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Journal of Virology, June 2007, p. 6632-6642, Vol. 81, No. 12
0022-538X/07/$08.00+0     doi:10.1128/JVI.02730-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of a Novel C-Terminal Cleavage of Crimean-Congo Hemorrhagic Fever Virus PreGN That Leads to Generation of an NSM Protein{triangledown}

Louis A. Altamura,1 Andrea Bertolotti-Ciarlet,1 Jeffrey Teigler,2 Jason Paragas,3,{dagger} Connie S. Schmaljohn,3 and Robert W. Doms1*

Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104,1 Ursinus College, Collegeville, Pennsylvania 19426,2 United States Army Medical Research Institute for Infectious Diseases, Fort Detrick, Frederick, Maryland 217023

Received 12 December 2006/ Accepted 29 March 2007


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ABSTRACT
 
The structural glycoproteins of Crimean-Congo hemorrhagic fever virus (CCHFV; genus Nairovirus, family Bunyaviridae) are derived through endoproteolytic cleavage of a 1,684-amino-acid M RNA segment-encoded polyprotein. This polyprotein is cotranslationally cleaved into the PreGN and PreGC precursors, which are then cleaved by SKI-1 and a SKI-1-like protease to generate the N termini of GN and GC, respectively. However, the resulting polypeptide defined by the N termini of GN and GC is predicted to be larger (58 kDa) than mature GN (37 kDa). By analogy to the topologically similar M segment-encoded polyproteins of viruses in the Orthobunyavirus genus, the C-terminal region of PreGN that contains four predicted transmembrane domains may also contain a nonstructural protein, NSM. To characterize potential PreGN C-terminal cleavage events, a panel of epitope-tagged PreGN truncation and internal deletion mutants was developed. These constructs allowed for the identification of a C-terminal endoproteolytic cleavage within, or very proximal to, the second predicted transmembrane domain following the GN ectodomain and the subsequent generation of a C-terminal fragment. Pulse-chase experiments showed that PreGN C-terminal cleavage occurred shortly after synthesis of the precursor and prior to generation of the GN glycoprotein. The resulting fragment trafficked to the Golgi compartment, the site of virus assembly. Development of an antiserum specific to the second cytoplasmic loop of PreGN allowed detection of cell-associated NSM proteins derived from transient expression of the complete CCHFV M segment and also in the context of virus infection.


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INTRODUCTION
 
Crimean-Congo hemorrhagic fever virus (CCHFV) causes a hemorrhagic syndrome in humans with fatality rates varying between 5 and 30% but generally causes subclinical disease in animals (12). CCHFV is a member of the genus Nairovirus within the family Bunyaviridae and, like all members of this family, has a tripartite genome consisting of large (L), medium (M), and small (S) negative-sense RNA segments that encode the viral RNA polymerase, the glycoproteins, and the nucleocapsid protein, respectively (6, 33). As with other Bunyaviridae, the GN and GC glycoproteins of CCHFV are derived from an M segment-encoded polyprotein. Unique to the Nairovirus genus, however, is the fact that the M polyprotein requires processing through a series of intermediate precursors prior to generation of the mature glycoproteins found in virions (4, 5, 27, 32, 40). For CCHFV, several seminal studies have illustrated the complexity of these endoproteolytic events (Fig. 1). First, the M polyprotein is cotranslationally cleaved into the PreGN (140-kDa) and PreGC (85-kDa) precursors, presumably by signal peptidase. PreGN is then cleaved by the protease SKI-1 in the endoplasmic reticulum/cis-Golgi to generate the N terminus of GN (39) and release a mucin-GP38 domain that is further processed by furin to generate the secreted glycoproteins GP38, GP85, and GP160 and possibly also a mucin-like protein (31). The PreGC precursor may also undergo trimming by a SKI-1-like protease to expose the N terminus of GC.


Figure 1
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  		FIG. 1. Processing of the M segment-encoded polyprotein of CCHFV strain IbAr10200. A schematic of the CCHFV M segment-encoded polyprotein is shown, with known and suspected cleavage sites indicated. Signal peptidase is thought to generate the N terminus of the polyprotein and may also liberate PreGC as indicated (32). The mucin-GP38 domains are liberated by SKI-1 cleavage following the RRLL cleavage site to generate the N terminus of GN at amino acid 520 (32, 39). A second cleavage event, perhaps also mediated by SKI-1 or a similar protease, produces the N terminus of GC at residue 1041 following the sequence RKPL (32). Further cleavage by furin following the RSKR motif separates the mucin-like domain from GP38 (31). The region defined by the N termini of GN and GC encodes a 58-kDa polypeptide having four predicted transmembrane domains, indicated by black bars. Since mature GN is approximately 37 kDa, an additional C-terminal processing site may exist between GN and GC, leading to the generation of an NSM protein. The uncertain boundaries of this putative NSM protein are indicated by a dashed line. The cylinders labeled TM 1 to TM 4 represent the four predicted transmembrane helices between the ectodomains of GN and GC. Amino acid boundaries for each helix were predicted with TMHMM 2.0 (37).

One uncertain aspect of CCHFV glycoprotein processing is the site of C-terminal cleavage for the GN glycoprotein. While GN migrates in sodium dodecyl sulfate (SDS)-polyacrylamide gels as a 37-kDa protein, the predicted mass of the polypeptide defined by its N-terminal SKI-1 cleavage site to the beginning of GC is approximately 58 kDa (Fig. 1). This discrepancy suggests that an additional cleavage event(s) is required to generate the GN protein. If so, then cleavage likely occurs somewhere within the topologically complex C-terminal portion of PreGN, which contains four predicted transmembrane domains, the last of which appears to function as a signal sequence for GC. A potential SKI-1-like protease substrate (RKLL) and a polybasic motif (KKRKK) within the cytoplasmic loops of the M polyprotein of CCHFV have been proposed for this putative cleavage event, but mutagenesis of these domains failed to influence GN maturation (39).

Cleavage within the C-terminal portion of PreGN could result in the generation of a discrete membrane protein. The M segments of viruses in the Orthobunyavirus genus, which have identical membrane topologies to those predicted for Nairovirus members such as CCHFV, encode a nonstructural membrane protein termed NSM within the region between their GN and GC glycoproteins (10, 14, 17, 24, 28, 41). These NSM proteins have been shown to accumulate in the Golgi compartment along with the viral glycoproteins, pointing to a possible role for NSM in virus assembly. Indeed, in Bunyamwera virus, deletions of the entire NSM or its N-terminal regions prevented the plasmid rescue of virus by reverse genetics (34). A similar NSM protein has been proposed to exist for CCHFV (18, 31). However, no such protein has yet been identified for this or for any other member of the genus Nairovirus.

To address the hypothesis that CCHFV PreGN undergoes a C-terminal cleavage event to generate an NSM protein, an extensive series of PreGN truncation and deletion constructs were designed, and new detection reagents were developed. Using these tools, we defined the topology of the C-terminal region of PreGN and found that it efficiently underwent an endoproteolytic cleavage event that liberated a C-terminal fragment that trafficked to the Golgi compartment. Furthermore, the chronology and independence of this cleavage event in relation to the N-terminal proteolytic events that define GN were addressed, and a probable location of this cleavage site was identified. This C-terminal fragment was also found to constitute a cell-associated NSM protein that was generated from the full-length M polyprotein, both by transient plasmid expression and by virus infection.


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MATERIALS AND METHODS
 
Cells and viruses. 293T/17, BHK-21, BSC-1, HeLa, Huh-7, and Vero E6 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA), 2 mM L-glutamine (Invitrogen), and 1% penicillin-streptomycin (P/S; Invitrogen). SW-13 cells were grown in Eagle's minimum essential medium (Invitrogen) supplemented with 5% FBS, 2 mM L-glutamine, and 1% P/S. Recombinant vaccinia virus vTF1.1 expressing bacteriophage T7 RNA polymerase (2) was propagated in HeLa cells, and titers were determined in BSC-1 cells according to standard protocols. CCHFV strain IbAr10200 was propagated and titers were determined in SW-13 cells. All CCHFV IbAr10200 infections were conducted in a biosafety level 3+ (BSL-3+) containment suite at the United States Army Medical Research Institute for Infectious Diseases (USAMRIID; Fort Detrick, Frederick, MD). While working with CCHFV, laboratory personnel wore standard personal protective equipment and powered air-purifying respirators.

Plasmids. An expression plasmid encoding the CCHFV IbAr10200 M segment (M [GCV5]) was generated by PCR amplification of the open reading frame, followed by ligation of the amplicon into the pcDNA3.1D/V5-His TOPO expression vector, which encodes an in-frame C-terminal V5-His6 epitope cassette. Thus, GC possessed a C-terminal epitope cassette in this context. To generate the panel of plasmids encoding C-terminal V5-His6 epitope-tagged PreGN truncation mutants and derivative mutants thereof, the CCHFV IbAr10200 M segment was used as a template for PCR amplification of DNA fragments beginning with the M segment start codon and ending with the codon of the terminal amino acid of each construct. These amplicons were also introduced into the pcDNA3.1D/V5-His TOPO expression vector. The PreGNV5(961)-NST mutant, as well as all of the PreGNV5(961) internal deletion mutants, was generated by using standard overlapping PCR approaches to introduce or delete nucleotides from the template plasmid. The cloning of the M segment of CCHFV strain IbAr10200 into the pCAGGS vector was described previously (3). All plasmids were propagated in MAX Efficiency Stbl2 competent cells (Invitrogen) and then confirmed by DNA sequencing. All primer sequences are available upon request.

Antibodies. Antipeptide antisera specific to the GN ectodomain (611TQEGRGHVKLSRGSE625) and to the second cytoplasmic domain of PreGN (926STDKEIHKLHLSIC939) from CCHFV strain IbAr10200 were generated in rabbits and affinity purified in collaboration with ProSci Inc. (Poway, CA). The anti-N monoclonal antibody 9D5-1-1A was produced by Jonathan Smith (formerly of USAMRIID) during previously described efforts to produce CCHFV-specific antibodies (3). A mouse anti-V5 monoclonal antibody (Invitrogen) was used for protein immunoblot assays, and a rabbit polyclonal serum specific to the V5 epitope was used for immunoprecipitations (16).

Transfection. For protein immunoblot analysis of cell lysates transiently transfected with pcDNA3.1 plasmids, confluent monolayers of 293T/17 cells in six-well cluster plates were first infected with vTF1.1 vaccinia virus diluted in DMEM (2.5% FBS, 1% P/S) at a multiplicity of infection (MOI) of 5 for 30 min at 37°C prior to transfection. The virus inoculum was then removed, and DMEM (10% FBS) was added to the cells. For pCAGGS vector-based expression of CCHFV glycoproteins, no vTF1.1 was needed. In both cases, the cells were transfected with 6 µg per well of each expression plasmid using Lipofectamine 2000 (Invitrogen) and then incubated for 18 to 24 h prior to subsequent analysis.

Preparation of transfected cell lysates. The cell culture medium was aspirated, and the cells were washed briefly with Dulbecco's phosphate-buffered saline (DPBS; Invitrogen) and then lysed with buffer consisting of 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl pH 7.4, and Complete protease inhibitor cocktail (Roche, Indianapolis, IN) for 10 min on ice. Insoluble material was removed from the lysates by centrifugation at 20,800 x g for 10 min at 4°C.

Enzymatic deglycosylation of proteins. To assess the N-linked glycosylation of CCHFV glycoproteins, transfected cell lysates were incubated overnight at 37°C with PNGase F, endoglycosidase H (Endo H), or no enzyme according to the manufacturer's instructions (New England Biolabs, Beverly, MA). In order to prevent complex glycosylation of proteins in vivo prior to enzymatic deglycosylation, transfected 293T/17 cells were cultured in DMEM (10% FBS) supplemented with 1 µM deoxymannojirimycin (DMJ; Sigma, St. Louis, MO) for 20 to 22 h prior to lysing the cells.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Protein electrophoresis was performed using the NuPAGE precast gel system (Invitrogen). Specifically, proteins were denatured under reducing conditions at 70°C for 10 min and then separated on 10% Bis-Tris gels using either morpholineethanesulfonic acid (MES) or morpholinepropanesulfonic acid (MOPS) running buffers. Protein molecular weights were estimated by comparison to Full-Range Rainbow molecular weight standards (GE Healthcare Biosciences, Piscataway, NJ). Protein gels were electroblotted onto an Immobilon-P polyvinylidene difluoride membrane (PVDF; Millipore, Billerica, MA) at 30 V for 1 h. The PVDF membranes were then incubated with blocking buffer (PBS, 5% [wt/vol] powdered milk, 0.1% [vol/vol] Tween 20, 0.1% [wt/vol] NaN3) for 30 min at room temperature. Primary antibodies were diluted in blocking buffer and used to probe the membranes overnight at 4°C. After washing with PBST buffer (PBS, 0.2% [vol/vol] Tween 20), horseradish peroxidase-conjugated sheep anti-mouse or sheep anti-rabbit secondary antibodies (GE Healthcare Biosciences) were diluted in blocking buffer lacking NaN3 and used to probe the membranes for an additional 1.5 h at room temperature. The secondary antibody solution was then washed away with additional PBST. Proteins were detected by chemiluminescence using the SuperSignal West Femto kit (Pierce, Rockford, IL), and the immunoblots were imaged using a LAS-1000 Plus gel documentation system (Fujifilm, Tokyo, Japan).

Metabolic radiolabeling of proteins and immunoprecipitation. To analyze the kinetics of CCHFV glycoprotein proteolytic cleavage events, pulse-chase experiments were performed. Briefly, 293T/17 cells expressing CCHFV glycoproteins were pulsed with [35S]cysteine-methionine labeling medium for 15 min, washed briefly with DPBS, and then chased for the indicated time periods with complete DMEM supplemented with 15 µg/ml unlabeled cysteine and methionine. CCHFV proteins were immunoprecipitated as previously described (32) and then separated by SDS-PAGE on NuPAGE 10% Bis-Tris gels. The 35S signal was enhanced with Amplify fluorographic reagent (GE Healthcare Biosciences), collected on storage phosphor screens, and detected using a Typhoon 9410 variable mode imager (GE Healthcare Biosciences). Protein bands were quantified using ImageQuant TL (GE Healthcare Biosciences). For each protein analyzed (PreGN, GN, or C-term961), the values were normalized as percentages of the peak time point for that protein.

CCHFV infections and preparation of viral material. Confluent monolayers of 293T/17 cells in 10-cm culture dishes were infected with CCHFV IbAr10200 at an MOI of 5. At 24 h postinfection, the culture medium was collected and clarified through 0.45-µm syringe filters, Complete protease inhibitor cocktail was added, and the pH was adjusted to 8.0 with 1 M Tris. Virions were semipurified by ultracentrifugation in a Beckman model SW41Ti rotor at 30,000 rpm for 3.5 h at 4°C. Afterwards, the virion pellet was resuspended in 150 µl 1% TX-100 lysis buffer (see above). The cell monolayers were washed once with DPBS and then lysed with 1.5 ml TX-100 lysis buffer as described above. Prior to removal from BSL-3+ containment, cell and virion lysates were inactivated in NuPAGE sample buffer containing 50 mM dithiothreitol for 10 min at 70°C.

Bioinformatics analysis of CCHFV glycoprotein sequences. The transmembrane domains for CCHFV M polyproteins were predicted using the TMHMM server, version 2.0 (37) available from the Center for Biological Sequence Analysis at the Technical University of Denmark. The molecular weights of polypeptide sequences were estimated using MacVector 9.0 (Accelerys, San Diego, CA).


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RESULTS
 
PreGN is cleaved at its C terminus. The structural glycoproteins of CCHFV are derived from the polyprotein precursor encoded by its M RNA segment through a series of endoproteolytic cleavage events. The M polyprotein is first cleaved into two intermediate precursors, PreGN and PreGC, though the precise boundary between them remains poorly defined. The PreGN precursor is subject to extensive proteolytic processing, with the N-terminal mucin-like and GP38 domains being removed by furin and SKI-1-mediated proteolysis, respectively (Fig. 1). SKI-1 cleavage generates the N terminus of mature GN at amino acid 520 (in strain IbAr10200), and a related protease is thought to cleave the N terminus of PreGC to yield a mature GC beginning at amino acid 1041. There are four predicted transmembrane helices between the GN and GC ectodomains, which define two relatively large (99 and 148 amino acids, respectively) cytoplasmic loops separated by a short (14-amino-acid) lumenal domain (Fig. 1). If GN were defined as comprising amino acids 520 to 1040, then this protein would have a predicted mass of 58 kDa, excluding posttranslational modifications. However, GN migrates with an approximate molecular mass of 37 kDa in SDS-polyacrylamide gels, suggesting that an additional cleavage event(s) in the topologically complex region between PreGN and PreGC must occur.

To test this hypothesis, a plasmid [PreGNV5(961)] was constructed that encoded amino acids 1 to 961 of the IbAr10200 M polyprotein and a C-terminal V5-His6 cassette (Fig. 2A). This version of PreGN, which included three of the four predicted transmembrane helices following the GN ectodomain and most of the second cytoplasmic loop, was designed based upon a previous report which operationally defined PreGC as amino acids 962 to 1684 of the polyprotein (32). Additionally, by truncating PreGN prior to the PreGC signal sequence, we hoped to avoid the complication of detecting additional product(s) generated from the PreGN-PreGC cleavage site.


Figure 2
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  		FIG. 2. PreGN is cleaved at its C terminus. (A) Schematic of CCHFV full-length M polyprotein and PreGN glycoprotein constructs. "M" and "961" refer to the constructs M[GCV5] and PreGNV5(961), respectively, that are described in the text. Each construct possessed a C-terminal V5-His6 epitope cassette. 293T/17 cells were transfected with the indicated constructs and then cell lysates were prepared at 18 to 22 h posttransfection. A plasmid expressing green fluorescent protein (GFP) was used as a negative control. The samples were separated by SDS-PAGE using 10% Bis-Tris NuPAGE gels and MOPS-based running buffer. Proteins in cell lysates were immunoblotted with an anti-GN ectodomain polyclonal antiserum (B) or a monoclonal antibody directed against the V5 epitope tag (C). The locations of PreGN, GN, and the C-terminal fragment are indicated.

When lysates of 293T/17 cells expressing PreGNV5(961) were analyzed by SDS-PAGE followed by immunoblotting with a polyclonal antiserum specific to the GN ectodomain, both PreGN (140 kDa) and GN (37 kDa) species were observed, as previously reported (1). These bands were of similar apparent size to those generated from a full-length IbAr10200 M polyprotein possessing a V5-His6 cassette at the C terminus of GC (M [GCV5]) (Fig. 2B), indicating that PreGN and GN were processed similarly in these two contexts.

When a blot of the PreGNV5(961) lysate was probed with an antibody specific to the C-terminal V5 epitope tag (Fig. 2C), PreGN was detected in addition to a band with a molecular mass of approximately 20 kDa, indicating that a cleavage event had occurred somewhere within the C-terminal region of PreGN. Although several species greater than 20 kDa were faintly visible, the absence of these bands in the anti-GN immunoblot suggests that these are not processing intermediates. It seems more likely that these are degradation products of PreGN or aggregates of the hydrophobic C-terminal fragment. Furthermore, the lack of processing intermediates demonstrated that C-terminal cleavage occurred efficiently and prior to the N-terminal cleavage events that liberate the mucin-like and GP38 glycoproteins from GN. To determine if this C-terminal cleavage event might be specific to 293T/17 cells, PreGNV5(961) was expressed in additional cell lines known to support CCHFV replication (BHK, CHO-K1, HeLa, Huh-7, Vero E6, and SW-13) (15, 32, 39) (personal observations). Indeed, C-terminal cleavage of PreGN occurred efficiently in all cell lines tested (data not shown). Thus, the addition of an epitope cassette at the C terminus of PreGN allowed for the identification of a novel, C-terminal proteolytic fragment.

Chronology of PreGN processing. Although the preceding data suggested that PreGN underwent an efficient C-terminal cleavage event, they did not formally show that a precursor-product relationship exists between PreGN and the fragment. Therefore, a pulse-chase experiment was performed to investigate the kinetics with which the C-terminal fragment was generated relative to other posttranslational processing events. Cells expressing PreGNV5(961) were metabolically radiolabeled with [35S]cysteine-methionine for 15 min and then placed in normal growth medium with excess unlabeled cysteine and methionine for the times indicated (Fig. 3). At each time point, cell lysates were prepared, divided equally, and then immunoprecipitated using antibodies specific to either the C-terminal V5 epitope tag or to the GN ectodomain. Using either antibody, the maximum amount of PreGN was immunoprecipitated after 5 min of chase and then decreased through the chase period. As shown in Fig. 3A, the C-terminal fragment was efficiently immunoprecipitated with the V5 antiserum after metabolic labeling for as little as 10 min, indicating that this processing event begins very shortly after the synthesis of PreGN, or perhaps even cotranslationally. However, C-terminal cleavage of PreGN appeared to be somewhat asynchronous, as evidenced by the fact that the fragment did not reach its peak abundance until approximately 1 hour into the chase (Fig. 3A). This variation could be due to a variety of factors, including limiting amounts of protease or asynchrony in the folding of this extensively disulfide-linked glycoprotein, both of which could be exacerbated by the levels of overexpression achieved in the vaccinia virus-T7 expression system used here. The greatest amount of mature GN was only precipitated after 3 to 4 hours (Fig. 3B), in agreement with previous reports (32, 39). Together, these data show that, in the context of this expression construct, PreGN is C-terminally cleaved shortly after its synthesis to generate the C-terminal fragment, followed by N-terminal SKI-1 cleavage to liberate GN.


Figure 3
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  		FIG. 3. Pulse-chase analysis of PreGN cleavage events. 293T/17 cells were transfected with a plasmid expressing PreGNV5(961), metabolically labeled with [35S]cysteine-methionine for 15 min, and then placed in normal growth medium containing excess cysteine-methionine for the times indicated at the tops of the gels. The cells were then lysed and immunoprecipitated with anti-V5 epitope polyclonal serum (A) or an anti-GN ectodomain polyclonal serum (B). Some cells were labeled for only 5 or 10 min prior to lysis (left side of gel, as indicated). The samples were separated by SDS-PAGE using 10% Bis-Tris NuPAGE gels and MOPS-based running buffer. An asterisk indicates the location of the unidentified coprecipitating protein described in the text. The abundance of PreGN, GN, and the C-terminal fragment at each time point was determined by storage phosphor screen image analysis. Relative protein abundance represents the percentage of the maximum signal intensity obtained for each protein over the course of the experiment.

Interestingly, the C-terminal fragment was also precipitated with the GN ectodomain antiserum (Fig. 3B), suggesting a possible interaction between this protein and either PreGN or the GN glycoprotein. Additional studies are warranted to confirm this observation. There was also a 60-kDa species that was immunoprecipitated throughout the pulse and chase periods using both antisera. The fact that this band was not detected by immunoblotting using either the V5 or GN ectodomain antiserum (Fig. 2B and C) suggests that this is not a PreGN processing intermediate but may instead be a coimmunoprecipitating cellular protein. However, further studies will be required to identify this protein.

The C-terminal fragment traffics to the Golgi compartment. If the C-terminal fragment constituted an NSM protein for CCHFV, then by analogy to Orthobunyavirus NSM proteins (24, 28, 34) one might expect it to accumulate in the Golgi compartment, the site of virus assembly (9, 11, 23). The presence of complex N-linked glycans on glycoproteins is one indicator of transport to the Golgi compartment. Thus, to characterize the intracellular trafficking of the C-terminal fragment, we introduced an N-linked glycosylation site within the lumenal loop of PreGN, which we hypothesized to be a part of the C-terminal fragment based upon amino acid mass predictions. However, since the glycosyltransferase machinery can only add N-linked sugars to sites greater than 12 to 14 amino acids away from the membrane (29), and since the predicted CCHFV lumenal domain is only 14 amino acids in length, an expression plasmid [PreGNV5(961)-NST] was produced in which the lumenal loop was duplicated and an NST glycosylation sequon was inserted at the middle of the loop (Fig. 4A).


Figure 4
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  		FIG. 4. Trafficking and topology of the PreGN C-terminal fragment. (A) Schematic of PreGNV5 lumenal loop constructs. Using the PreGNV5(961) construct as a template, the lumenal loop was duplicated and an NST glycosylation sequon was added at the junction to make PreGNV5(961)-NST. Each construct possessed a C-terminal V5-His6 epitope cassette. (B) 293T/17 cells were transfected with the indicated constructs and cultured in the absence (top panel) or presence (bottom panel) of 1 µM DMJ, and then cell lysates were prepared at 18 to 22 h posttransfection. The samples were then treated with PNGase F, Endo H, or mock digested as indicated prior to separation by SDS-PAGE using 10% Bis-Tris NuPAGE gels and MOPS-based running buffer. Proteins in cell lysates were immunoblotted with a monoclonal antibody directed against the V5 epitope. The locations of PreGN, the C-terminal fragment of PreGNV5(961) (C-term961), and the deglycosylated (C-term961-NST) and glycosylated (C-term961-NSTglyc) C-terminal fragments of PreGNV5(961)-NST are indicated.

Analysis of PreGNV5(961)- and PreGNV5(961)-NST-transfected 293T/17 cell lysates separated by SDS-PAGE and immunoblotted with anti-V5 antibody showed that both constructs generated PreGN and C-terminal fragments (Fig. 4B, top panel). Immunoblotting with the GN ectodomain antiserum demonstrated that the mature GN glycoprotein was also generated in both contexts (data not shown). Untreated lysates showed that the PreGNV5(961)-NST C-terminal fragment migrated more slowly than that from the parental construct. Treatment of these cell lysates with PNGase F had no effect on the parental C-terminal fragment but increased the mobility of the PreGNV5(961)-NST fragment, confirming the utilization of the introduced glycosylation site. The deglycosylated PreGNV5(961)-NST C-terminal fragment still migrated more slowly than the parental fragment, consistent with the increased length of the lumenal domain in the mutant. When samples were treated with Endo H, an enzyme that only removes immature N-linked carbohydrate domains, deglycosylation of the PreGNV5(961)-NST C-terminal fragment was incomplete. To control for inefficient deglycosylation by Endo H, these constructs were also expressed in 293T/17 cells treated with DMJ, which prevents trimming of high-mannose sugars to more complex glycans. Incubation of DMJ-treated PreGNV5(961)-NST lysates with Endo H led to complete deglycosylation of the C-terminal fragment (Fig. 4B, bottom panel). Thus, we concluded that a fraction of the PreGNV5(961)-NST C-terminal fragment pool contained a complex N-linked glycan indicative of transport to the medial Golgi or beyond. Furthermore, the utilization of the introduced glycosylation site confirmed the predicted lumenal membrane topology of this domain within PreGN, as well as its presence within the C-terminal fragment.

Mapping of the GN C terminus. To more precisely define the GN C-terminal cleavage site, a series of plasmids was constructed that encoded PreGN molecules of varying lengths truncated at their C termini and each appended with a V5-His6 epitope cassette (Fig. 5A). These plasmids were transiently transfected into 293T/17 cells, and the expressed proteins were analyzed by SDS-PAGE and immunoblotting using antibodies specific to the GN ectodomain or to the V5 epitope tag. When the immunoblot was probed with the GN antiserum (Fig. 5B), PreGN species were detected in each of the lysates, but efficient processing of PreGN to mature GN was only observed from the four largest constructs, PreGNV5(881) to PreGNV5(1036).


Figure 5
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  		FIG. 5. PreGN truncation mutants. (A) A schematic of the PreGNV5 truncation constructs is shown, with the predicted transmembrane domains indicated by black bars. The terminal amino acid for each construct (relative to the IbAr10200 M polyprotein) is indicated at the left, and each construct possessed a C-terminal V5-His6 epitope cassette. 293T/17 cells were transfected with the indicated constructs or with a plasmid expressing GFP, and then cell lysates were prepared at 18 to 22 h posttransfection. The samples were separated by SDS-PAGE using 10% Bis-Tris NuPAGE gels and MOPS-based running buffer. Proteins in the cell lysates were immunoblotted with an anti-GN ectodomain polyclonal antiserum (B) or a monoclonal antibody directed against the V5 epitope (C). An asterisk indicates a putative product of PreGN-PreGC cleavage in the PreGNV5(1036) construct. (D) For cells transfected with PreGNV5(842) and PreGNV5(856), proteins were also immunoprecipitated from the culture medium above these cells using a polyclonal antiserum directed against the V5 epitope. Afterwards, the precipitated proteins were separated by SDS-PAGE using 10% Bis-Tris NuPAGE gels and MES-based running buffer, followed by immunoblotting with a monoclonal antibody directed against the V5 epitope. The locations of PreGN and the C-terminal fragments (C-termxxx) are indicated ("xxx" refers to the terminal amino acid of the PreGNV5 truncation construct from which each fragment is derived).

When the immunoblot of these constructs was probed with an antibody to the C-terminal V5 epitope tag (Fig. 5C), PreGN species were again observed in all of the lysates. The molecular weights of these V5-reactive species varied in proportion to their total length, whereas PreGN species detected with the GN antiserum migrated more similarly (Fig. 5B). This discrepancy could reflect differences in epitope accessibility between the two antisera and that these two antisera might detect slightly different PreGN species. The V5-specific antibody also recognized C-terminal fragments of constructs PreGNV5(881) and larger, though these migrated at different positions, consistent with their variable lengths. In the Bis-Tris-MOPS gel system used here, proteins smaller than 7 kDa could not be detected. To resolve proteins less than 7 kDa in size, we also employed Bis-Tris-MES and Tris-Tricine SDS-PAGE systems, which can resolve proteins as small as 2 kDa. Even with these systems, no C-terminal fragments were detected in the lysates of cells expressing the four shortest constructs. Given the predicted membrane topology of PreGNV5(842) and PreGNV5(856) (Fig. 1), we reasoned that C-terminal cleavage might liberate a soluble, secreted C-terminal fragment. To address this, supernatants of cells transfected with these constructs were immunoprecipitated with an anti-V5 polyclonal antiserum and then immunoblotted for the V5 epitope tag. As shown in Fig. 5D, appropriately sized C-terminal fragments of PreGNV5(842) and PreGNV5(856) were found in the supernatants of these cells. Together, the increasing size of C-terminal fragments generated by this panel of PreGN truncation mutants suggested that they were all generated by proteolysis at a single common cleavage site.

The PreGNV5(1036) construct was unique in that two V5-reactive species were generated from PreGN. Amino acid 1036 is the last residue before the RKPL cleavage motif preceding the N terminus of mature GC. This motif was omitted so as to prevent proteolytic removal of the V5-His6 epitope cassette. Thus, PreGNV5(1036) includes some fraction of PreGC, although the precise number of amino acids is unknown because the N terminus of PreGC has yet to be identified. Sanchez et al. proposed that signal peptidase might cotranslationally cleave the nascent M segment-encoded polyprotein into PreGN and PreGC during its translocation into the ER lumen (32). This cleavage site is thought to exist proximal to the fourth predicted transmembrane helix (amino acids 969 to 991) following the GN ectodomain, though no direct evidence for this location exists. The predicted mass of the polypeptide spanning residues 991 to 1036 and including the V5-His6 cassette is 10.3 kDa. Thus, the smaller (<15 kDa) C-terminal fragment generated from PreGNV5(1036) is appropriately sized to confirm the presence of the PreGN-PreGC cleavage site within, or C-terminal to, the fourth predicted transmembrane domain.

From these data, we concluded that the first three predicted transmembrane domains following the GN ectodomain, along with their associated lumenal and cytoplasmic loops, are minimally required for efficient SKI-1 processing of PreGN. Truncation beyond this point (i.e., PreGNV5 856, 842, 805, and 719) resulted in proteins that did not undergo the SKI-1 cleavage event, or at least did so very inefficiently. In addition, the C-terminal cleavage event did not occur in the two shortest truncations, suggesting that cleavage occurs C-terminal to amino acid 805 of PreGN.

Next, a panel of internal deletion mutants was generated based upon the PreGNV5(961) vector (Fig. 6A). The goal of this approach was to delete the putative cleavage site and to prevent the generation of a C-terminal fragment. Sequential deletions of 18 to 22 amino acids were made in the first and second cytoplasmic loops (amino acids 720 to 819 and 880 to 961, respectively). Additionally, a mutant was constructed in which the lumenal loop (amino acids 843 to 856) was deleted and replaced with six glycine residues in order to maintain the protein's membrane topology. These constructs were then expressed in 293T/17 cells, separated by SDS-PAGE, and immunoblotted with the GN- and V5-specific antibodies. As shown in Fig. 6B (top panel), GN generated from mutants of the first cytoplasmic loop migrated faster than GN derived from PreGNV5(961), whereas GN molecules derived from the lumenal and second cytoplasmic loop deletions (Fig. 6C and D, top panels) were unaffected in comparison to PreGNV5(961). Conversely, when these same constructs were immunoblotted for the V5 epitope tag at their C termini, only deletion mutants within the first cytoplasmic loop generated full-length 20-kDa C-terminal fragments (Fig. 6B, bottom panel), whereas all other deletion mutants gave rise to smaller C-terminal fragments (Fig. 6C and D, bottom panels). Despite having deletions of similar lengths in the first and second cytoplasmic loops, the migration of GN and the C-terminal fragment was not uniform among the respective panels of mutants (Fig. 6B, top panel, and D, bottom panel). Although subtle differences in the predicted masses of these proteins exist, it is more likely that these migration discrepancies are due to differences in amino acid composition or loss of posttranslational modification sites among these proteins. Nevertheless, these data indicate that the entire first cytoplasmic loop is contained within GN, whereas the lumenal and second cytoplasmic loops are within the C-terminal fragment. Furthermore, since none of the deletions blocked PreGN C-terminal cleavage, the cleavage site responsible for the fragment is probably not contained within any of the loops of PreGN. Rather, these results point to a cleavage event occurring within or very near to the second predicted transmembrane domain of PreGN (amino acids 820 to 842).


Figure 6
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  		FIG. 6. PreGN internal deletion mutants. (A) A schematic of the PreGNV5 internal deletion constructs is shown, with the predicted transmembrane domains indicated by black bars. Using the PreGNV5(961) construct as a template, 18 to 22 amino acid deletions were made in cytoplasmic loops 1 and 2 as indicated. The lumenal loop was also deleted but then replaced with six glycines in order to maintain membrane topology of the protein. 293T/17 cells were transfected with the indicated constructs and then cell lysates were prepared at 18 to 22 h posttransfection. The samples were separated by SDS-PAGE using 10% Bis-Tris NuPAGE gels and MOPS-based running buffer. Proteins in the cell lysates were immunoblotted with anti-GN ectodomain polyclonal antiserum (B to D, top panels) or a monoclonal antibody directed against the V5 epitope (B to D, bottom panels). The locations of PreGN, GN, and the C-terminal fragment of PreGNV5(961) are indicated. GN and C-terminal fragment species having internal deletions are indicated by GN{Delta} and C-term961{Delta}, respectively.

The CCHFV M segment encodes an NSM protein. Although the preceding set of experiments clearly demonstrated the C-terminal cleavage of PreGN, the possibility remained that this fragment might in some way be an artifact of these constructs and of the expression system used. Furthermore, to confirm the hypothesis that this fragment represents a true NSM protein for CCHFV, this species would have to be detected as a product of a complete M polyprotein. To address these issues, a polyclonal antipeptide antiserum, specific to a sequence within the second cytoplasmic domain of PreGN (amino acids 926 to 939), was developed that would allow for immunoblotting of the putative NSM domain in the context of both PreGN and full-length M polyprotein constructs.

This NSM antiserum was used to probe an immunoblot of 293T/17 lysates expressing M[GCV5], PreGNV5(961), and PreGNV5(1036). Using this antiserum, an approximately 15-kDa species was detected in the M[GCV5] and PreGNV5(1036) lysates (Fig. 7A). This species is a likely candidate for the NSM protein, as a polypeptide including all of the lumenal loop, the third transmembrane domain, and the entire second cytoplasmic loop (amino acids 843 to 968 [Fig. 1]) has a predicted molecular mass of 14 kDa. In the PreGNV5(961) lysate, the NSM antiserum reacted with a 20-kDa species and, in the PreGNV5(1036) lysate, a band of about 30 kDa was also detected. Based on their sizes, we concluded that these proteins were likely the PreGN C-terminal fragments previously detected by immunoblotting with the anti-V5 antibody (Fig. 5C). The increased mass of the species derived from PreGNV5(961) can be explained by the presence of the V5-His6 cassette encoded by the expression vector, which adds approximately 3.6 kDa to the mass of the protein. The probable C-terminal signal peptidase processing of the PreGNV5(1036) C-terminal fragment likely allows for proper N- and C-terminal cleavage of the NSM such that it migrates identically to NSM derived from a complete M polyprotein. However, the presence of the 30-kDa C-terminal fragment suggests that these processing events may not occur as efficiently as in the native M polyprotein.


Figure 7
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  		FIG. 7. Identification of an NSM protein by transient expression and virus infection. (A) 293T/17 cells were transfected with pcDNA3.1 CCHFV glycoprotein expression constructs M[GCV5], PreGNV5(961), and PreGNV5(1036). The 30-kDa band present in all lanes is background, as it was also present in the GFP negative control. (B) 293T/17 cells were infected with CCHFV strain IbAr10200 at an MOI of 5, and cell lysates were prepared at approximately 24 h postinfection. In parallel, cells were also mock infected or transfected with a pCAGGS plasmid expressing the IbAr10200 M segment. Infected and mock culture supernatants were clarified and overlaid on a 20% sucrose-PBS cushion, and virions were semipurified by ultracentrifugation. C, cell lysate; P, virion pellet. The samples for panels A and B were separated by SDS-PAGE using 10% Bis-Tris NuPAGE gels and either MES-based (NSM) or MOPS-based (N, GN) running buffers. Proteins in cell and virion lysates were immunoblotted with anti-NSM cytoplasmic loop polyclonal antiserum (A and B, top panels), anti-N monoclonal antibody 9D5-1-1A (B, top panel), or anti-GN ectodomain polyclonal antiserum (B, middle panel).

To demonstrate the presence of an NSM protein in the context of CCHFV infection, 293T/17 cells were infected with CCHFV strain IbAr10200 and then cellular lysates were prepared at 24 h postinfection. Additionally, virions released into the culture supernatant were semipurified by ultracentrifugation through a 20% sucrose cushion. When these lysates were separated by SDS-PAGE and immunoblotted using an anti-N monoclonal antibody (Fig. 7B, top panel), the nucleoprotein was detected specifically in the CCHFV-infected lysate and in the virion pellet. Similarly, GN was present in infected lysates and in virion pellets, as well as in cells transiently expressing the IbAr10200 M segment. PreGN was detected only in the cell lysates and not in the virion pellet, confirming that the virion pellet was relatively free of cell-associated viral proteins such as the glycoprotein precursors. Interestingly, an approximately 80-kDa band was detected in addition to monomeric GN in the virion pellet. We assumed that this larger species was an oligomeric form of GN, as suggested by a previous study (31).

When these lysates were immunoblotted using the NSM antiserum, NSM was detected specifically in CCHFV-infected cell lysates. The virus-derived NSM migrated at the same molecular weight as plasmid-derived NSM. No NSM band was detected in the virion lysates, suggesting that this protein is nonstructural. However, this result does not exclude the possibility that NSM is incorporated into virions in such low abundance that it is undetectable using this immunoblotting system. In summary, these data provide the first direct evidence for a CCHFV-encoded NSM protein as a product of C-terminal proteolytic cleavage of PreGN.


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DISCUSSION
 
In addition to the GN and GC glycoproteins, the M RNA genome segment of viruses in some genera within the Bunyaviridae family encodes a nonstructural (NSM) protein by a variety of different strategies. The plant-infecting viruses of the Tospovirus genus employ an ambisense coding strategy to encode an NSM protein in the M segment viral RNA (21, 25), while the phleboviruses Rift Valley fever virus (RVFV), Punta Toro virus, and Toscana virus encode NSM proteins in the same open reading frame as the viral glycoproteins (7, 8, 20). The NSM proteins of orthobunyaviruses are encoded between GN and GC and are liberated by endoproteolysis of the M segment-encoded polyprotein (10, 14). With regards to function, the NSM protein of the tospovirus Tomato spotted wilt virus acts as a viral movement protein early during infection, enabling viral nucleocapsids to be transported through plasmodesmata prior to assembly of viral particles (22, 36, 38). For the phleboviruses and orthobunyaviruses, however, relatively little is known about the function of their NSM proteins. The RVFV NSM1 protein can be coimmunoprecipitated with the GC glycoprotein, though NSM-deficient RVFV replicates in cultured mammalian cells with no apparent growth defects (19, 43). For Orthobunyavirus genus members, the NSM proteins are known to traffic to the Golgi compartment, where they may interact with the glycoproteins and nucleoprotein (34). A Maguari virus mutant lacking the C-terminal two-thirds of the NSM was still viable (30). However, for Bunyamwera virus (BUNV), it was impossible to rescue viruses lacking the entire NSM or having deletions within the N-terminal regions, suggesting a role for the NSM in viral assembly (34). No NSM proteins have been identified for members of the Hantavirus and Nairovirus genera.

The M polyproteins of nairoviruses possess four predicted transmembrane domains in the region between the ectodomains of the GN and GC glycoproteins (Fig. 1). We introduced an N-linked glycosylation site in the lumenal loop that is predicted to exist between the second and third putative transmembrane segments. The fact that this site was glycosylated, in addition to a recent report (13) showing that an N-linked glycosylation site within the GN ectodomain was utilized, but that a site within the first cytoplasmic loop was not, confirms the topology of this domain, making it identical to that found in the M polyproteins of orthobunyaviruses (34). In addition to a shared topology, we found that this domain is also subject to endoproteolysis, releasing a previously unidentified integral membrane protein whose size is similar to that of the NSM proteins of the orthobunyaviruses (10, 17).

If the integral membrane protein liberated from the C-terminal region of PreGN constituted an NSM protein that was relevant for CCHFV biology, we predict that it would exhibit several characteristics. First, it should be generated through proteolytic cleavage at a defined site, utilizing a protease(s) common to its mammalian and arthropod hosts. Second, an NSM protein should have a reasonable half-life and perhaps be targeted to the Golgi in a manner analogous to the NSM protein of the orthobunyaviruses (24, 28, 34). Third, an NSM protein would likely be absent from virus particles or exist in very low abundance, as there is no evidence from previous studies with semipurified virions to suggest the presence of additional proteins (31, 32, 39).

We found that the novel protein described here fulfilled all three of these criteria, and we therefore conclude that it constitutes an NSM protein. We identified NSM in cells infected with CCHFV but could not detect NSM in partially purified virus preparations. Further, the NSM protein was produced in a number of mammalian cell lines known to support CCHFV replication, and an extensive panel of PreGN mutants all suggested that cleavage occurs at a common site. It will be interesting to test if this event also occurs in cell lines derived from ticks, the primary vector for CCHFV. Pulse-chase analysis confirmed that PreGN C-terminal cleavage occurred efficiently, shortly after its synthesis or cotranslationally, and prior to N-terminal cleavage of the GN ectodomain.

Utilization of a glycosylation site introduced within the lumenal loop of NSM confirmed the topology of this domain relative to the membrane. The partial Endo H resistance of the C-terminal fragment in this mutant provided evidence that at least a fraction of the NSM protein pool traffics to the Golgi compartment, in agreement with data obtained with the NSM protein of BUNV (24, 28, 34). However, further studies are warranted to examine the trafficking of the CCHFV NSM when expressed alone or in the context of a complete M segment. If the NSM possesses its own Golgi targeting signal, then it may contribute to targeting of GN and GC to the Golgi or retention therein. Although the NSM of BUNV could not rescue Golgi localization of GC (24), when NSM was deleted from the M polyprotein, the acquisition of Endo H-resistant glycans on GC was much less efficient, suggesting that NSM may play a role in maturation of BUNV glycoproteins (35). In this study, deletion of the entire NSM cytoplasmic domain from PreGN did not impair SKI-1 cleavage of the GN ectodomain, but further truncation of PreGN prevented efficient processing by SKI-1 [Fig. 5, compare PreGNV5(719-856) with 881]. These data suggest that the lumenal loop and third transmembrane domain contained within the NSM may play a critical role in GN maturation. Additional studies will be required to test this hypothesis in the context of the M polyprotein.

Perhaps because of its multiple membrane-spanning domains, we have been unable to purify NSM in sufficient quantity or purity to identify its N-terminal residue. However, using a panel of PreGN truncation and internal deletion mutants, GN was defined to include the entire first cytoplasmic loop of PreGN, whereas NSM was determined to include the lumenal domain and second cytoplasmic loop. Thus, the second predicted transmembrane helix (TM 2) following the GN ectodomain (amino acids 820 to 842) appears to be a likely candidate for the PreGN-NSM cleavage site. Interestingly, alignment of the five predicted transmembrane domains of CCHFV with their cognate domains in the Dugbe virus polyprotein showed that TM 2 shared 56% identity between these viruses, whereas the other transmembrane domains were much less conserved (18 to 34% identity). The C-terminal portion of TM 2, which immediately precedes the lumenal domain of NSM, contains the sequence SxSPVQSAP. Its perfect conservation in CCHFV isolates suggests that it may be a possible cleavage motif. Most of these residues are also conserved within the second transmembrane domain of the M polyprotein of Dugbe virus, suggesting a more generalized role for this domain in the processing of nairovirus M polyproteins. Within this motif, there are a number of serine and proline residues, which are known to disfavor helix formation in lipid membranes (26). The transmembrane helix substrates of intramembrane cleaving proteases (I-CLiPs), such as site 2 protease, Rhomboid, and signal peptide peptidase, often possess helix-breaking residues, which may loosen the helix sufficiently for these I-CLiPs to gain access to the polypeptide backbone (42). Together, these data suggest a role for I-CLiPs in the processing of CCHFV PreGN, and efforts are currently under way to address this hypothesis.

Ideally, the function of the NSM protein would best be addressed in the context of infections with recombinant CCHFV lacking the NSM or possessing mutations within it, as has been recently done for BUNV (34), Maguari virus (30), and RVFV (19, 43). Although a reverse genetics system exists for CCHFV (15), it requires superinfection with wild-type CCHFV to drive expression of plasmid-based minigenomes. Refinement of this system to allow rescue of virus solely from plasmids would greatly benefit the study of CCHFV proteins in vitro and in vivo. In the meantime, it may be possible to engineer mutant NSM proteins and to supply them during wild-type CCHFV infection in hopes of mediating a transdominant effect against the virus.

In summary, this report provides the first direct evidence of an NSM protein for a Nairovirus. Further study of the CCHFV-encoded NSM and the proteases involved in its generation may lead to better understanding of CCHFV assembly events, development of attenuated CCHFV vaccines, or inhibitors of CCHFV replication.


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ACKNOWLEDGMENTS
 
L.A. thanks Aura Garrison, Wendy Grace, Kristin Spik, and the members of the Schmaljohn and Paragas laboratories for training and excellent technical assistance while using BSL-3+ facilities at USAMRIID. Donald Pijak, Val Hardy, and other members of the Doms laboratory provided invaluable support throughout this work. We also thank Stuart Nichol, Martin Vincent, and Eric Bergeron at the CDC for providing reagents and helpful discussions.

This work was funded by Department of Defense Peer Reviewed Medical Research Program grant PRMRP PR033269. L.A. and A.B.-C. were supported in part by NIH T32 AI055400.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, University of Pennsylvania, 225 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104. Phone: (215) 898-0890. Fax: (215) 898-9557. E-mail: doms{at}mail.med.upenn.edu Back

{triangledown} Published ahead of print on 11 April 2007. Back

{dagger} Present address: National Institute for Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892. Back


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Journal of Virology, June 2007, p. 6632-6642, Vol. 81, No. 12
0022-538X/07/$08.00+0     doi:10.1128/JVI.02730-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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