers at the top indicate the periods of sampling, and those on the right-hand side indicate the position of molecular size markers. PreGn and PreGc denote precursors of Gn and Gc, respectively. A darker exposure of the proteins ranging from 50 kDa to the bottom of the gel is presented as the bottom panel to better visualize the Gn protein.

FIG. 6.

Processing of Gc and effects of RKPL>IKPL mutation. SW13 cells were transfected with plasmids encoding the wt glycoprotein or a mutant (1037 IKPL) at the Gc processing site (1037R in the RKPL tetrapeptide to I) and Gc or a mutant form of Gc (Gc-IKPL). Proteins were immunoprecipitated with HMAF and were resolved in a 10% NuPAGE gel. In each batch, cells were labeled for 30 min (designated 0) and were chased for 3 h (designated 3). Lanes 1 and 2, wt protein; lanes 3 and 4, 1037 IKPL mutant in the context of full-length protein; lanes 5 and 6, Gc; lanes 7 and 8, IKPL mutant in the context of Gc. PreGn and PreGc denote the precursors of Gn and Gc, respectively. The numbers at the right-hand side indicate the position of molecular size markers.

FIG. 7.

Proposed model of CCHF virus glycoprotein processing in mammalian cells. CCHF virus glycoprotein is likely synthesized as a precursor (its molecular size is not yet known). Upon translation, PreGn and PreGc are generated possibly after the hydrophobic domain and before RKPL, and this cleavage may be mediated by signal peptidase. From PreGc, mature Gc is processed following the RKPL (amino acid 1040) site, and this process is mediated by a protease other than SKI-1. This process is rapid as mature Gc can be visualized even during 20- or 30-min pulse periods. Mature Gc (but not PreGc) is recovered from the extracellular medium and becomes the structural element of the virus. Gn is generated from PreGn following the RRLL (amino acid 519) site, and this process is mediated by SKI-1. The processing of Gn appears to be a rather slow process, as Gn is visualized only in 2- to 3-h chase samples. Processing of Gn and Gc are not affected by BFA treatment, suggesting their occurrence in the ER-cis Golgi compartments. A 160-kDa protein (PreGn with terminal O-glycosidic modifications) and Gn are recovered from the extracellular media. CCHF virus contains predominantly mature forms of Gn and Gc as the glycoprotein elements. It remains to be seen if furin-mediated cleavage occurs at the RSKR (amino acid 247) site. If that happens, it will result in the separation of a mucin-like region and a protein with an expected molecular size of approximately 35 kDa (P35). The predicted O-glycosylation sites are indicated by long Y bars, and the predicted N-glycosylation sites are indicated by short Y bars.