Crimean-congo hemorrhagic fever virus glycoprotein precursor is cleaved by Furin-like and SKI-1 proteases to generate a novel 38-kilodalton glycoprotein

Abstract

Crimean-Congo hemorrhagic fever virus (genus Nairovirus, family Bunyaviridae) genome M segment encodes an unusually large (in comparison to members of other genera) polyprotein (1,684 amino acids in length) containing the two major structural glycoproteins, Gn and Gc, that are posttranslationally processed from precursors PreGn and PreGc by SKI-1 and SKI-1-like proteases, respectively. The characteristics of the N-terminal 519 amino acids located upstream of the mature Gn are unknown. A highly conserved furin/proprotein convertase (PC) cleavage site motif (RSKR247) is located between the variable N-terminal region that is predicted to have mucin-like properties and the rest of PreGn. Mutational analysis of the RSKR247 motif and use of a specific furin/PC inhibitor and brefeldin A demonstrate that furin/PC cleavage occurs at the RSKR247 motif of PreGn as the protein transits the trans Golgi network and generates a novel glycoprotein designated GP38. Immunoprecipitation analysis identified two additional proteins, GP85 and GP160, which contain both mucin and GP38 domain regions, and whose generation does not involve furin/PC cleavage. Consistent with glycosylation predictions, heavy O-linked glycosylation and moderate levels of N-glycans were detected in the GP85 and GP160 proteins, both of which contain the mucin domain. GP38, GP85, and GP160 are likely soluble proteins based on the lack of predicted transmembrane domains, their detection in virus-infected cell supernatants, and the apparent absence from virions. Analogy with soluble glycoproteins and mucin-like proteins encoded by other hemorrhagic fever-associated RNA viruses suggests these proteins could play an important role in viral pathogenesis.

FIG. 1.

Identification of novel CCHF viral glycoproteins expressed from the N-terminal region of the CCHF virus glycoprotein ORF. (A) Autoradiograph of immunoprecipitated viral proteins from the supernatants of CCHF virus-infected SW-13 cells. At 24 h after infection, proteins were pulse-labeled with [³⁵S]cysteine for 30 min and chased for 4 h. Immunoprecipitated proteins were run on a 3 to 8% NuPAGE gel. Proteins were immunoprecipitated with the antibodies GP38/379-392 (lanes 1 and 3) and Gn/540-551 (lanes 2 and 4). Inset shows samples from lanes 1 and 2 run on a 10% NuPAGE gel. (B) Schematic of the WT construct of CCHF virus and a truncation mutant generated that ends at the motif RRLL₅₁₉ (RRLL↓). (C) Autoradiograph of viral proteins immunoprecipitated from the supernatants of SW-13 cells transfected with WT and RRLL↓ constructs with the indicated antibodies and run on a 3 to 8% NuPAGE gel. WT, RRLL↓, and untransfected (control) samples were pulse-labeled with [³⁵S]cysteine for 30 min and were either chased for 3 h and precipitated with the antibody GP38/379-392 (lanes 1 to 3) or chased for 5 h and precipitated with MAb 6C11 (lanes 4 to 6). Proteins expressed from the RRLL↓ construct were pulse-labeled with [³⁵S]cysteine for 30 min, chased for 3.5 h, and precipitated with MAb 7A7 (lane 7). (D) Lanes 1 to 3, WT and RRLL↓ constructs and untransfected control expressed in SW-13 cells. Proteins were pulse-labeled with [³⁵S]cysteine for 45 min and chased for 3 h. Lysed cell monolayers were immunoprecipitated with Gn/540-551 antibody and run on a 3 to 8% NuPAGE gel.

FIG. 2.

Western blot analyses of novel CCHF virus glycoproteins. SW-13 cells were infected with CCHF virus. Supernatants were harvested and concentrated. (A) Supernatants were run on a 3 to 8% NuPAGE gel, and antibody GP38/379-392 against aa 379 to 392 within the GP38-coding region of the CCHF virus glycoprotein precursor was used to probe the proteins. (B) Proteins were probed with a polyclonal monospecific antibody to the mucin region of CCHF virus strain IbAr10200. Lanes 1 and 2, supernatants were run on a 3 to 8% NuPAGE gel. Lane 3, supernatant was run on a 5% SDS-PAGE gel containing urea.

FIG. 3.

Characterization of the newly identified CCHF virus glycoproteins. (A) After SW-13 cells were transfected with the RRLL↓ truncation construct, proteins were pulse-labeled with [³⁵S]cysteine for 30 min and chased for 3 h, and the supernatant proteins were immunoprecipitated with hyperimmune mouse ascitic fluid generated against CCHF virus. Proteins were then treated with various combinations of enzymes. Lane 1, untreated; lane 2, PNGase F; lane 3, O-glycanase (endo-α-N-acetylgalactosaminidase) and sialidase; lane 4, O-glycanase, sialidase, β(1-4)-galactosidase, and β-N-acetylglucosaminidase; lane 5, O-glycanase, sialidase, β(1-4)-galactosidase, β-N-acetylglucosaminidase, and PNGase F. (B) SW-13 cells were infected with CCHF virus and proteins were labeled overnight with [³H]glucosamine. Virions were partially purified by pelleting through a 20% sucrose cushion and then run on a 10% SDS-PAGE gel containing urea.

FIG. 4.

Pulse-chase analysis of GP38-associated proteins in cell and supernatant. SW-13 cells were infected with CCHF virus, proteins were pulse-labeled with [³⁵S]cysteine for 20 min and chased for various times indicated at the top of the gels. Brefeldin A (BFA) and 32 μM dec-RVKR-CMK were added during starvation, labeling, and chase periods. The samples were run on a 7% NuPAGE gel system. (A) Proteins in cell lysates were immunoprecipitated with MAb 6C11. (B) Proteins in supernatants were immunoprecipitated with MAb 6C11. (C) Supernatant proteins were precipitated with antibody GP38/379-392.

FIG. 5.

Effect of mutations on the generation of GP38. (A) Schematic of the mutations generated in the CCHF virus glycoprotein ORF. (B) SW-13 cells were transfected with the indicated constructs, and proteins were pulse-labeled with [³⁵S]cysteine for 30 min and chased for 5 h. The supernatant proteins were immunoprecipitated with MAb 6C11. WT, RRLL↓, and untransfected control were also treated with 50 μM dec-RVKR-CMK. Proteins were run on a 3 to 8% NuPAGE gel. (C) SW-13 cells were transfected with the indicated constructs, and proteins were pulse-labeled with [³⁵S]cysteine for 30 min and chased for 3 h. Proteins were immunoprecipitated with the antibody GP38/379-392 and then run on a 3 to 8% NuPAGE gel.

FIG. 6.

Inhibition of GP38 processing does not prevent the processing of mature Gn. (A) SW-13 cells were infected with CCHF virus, pulse-labeled with [³⁵S]cysteine for 30 min, chased for 4 h and the cell lysates/supernatants were immunoprecipitated with antibody Gn/540-551, and run on a 3 to 8% NuPAGE gel. Samples were either untreated or treated with 30 μM dec-RVKR-CMK. (B) SW-13 cells were transfected with the indicated constructs, and proteins were pulse-labeled with [³⁵S]cysteine for 30 min, chased for 3 h, immunoprecipitated with antibody Gn/540-551, and run on a 3 to 8% NuPAGE gel.

FIG. 7.

Current model of CCHF virus glycoprotein processing.