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Journal of Virology, November 2005, p. 13725-13734, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13725-13734.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Role of N-Linked Glycans on Bunyamwera Virus Glycoproteins in Intracellular Trafficking, Protein Folding, and Virus Infectivity

Xiaohong Shi, Kristina Brauburger, and Richard M. Elliott*

Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 5JR, Scotland, United Kingdom

Received 22 June 2005/ Accepted 8 August 2005


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ABSTRACT
 
The membrane glycoproteins (Gn and Gc) of Bunyamwera virus (BUN, family Bunyaviridae) contain three potential sites for the attachment of N-linked glycans: one site (N60) on Gn and two (N624 and N1169) on Gc. We determined that all three sites are glycosylated. Digestion of the glycoproteins with endo-ß-N-acetylglucosaminidase H (endo H) or peptide:N-glycosidase F revealed that Gn and Gc differ significantly in their glycan status and that late in infection Gc glycans remain endo H sensitive. The roles of the N-glycans in intracellular trafficking of the glycoproteins to the Golgi, protein folding, and virus replication were investigated by mutational analysis and confocal immunofluorescence. Elimination of the glycan on Gn, by changing N60 to a Q residue, resulted in the protein misfolding and failure of both Gn and Gc proteins to traffic to the Golgi complex. We were unable to rescue a viable virus by reverse genetics from a cDNA containing the N60Q mutation. In contrast, mutant Gc proteins lacking glycans on either N624 or N1169, or both sites, were able to target to the Golgi. Gc proteins containing mutations N624Q and N1169Q acquired endo H resistance. Three viable N glycosylation-site-deficient viruses, lacking glycans on one site or both sites on Gc, were created by reverse genetics. The viability of these recombinant viruses and analysis of growth kinetics indicates that the glycans on Gc are not essential for BUN replication, but they do contribute to the efficiency of virus infection.


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INTRODUCTION
 
The family Bunyaviridae contains more than 300 mostly arthropod-borne viruses that share biochemical and morphological characteristics; the family is classified into five genera (Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus) (7, 12). Several members of the family cause encephalitis or hemorrhagic fever in humans, e.g., La Crosse, Hantaan, Rift Valley fever, and Crimean-Congo hemorrhagic fever viruses, and are recognized as posing an increasing threat to human health (14).

Bunyamwera virus (BUN) is the prototype of both the family Bunyaviridae and the genus Orthobunyavirus and has a tripartite, single-stranded negative-sense RNA genome. The largest segment (L) codes for an RNA polymerase (L protein), the medium segment (M) codes for a polyprotein precursor which is cotranslationally cleaved to yield the two virion glycoproteins (Gn and Gc) and a nonstructural protein called NSm, and the smallest segment (S) codes for the nucleocapsid protein, N, and a second nonstructural protein NSs (7, 12-14). NSs plays a role in viral pathogenesis and shutoff of host cell protein synthesis (3, 24, 48, 50, 52). The function of NSm is still unknown; the fact that it localizes to the Golgi in BUN-infected cells suggests that it may have a role in virus morphogenesis (26, 33, 46).

In accord with a characteristic of the Bunyaviridae family, BUN Gn and Gc accumulate in the Golgi complex where virus assembly and budding occurs (33, 43). When expressed alone, Gn localizes to the Golgi, but Gc is dependent on its association with Gn protein, in the form of a heterodimer, for Golgi trafficking (26, 46). We recently mapped the signal for Golgi targeting and retention to the transmembrane domain of Gn (46). The requirement for Gn-Gc heterodimerization for efficient trafficking to the Golgi has also been documented for other members of family, such as Uukuniemi, Punta Toro and Rift Valley Fever viruses of the Phlebovirus genus (9, 17, 31, 40), La Crosse virus of the Orthobunyavirus genus (6), Hantaan and Sin Nombre viruses of the Hantavirus genus (42, 45, 47), and tomato spotted wilt virus of the Tospovirus genus (23).

Both Gn and Gc of BUN are type I transmembrane glycoproteins and are modified by N-linked glycosylation (33, 46). They possess a total of three potential N-linked glycosylation sites (Fig. 1): one on Gn (at N residue 60) and two on Gc (N624 and N1169) (27). Alignment of the amino acid sequences of the glycoproteins encoded by members of the Orthobunyavirus genus revealed that the N glycosylation site on Gn and the second site on Gc (N60 and N1169 in BUN) are conserved in all members of the genus while the first site in Gc (N624 in BUN) is conserved only among Bunyamwera serogroup viruses (4). The strict conservation of N glycosylation sites in orthobunyavirus glycoproteins suggests that N-glycans are likely required for protein folding and biological functions of the viral glycoproteins. Mature Gc protein expressed from transfected cDNAs in mammalian cells was shown to be resistant to endoglycosidase H digestion, indicating that the glycans are of the complex type (26, 33, 46). The glycosylation state of Gn has not yet been defined, although immunofluorescence assays revealed that Gn was able to transport to Golgi complex on its own or in association with Gc.



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  		FIG. 1. Bunyamwera virus glycoprotein N-glycosylation-site mutants. The bar represents a schematic of the BUN glycoprotein precursor, with gene order of Gn (residues 1 to 302), NSm (residues 303 to 476), and Gc (residues 477 to 1433). The predicated signal sequences (SS) and transmembrane domains (TMD) are shown as open and filled boxes, respectively. Lollipops indicate the locations of the potential N glycosylation sites. The lines below indicate the mutant constructs and the predicted N glycosylation sites they contain. Mutants were generated by substitution of the asparagines (N) residues at an N glycosylation site with glutamine (Q) PCR-mediated mutagenesis.

Enveloped viruses usually contain one or more types of integral membrane proteins, the majority of which undergo N-linked glycosylation (11). N-linked glycosylation is important for both correct protein folding and protein function (20, 35, 53); for viral glycoproteins these functions include receptor binding, membrane fusion and penetration into cells, virulence, directing virus morphogenesis at the budding site and immune evasion (1, 11, 34, 39).

In the present study, we determined the usage of each individual N glycosylation site and assessed the roles of N-glycans in protein folding and intracellular trafficking of the BUN glycoproteins. Furthermore, we generated N glycosylation site deficient viruses by reverse genetics (2, 28) to evaluate the role of N-glycans in virus replication and infectivity. Our results indicate that the glycan on Gn (N60) is crucial for correct folding of both Gn and Gc proteins and thus essential for virus viability. The two glycans on Gc are dispensable for virus replication but contribute to efficient virus growth.


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MATERIALS AND METHODS
 
Cells and viruses. HeLaT4+cells (29) and Vero E6 cells (ATCC C1008) were grown in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). BHK-21 and BSR-T7/5 (a BHK derivative that stably expresses T7 RNA polymerase [5], kindly provided by K. K. Conzelmann, Max-von-Pettenkofer Institut, Munich, Germany) cells were maintained in Glasgow minimal essential medium supplemented with 10% tryptose phosphate broth, 10% FBS, and (for BSR-T7/5 cells only) 1 mg of Geneticin/ml. A recombinant vaccinia virus, vTF7-3, which expresses T7 RNA polymerase (16), was a gift from B. Moss (NIH, Bethesda, MD). Wild-type (wt) BUN was grown in BHK-21 cells as previously described (2, 51).

Antibodies. A rabbit antiserum against purified BUN virions (anti-BUN) and a Gc-specific mouse monoclonal antibody (MAb) 742 have been described previously (26, 51). A rabbit polyclonal antibody against GM130, a cis-Golgi matrix protein (32), was provided by M. Lowe (School of Biological Sciences, University of Manchester, Manchester, United Kingdom). An MAb against human golgin 97, a novel 97-kDa Golgi complex autoantigen (18), was purchased from Molecular Probes, Inc. (Leiden, The Netherlands). Goat anti-rabbit antibody conjugated with fluorescein isothiocyanate was purchased from Sigma, and goat anti-mouse antibody conjugated with Cy5 was purchased from Amersham Pharmacia Biotech (Buckingham, United Kingdom).

Plasmids and mutagenesis. Plasmids pT7riboBUNL(+), pT7riboBUNM(+), and pT7riboBUNS(+), which generate full-length antigenome RNA transcripts, were used for virus rescue as described previously (2, 28). Three N glycosylation site mutant constructs pT7riboBUNM-N60Q, pT7riboBUNM-N624Q, and pT7riboBUNM-N1169Q, in which the asparagine residue (N) at a potential N glycosylation site was substituted with glutamine (Q), were generated from pT7riboBUNM(+) (Fig. 1), using a PCR mutagenesis approach (44, 45). pT7riboBUNM-D61E was constructed from pT7riboBUNM-N60Q by changing Q60 and aspartic acid D61 to N and glutamic acid (E) to restore the N glycosylation site at the same position on Gn. Incorporation of E at residue 61 in pT7riboBUNM-N60Q acted as a genetic marker to distinguish this mutant from wt Gn. We also constructed a double N glycosylation site mutant, pT7riboBUNM-N624/1169Q, by replacing a BglII-XbaI fragment of pT7riboBUNM-N624Q with the corresponding fragment from pT7riboBUNM-N1169Q.

Infection and transfection of cells. Subconfluent monolayers of Vero E6 cells were grown in 35-mm-diameter petri dishes for immunoprecipitation experiments; BSR-T7/5 and HeLaT4+ cells were grown on 13-mm-diameter glass coverslips for immunofluorescence assays. Cells were infected with vTF7-3 at 5 PFU/cell for 60 min and then transfected with plasmid DNA as described previously (26) with minor modifications. (Preinfection with vTF7-3 was not required for BSR-T7/5 cells.) Briefly, for cells grown on 35-mm-diameter dishes 2 µg of DNA and 5 µl of DAC-30 (Eurogentec) were diluted in 500 µl of Opti-MEM (BRL/Life Technologies, Paisley, United Kingdom), and for cells grown on coverslips 0.5 µg of plasmid DNA and 2 µl of DAC-30 were diluted in 250 µl of Opti-MEM. The DNA-liposome mixtures were incubated for 30 min at room temperature before being added to the cells that had been washed previously with Opti-MEM. At 3 h posttransfection, DMEM containing 10% FBS was added, and incubation continued at 37°C.

Metabolic radiolabeling of cells and virus particles. Cells either transfected with cDNA constructs or infected with virus were incubated for 1 h in starvation medium lacking methionine, washed, and then labeled with [35S]methionine (Amersham Pharmacia Biotech) for 20 min to 15 h. The radiolabeled cells were then incubated in chase medium (DMEM containing 10% FBS and 15 µg of methionine/ml) for 2 h before harvest. For labeling virus particles, infected cells grown in 175-cm2 flasks were incubated with [35S]methionine (20 µCi/ml in 10 ml of methionine-free medium) for 6 h at 24 h postinfection (p.i.). The supernatant was clarified by low-speed centrifugation (3,000 rpm for 10 min at 4°C), and the virus particles were collected by ultracentrifugation (26,000 rpm for 1 h in an SW28 rotor). Virus pellets were suspended in 100 µl of 0.5% sodium dodecyl sulfate (SDS)-1% ß-mercaptoethanol for endo-ß-N-acetylglucosaminidase H (endo H) or N-glycosidase F (PNGase F).

Immunoprecipitation of viral proteins. Radiolabeled infected cells were lysed on ice with 300 µl of nondenaturing radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.4], 1% Triton X-100, 300 mM NaCl, 5 mM EDTA) containing a cocktail of protease inhibitors (Roche). BUN glycoproteins were immunoprecipitated with anti-BUN serum or MAb 742 that had been conjugated to protein A-agarose (Sigma). The beads were washed four times with radioimmunoprecipitation assay buffer containing 0.1% Triton X-100 and once with ice-cold phosphate-buffered saline (PBS), and the bound proteins were either analyzed by SDS-PAGE under reducing conditions or subjected to digestion with endo H or PNGase F.

Endo H and PNGase F digestion. Immunoprecipitates were denatured in 30 µl of denaturing buffer (0.5% SDS and 1% ß-mercaptoethanol) at 100°C for 10 min and cooled to room temperature. The denatured samples were then digested with 150 mU of endo H (New England Biolabs) in 40 µl of reaction buffer (50 mM sodium citrate [pH 5.5], 0.5% SDS, 1% ß-mercaptoethanol) or 4 mU of PNGase F (New England Biolabs) in 40-µl reaction buffer (50 mM sodium phosphate [pH 7.5], 0.5% SDS, 1% ß-mercaptoethanol) for 20 h at 37°C. The treated samples were analyzed on SDS-12.5% polyacrylamide gel electrophoresis (PAGE) under reducing conditions.

Indirect immunofluorescence staining. Immunofluorescence assays were performed as previously described (45). Briefly, at 5 h posttransfection of vTF7-3-infected HeLaT4+ cells or at 24 h posttransfection of BSR-T7/5 cells, cycloheximide was added to a final concentration of 50 µg/ml, and incubation continued for 4 h. Cells were then fixed for 20 min with 4% paraformaldehyde and permeabilized by incubation in PBS containing 0.1% Triton X-100 for 30 min. The cells were reacted for 30 min with antibodies against BUN glycoproteins (anti-BUN serum or anti-Gc MAb 742) and Golgi markers (GM130 antiserum or anti-golgin 97 MAb). After a thorough wash with PBS, the cells were stained for 30 min with Cy5-conjugated anti-mouse immunoglobulin G or fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G. Localization of fluorescence-labeled proteins was examined by using a Zeiss LSM confocal microscope.

Generation of recombinant viruses by reverse genetics. The recently described three-plasmid rescue protocol was used (28). Briefly, BSR-T7/5 cells grown in 60-mm-diameter dishes were transfected with mixtures of plasmid DNAs comprising 1.5 µg each of pT7riboBUNL(+), pT7riboBUNS(+), and either pT7riboBUNM(+) or one of the N glycosylation site mutant constructs using 10 µl of DAC-30 as transfectant in a total volume of 0.7 ml of Opti-MEM. After 5 h, 4 ml of supplemented DMEM were added, and incubation was continued for 5 to 11 days. The supernatants were collected, and recombinant viruses were isolated by plaque formation in Vero E6 cells.

Virus growth curves. Vero E6 cells seeded in 35-mm-diameter dishes were infected at a multiplicity of infection (MOI) of either 0.1 or 1.0 PFU/cell. The inoculum was removed after 1 h, and cells were washed twice with PBS to remove unattached viruses. Supernatants were harvested at the indicated time points, and virus amounts were titrated by plaque assay on Vero E6 cells.


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RESULTS
 
Determination of the N-linked glycosylation sites used on BUN glycoproteins. Three potential sites for the attachment of N-linked oligosaccharides are predicted on BUN glycoproteins: one site on Gn (N60) and two sites on Gc (N624 and N1169) (27) (Fig. 1). To determine the utilization of each individual site for the attachment of oligosaccharide side chains, we generated four N glycosylation site mutations in the BUN M cDNA by replacing the asparagine (N) residue of the N glycosylation motif with glutamine (Q). The plasmids were transfected into cells infected with vaccinia virus vTF7-3 (to provide T7 RNA polymerase), and the expressed glycoproteins were labeled with [35S]methionine, immunoprecipitated with anti-BUN antibodies, and analyzed by SDS-PAGE. The sizes of the glycosylated wt Gc and Gn proteins were estimated to be 110,000 and 41,000, respectively, according to their electrophoretic migration (Fig. 2). Mutation of the N glycosylation site on Gn (N60) resulted in an increased electrophoretic mobility corresponding to a molecular weight loss of approximately 3,000 compared to wt Gn protein (Fig. 2A, lanes 1 and 2), indicating that this site on Gn was modified by N-glycan attachment. The glycosylation site on Gn was restored by back mutation of the N60Q clone, together with introduction of a genetic marker such that the site had a different sequence (NDT to NET) that would allow distinction from the wt construct. The Gn band produced by this construct (D61E) had a electrophoretic mobility similar to that expressed by the wt construct (Fig. 2A, lane 3). The usage of site N60 was also validated by the increased electrophoretic mobility observed for Gn proteins in either BUN-infected cells (Fig. 2B, lanes 1 to 3) or purified virus particles (Fig. 2B, lanes 4 to 6) after endo H and PNGase F digestion.



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  		FIG. 2. Determination of N-glycosylation-site usage by mutagenesis and endoglycosidase digestion. (A) Immunoprecipitation of wt and mutant glycoproteins expressed from M segment cDNAs transfected into Vero E6 cells. Glycoproteins were labeled with 50 µCi of [35S]methionine for 15 h and then immunoprecipitated with anti-BUN serum. Immunoprecipitates were analyzed by SDS-12.5% PAGE under reducing conditions. (B) Endo H and PNGase F digestions of radiolabeled glycoproteins either immunoprecipitated from BUN-infected Vero E6 cells (Anti-BUN) or in purified virus particles (Virion). Glycoproteins were subjected to endo H (H) or PNGase F (F) digestion for 20 h or left undigested and then analyzed by SDS-PAGE. The positions of viral proteins Gc and Gn and the nucleocapsid protein (N) are marked. Deglycosylated Gn, dGn, is indicated by asterisks. Migration of molecular weight standards is also shown. Below is shown an enlargement of lanes 1 to 3 to show more clearly the mobility difference between glycosylated and deglycosylated Gc.

Unglycosylated Gc, for example, synthesized in the presence of tunicamycin (27) or after treatment with endoglycosidases (46), shows only a small electrophoretic mobility difference from glycosylated Gc, suggesting that the carbohydrate addition contributes little to the overall size of the protein. However, it is possible to determine a slight size difference between wt Gc and mutant Gc that lack either of the two potential N glycosylation sites (Fig. 2A, lanes 4 and 5). That both sites were modified by glycan attachment was confirmed by analysis of the double N glycosylation site mutant (N624/1169Q), where unglycosylated Gc is smaller than Gc produced by either of the single mutants (Fig. 2A, lane 6). We also observed similar differences in the mobilities of the deglycosylated Gn and Gc proteins from either infected cells (Fig. 2B, lane 3) or purified virus particles (Fig. 2B, lane 6) after the treatment with PNGase F. In conclusion, these data indicate that all three potential N glycosylation sites on the ectodomains of Gn and Gc proteins are utilized.

Analysis of N glycosylation status of Gn and Gc. Comparison of the electrophoretic mobilities of Gn and Gc proteins after endo H digestion suggested a significant difference in endo H resistance between the glycoproteins in BUN virus-infected cells and those in purified virus particles (Fig. 2B). Both Gn and Gc immunoprecipitated from infected Vero E6 cells were shown to be predominantly endo H sensitive (Fig. 2B, lanes 1 to 3), whereas Gc and approximately half of Gn in virus particles appeared endo H resistant (lane 5 to 6). The presence of predominantly endo H sensitive glycoproteins in virus-infected cells suggests that the majority of glycoproteins, especially Gc, were either immature or their glycans remained unconverted; however, Gc proteins embedded in virions were primarily of the mature, complex type.

To investigate the apparent discrepancy between the glycosylation status of the glycoproteins in virus-infected cells and in purified virus particles, we pulse-labeled BUN-infected cells for 30 min at 6 hourly intervals over 36 h of infection. Aliquots of each sample were immunoprecipitated with either MAb 742 or anti-BUN antibodies. The Gc specific MAb 742 only recognizes correctly folded and heterodimerized Gc protein (46), whereas the polyclonal anti-BUN serum recognizes Gn and Gc whether correctly folded or not. Gc precipitated by anti-BUN serum at 12 h p.i. was partially endo H resistant (Fig. 3, lanes 4 to 6), but Gc synthesized from 18 h p.i. onward was endo H sensitive (lanes 7 to 15). Precipitation by MAb 742 showed Gc to be endo H resistant up to 24 h p.i. (lanes 19 to 27), but Gc synthesized at late stage of infection (i.e., 36 h p.i.) was mainly endo H sensitive (lanes 28 to 30). Gn was predominantly endo H sensitive, with the endo H-resistant form of Gn faintly observable in the sample coprecipitated with MAb 742 at 24 h p.i. (lane 26). These results indicate that the glycosylation status of BUN glycoproteins, especially Gc, was affected by the stage of virus replication; the glycans on glycoproteins synthesized at later stage of infection mostly remained of the high-mannose type. Even in early stages of infection, the glycoproteins in infected Vero E6 cells comprised a mixture of those containing glycans of both high-mannose and complex types.



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  		FIG. 3. Analysis of N glycosylation of BUN glycoproteins. BUN-infected Vero E6 cells were labeled with [35S]methionine for 30 min at different time points p.i. as indicated, and equal volumes of cell lysate were immunoprecipitated with anti-BUN serum (lanes 1 to 15) or anti-Gc MAb 742 (lanes 16 to 30). The resulting precipitates were subjected to digestion with endo H (H) or PNGase F (F) or left undigested and then analyzed by SDS-12.5% PAGE under reducing conditions. The positions of viral proteins Gc, Gn, and N are marked. Below is shown an enlargement of lanes 4 to 15 to show more clearly the mobility difference between glycosylated and deglycosylated Gc.

Role of N glycosylation in BUN glycoprotein intracellular trafficking. Gn and Gc accumulate in the Golgi complex (26, 33, 46), where virus assembly and budding occurs (43), and this feature can be exploited to investigate the role of N-linked glycosylation in protein folding and intracellular trafficking. We expressed the mutant glycoproteins containing mutations at N glycosylation sites in either BSR-T7/5 cells or vTF7-3-infected HeLa T4+ cells after transfection with the appropriate cDNAs. The ability of each mutant glycoprotein to localize to the Golgi was assessed by immunofluorescence microscopy using antibodies to the BUN proteins and the Golgi-specific markers GM130 or golgin 97. As shown in Fig. 4, loss of the N-glycan chains from different sites had different effects on intracellular translocation of the modified glycoproteins. Abolition of the N-glycan on Gn (N60) had the most drastic effect. Staining with anti-BUN serum showed that the glycoproteins failed to target to the Golgi complex but instead exhibited a typical endoplasmic reticulum (ER) staining pattern (Fig. 4H), and the Gc protein expressed from this construct did not react with MAb 742 (Fig. 4B). When we recreated the N glycosylation site on Gn, as shown by construct D61E, Golgi localization and reactivity with MAb 742 of Gc protein was restored (Fig. 4C and I). This indicates that glycosylation on N60 is crucial for the correct folding of both Gn and Gc proteins.



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  		FIG. 4. Intracellular localization of wt BUN and mutant glycoproteins. BSR-T7/5 cells (A to F) or HeLa T4+ cells (G to L) were transfected with BUN M segment cDNAs and stained with a mixture of either mouse anti-Gc MAb 742 and a rabbit anti-GM130 antibody (A to F) or rabbit anti-BUN serum and mouse anti-golgin 97 MAb (G to L). In panels A to F, BUN Gc stains red and Golgi stains green, whereas in panels G to L BUN glycoproteins are stained green and the Golgi red. Merged confocal microscopic images are also shown, with colocalization of BUN glycoproteins and Golgi showing as yellow.

We next examined the effect on intracellular localization of elimination of either individual site or both sites in combination on Gc (at residues N624 and N1169). Immunofluorescence staining showed that MAb 742 did not recognize Gc expressed from either the single-site mutant N624Q or the double-site mutant N624/1169Q (Fig. 4D and F), whereas staining with the anti-BUN serum revealed partial colocalization of the glycoproteins with the Golgi marker golgin 97 (Fig. 4J and L). In contrast, loss of the site at N1169 (second site on Gc) had no obvious effect on Golgi trafficking of BUN glycoproteins (Fig. 4E and K). This suggests that N glycosylation of the site at N624 affected the efficiency of Golgi trafficking of the two mutant proteins. It was noticeable that the ER-like staining pattern was more intense in cells transfected with the double-mutant N624/1169Q construct than in cells transfected with the single N glycosylation site mutant N624Q, perhaps indicating a slower transport of glycoproteins from the ER to Golgi (the cells were treated with cycloheximide for 4 h before immunostaining).

To further examine the effect of N-glycosylation-site mutation on intracellular trafficking from ER to Golgi, we compared their resistance to endo H, which cleaves N-linked oligosaccharides in the high-mannose form but not those that have been converted, in the medial Golgi, to the complex form (25). A pulse-chase experiment was performed. The glycoproteins, expressed in vTF7-3-infected Vero E6 cells transfected with wt and mutant M cDNA constructs, were radiolabeled with [35S]methionine, and samples were harvested over 2 h of chase in the presence of excess unlabeled methionine. As shown in Fig. 5, Gc expressed by N624Q and N1169Q acquired endo H resistance in 60 min, similar to wt Gc (panels A, C, and D). This suggests that the loss of either site on Gc had no obvious effect on protein trafficking out of ER to Golgi. However, Gc expressed by N60Q remained endo H sensitive throughout the 2 h chase (panel B), confirming that the protein was retained in the ER and failed to translocate to the Golgi. In addition, the data also showed that Gn proteins expressed by both wt and mutant constructs were all predominantly endo H-sensitive (Fig. 5A, C, D, and E). Since there were no glycans on Gn protein expressed from N60Q or on Gc expressed by the double-site mutant N624/1169Q, we could not monitor transport of these protein by using endo H treatment since they did not show gel mobility differences (Fig. 5B and E).



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  		FIG. 5. Pulse-chase analysis of endo H resistance of wt and mutant glycoproteins. Vero E6 cells were infected with vTF7-3 and subsequently transfected with wt or mutant BUN M cDNA constructs. Cells were labeled with [35S]methionine for 20 min and chased in the presence of additional unlabeled methionine for different lengths of time as indicated (in minutes). Radiolabeled proteins were immunoprecipitated with anti-BUN serum, subjected to Endo H digestion (+) or left undigested (–) as indicated, and analyzed by SDS-8% PAGE under reducing conditions. Viral protein Gc and Gn and deglycosylated Gn, dGn, are indicated at the side. On the gels Gn and deglycosylated Gn positions are marked by asterisks.

Effect of N-glycans on BUN glycoprotein folding. To investigate further the effect of N-glycans on glycoprotein folding, we compared the immunoreactivity of wt and N glycosylation site mutant proteins with polyclonal anti-BUN serum and the conformation-sensitive anti-Gc MAb 742. The anti-BUN serum (lanes designated P in Fig. 6) precipitated Gn and Gc expressed by wt and all mutant constructs. MAb 742 (lanes designated M in Fig. 6) precipitated Gn and Gc expressed by wt, D61E and N1169Q constructs, validating the heterodimerization between the two proteins (Fig. 6, lanes 2, 6, and 10). Abolition of the N-glycan at N60 on Gn led to the loss of reactivity of Gc with MAb 742 (Fig. 6, lane 4), resulting in no coimmunoprecipitation of Gn. Restoration of the site on N60, as shown by construct D61E, resulted in an immunoprecipitation pattern identical to that of wt glycoproteins (lanes 1 to 2 and 5 to 6).



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  		FIG. 6. Immunoreactivity of wt and mutant BUN glycoproteins with anti-BUN polyclonal serum and anti-Gc MAb 742. Vero E6 cells were infected with vTF7-3, followed by transfection with wt or mutant cDNA constructs as indicated. Cells were labeled with [35S]methionine for 15 h and immunoprecipitated with anti-BUN (P) or MAb 742 (M). The resulting immunoprecipitates were analyzed by SDS-12.5% PAGE under reducing conditions.

Consistent with the immunofluorescence observations (Fig. 4), the immunoprecipitation assay confirmed that mutation of the first glycosylation site (N624) on Gc also resulted in loss of reactivity of Gc with MAb 742, and thus no Gn was coimmunoprecipitated when expressed by constructs N624Q and N624/1169Q (Fig. 6, lanes 8 and 12). However, since we demonstrated above that Gc expressed by N624Q acquired endo H resistance, this suggests that the Gc proteins of the two mutants maintain their overall correct conformation and function. This presumption is supported by the successful recovery of the recombinant viruses vQ624 and vQ624/1169 (see below) that lack the N glycosylation site at N624 on Gc. Gc lacking the N-glycan at N1169 reacted with MAb 742 and was able to coprecipitate Gn (Fig. 6, lane 10). These data indicate that the glycan on Gn is crucial for protein folding and heterodimerization of BUN glycoproteins and furthermore that the Gc protein depends on its Gn counterpart for proper folding and maturation, most likely through oligomerization. The lack of other MAbs prevented us from conducting further protein folding analyses.

Generation of mutant BUN viruses lacking N glycosylation sites. The availability of an efficient reverse genetics system to rescue transfectant bunyaviruses (2, 28) enabled us to evaluate the role of N-linked glycosylation on virus replication and infectivity. To generate recombinant N glycosylation-site deficient viruses, the constructs pT7riboBUNM-N60Q, pT7riboBUNM-N624Q, pT7riboBUNM-N1169Q, and pT7riboBUNM-N624/1169Q were transfected into BSR-T7/5 cells, together with plasmids pT7riboBUNL and pT7riboBUNS that contain wt L- and S-segment cDNAs. No virus was recovered from repeated rescue experiments using pT7riboBUNM-N60Q as source of the M-segment cDNA. However, we generated four recombinant viruses with mutations at the N glycosylation sites designated vD61E, vQ624, vQ1169, and vQ624/1169. The presence of the introduced mutations at the N glycosylation sites in the recombinant viruses was confirmed by reverse transcription-PCR and subsequent DNA sequencing analysis (data not shown).

Compared to the rescued wt BUN, there was little difference in plaque morphology of the rescued virus vD61E, which possesses all three N glycosylation sites, or vQ624 and vQ1169, which contain single-site mutations. However, plaques produced by vQ624/1169, the virus containing the double mutation in Gc, were smaller (Fig. 7A). The titers of the rescued viruses from the initial transfection are compared in Fig. 7B. Consistently, the titers of vD61E and vQ1169 were similar to those of wt BUN, whereas titers were markedly lower for vQ624 and vQ624/1169. The protein profiles of wt BUN and mutant viruses were compared by infecting cells at a high MOI (5 PFU/cell) to ensure good shutoff of host cell protein synthesis. As seen in Fig. 7C, the electrophoretic mobilities of Gc synthesized by vQ624, vQ1169, and vQ624/1169 were increased, as expected, due to the loss of one or two N-linked oligosaccharide side chains.



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  		FIG. 7. Plaque phenotypes and protein profiles of transfectant BUN viruses containing mutations at N glycosylation-site. (A). Comparison of plaques produced on Vero E6 cells. Cell monolayers were fixed with 4% formaldehyde and stained with Giemsa solution 4 days after infection. (B) Yields of rescued viruses. Supernatants from the initial transfection dishes were titrated by plaque formation on Vero E6 cells, and the results are the average of two independent titrations. (C) Protein profiles of cells infected with rescued wt BUN and mutant viruses. Vero E6 cells were infected with wt BUN (lane 2), vD61E (lane 3), vQ624 (lane 4), vQ1169 (lane 5), and vQ624/1169 (lane 6) as indicated at 5 PFU/cell. At 24 h p.i., cells were labeled with 100 µCi of [35S]methionine for 2 h, and then equal amounts of cell lysate were analyzed by SDS-12.5% PAGE under reducing conditions. Positions of viral proteins are indicated.

To check the stability of each mutation in the recombinant viruses, they were passaged for 10 generations on BHK-21 cells, and then the sequences of M segment were determined. No changes were found at any of N glycosylation sites compared to the sequence of the originally rescued viruses (data not shown).

Effect of N-glycans on BUN virus replication and infectivity. The roles of the N-glycans in BUN virus replication and infectivity were evaluated by comparing the kinetics of growth and effects on host protein synthesis in cells infected at MOIs of either 0.1 or 1.0 PFU/cell. The growth curves of mutant viruses in cell infected at 1.0 PFU/cell were mostly comparable to that of wt BUN after 12 h p.i. (Fig. 8A), indicating that glycosylation of Gc protein is not necessary for virus replication. However, at the lower multiplicity of 0.1 PFU/cell distinct differences in the patterns of growth were observed between mutant and wt viruses (Fig. 8B). The growth of the three mutant viruses was delayed, but to different extents, and the yields of virus at 54 h p.i. were consistent with the yields from the initial rescue transfection (Fig. 7B). The difference in growth kinetics at the two MOIs suggests that the N-glycans on Gc, although not required for virus replication, play a role in the virus spread from one infected cell to neighboring ones and that two glycans function "globally" for efficiency of virus infection.



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  		FIG. 8. Growth kinetics and protein profiles of wt and mutant BUN viruses. (A and B) Viral growth curves. Vero E6 cells were infected with either wt or mutant viruses at an MOI of either 0.1 (A) or 1.0 (B) PFU/cell. Virus released from the cells was titrated by plaque assay at the time points indicated, and the results shown are the average of two independent titrations. (C) Time course of protein synthesis. Vero E6 cells infected at an MOI of 1.0 PFU/cell were labeled with 100 µCi of [35S]methionine for 20 min at the time points indicated and cell lysates analyzed by SDS-12.5% PAGE. The positions of the viral proteins are indicated at the right.

A high MOI with wt BUN results in rapid shutoff of host cell proteins synthesis in mammalian cells (3), but at low MOIs shutoff takes longer to manifest. To determine whether there was a difference in the ability of the mutant viruses to shut off host protein synthesis, Vero E6 cells infected at 1.0 PFU/cell were radiolabeled at 6-h intervals, and equal amounts of cell lysate were analyzed by SDS-PAGE (Fig. 8C). Comparison of these time courses revealed that host protein synthesis shut off by vQ624 and vQ1169 was comparable to that mediated by wt BUN, in which shutoff was only apparent at 36 h p.i. (Fig. 8C). In contrast, shutoff of host protein synthesis in cells infected with vQ624/1169 was not observed at this time point, even though the amounts of viral protein synthesized in infected cells (as monitored by the N protein band on the gel) appeared similar for all viruses.


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DISCUSSION
 
BUN glycoproteins possess three potential N-linked glycosylation sites: one site on Gn (at N residue 60) and two sites on Gc (N624 and N1169) (Fig. 1). Alignment of the glycoprotein gene sequences of 20 orthobunyaviruses indicates that site 1 (N60 for BUN) on Gn and site 3 (N1169 for BUN) on Gc are conserved for all viruses, whereas site 2 (N624 for BUN) on Gc is conserved among viruses in the Bunyamwera serogroup (4, 15). The strict conservation of N glycosylation sites suggests that the presence of N-glycans plays an important role in protein function, perhaps in protein folding, intracellular protein trafficking, or virus infection.

In the present study, we confirmed that all three predicted sites are modified by N-linked glycosylation. Interestingly, we found that the N-glycan status on Gn and Gc were significantly different and influenced by the stage of virus replication. wt Gc protein expressed from transfected M segment cDNA was able to translocate to the Golgi complex, together with Gn, and acquired endo H resistance, as demonstrated previously (26, 33, 46). However, the glycans on Gc synthesized in BUN-infected cells at late stages of virus infection (36 h p.i.) did not acquire endo H resistance, but they could fold correctly, as monitored by their reactivity with MAb 742. It is likely that the N-glycans did not undergo terminal modification due to impaired glycosylation processing in the Golgi complex at later stages in the infection cycle, perhaps an effect of the shutoff of host cell protein synthesis. We showed that Gc and about half of the Gn protein present in virus particles was endo H resistant, although both Gc and Gn proteins in infected cells were largely endo H sensitive. This suggests that only mature and heterodimerized glycoproteins, which were probably synthesized in the early stage of virus infection, are used for virus assembly in the Golgi complex.

Similar to BUN, the N-glycans on Gn of La Crosse (LAC) and Inkoo orthobunyaviruses are mostly of the high-mannose type, whereas Gc contains both complex and an intermediate-type of oligosaccharide (30). Uukuniemi virus (UUK; Phlebovirus genus) also contains two types of N glycans, with glycans on Gn being mainly endo H resistant and those on Gc being endo H sensitive (37). It is not known why one glycoprotein should be modified by predominantly the complex form of sugar and the other by the high-mannose type. It is generally accepted that heterodimerization between bunyavirus Gn and Gc proteins is essential for correct folding and efficient intracellular transport to the Golgi, where viral assembly and budding occurs (8, 36, 38, 44, 46), and indeed oligomerization seems to be an important prerequisite for exit of integral membrane proteins in general from the ER compartment (21, 22, 41). We postulate that the glycan on Gn is somehow shielded or hidden by the Gc protein in the Gn-Gc heterodimer and may not be easily accessed by Golgi enzymes for terminal modification. The first N-glycan on abrin-a, a type II ribosome-inactivating protein from the seeds of Abrus precatorius, works as a bridge between abrin-a molecules, connecting them as a linear polymer (49); perhaps the glycan on N60 acts in the same way to promote formation of the Gn-Gc heterodimer.

One of the key roles of N-linked glycosylation is to promoter proper protein folding of glycoproteins (20, 21, 35). It is evident that the N-glycan (N60) on Gn protein, which is nearest to the N terminus of the BUN glycoprotein precursor, is crucial for achieving the correct protein conformation and targeting to the Golgi of both Gn and Gc. Elimination of the site (N60) resulted in severe misfolding of Gc protein, shown by its retention in the ER and lost reactivity with MAb 742. This suggests that the larger Gc protein depends on its smaller counterpart Gn for proper folding and maturation via heterodimerization. Indeed, we noted that Gn was coimmunoprecipitated by the anti-Gc MAb, evidence of heterodimerization between Gn and Gc. The loss of Golgi targeting of N60Q Gn itself and the chaperone-like function in Gc folding suggests that the conformation of Gn was also seriously compromised, although the lack of Gn-specific MAb prevented us from directly detecting conformational alterations of the unglycosylated Gn protein. The importance of N-glycan at N60 was further confirmed by the fact that no virus could be rescued from the mutant M cDNA clone carrying a mutation at that site. Others have also reported that glycoproteins are most sensitive to removal of glycosylation sites near their N termini, since glycans at these sites likely first recruit the protein folding machinery to initiate the folding process (10, 19, 20, 44).

BUN glycoproteins were more tolerant to elimination of the two N glycosylation sites on Gc protein. Mutant glycoproteins lacking either the glycan on N624 or on N1169 were able to fold properly, target to the Golgi, and acquire endo H resistance. This indicates that the proper folding of Gc mainly depends on its association with Gn rather than on N glycosylation at these two sites. It was noted that Gc lacking N-glycan at N624, in the case of N624Q and double mutant N624/1169Q, was not recognized by the anti-Gc MAb 742; whether this reflects the consequence of a conformational change of Gc or a minor alteration of the epitope that binds the antibody is unclear. However, the acquisition of endo H resistance by Gc expressed from mutant N624Q suggests that the protein was properly folded, although at lower efficiency. (We suggest that the non-Golgi immunofluorescence seen in Fig. 4J, represents misfolded/aggregated protein that is removed during sample preparation for immunoprecipitation and hence is not detected by this technique.) It is likely that the epitope recognized by MAb 742 might span, or be close to, the glycosylation site. The recovery of recombinant viruses lacking N-glycans at either or both sites on Gc confirmed that the N-glycans are not strictly required for protein folding and virus morphogenesis.

The role of N glycans of BUN glycoproteins in viral replication was assessed by using reverse genetics to rescue viable mutant viruses. The fact that no viable virus was rescued from mutant M segment that lacks the first N glycosylation motif (N60) stressed the importance of that glycan side chain of Gn protein for BUN virus replication and corroborates our other findings using transiently expressed proteins. Upon restoration of the N glycosylation site at N60 the full function of glycoproteins resumed and consequently enabled rescue of the virus named vD61E. This displayed a plaque phenotype identical to that of wt BUN. The successful recovery of the recombinant viruses that lack either one (vQ624 or vQ1169) or both glycosylation sites (vQ624/1169) on Gc indicated that the two sites are dispensable for virus replication. However, the phenotypes of the mutant viruses indicate that N glycosylation of Gc affects plaque phenotype, growth kinetics, and ability to shut off host protein synthesis. The single-site mutants vQ624 and vQ1169 had plaque sizes similar to that of wt BUN, whereas the double-site mutant vQ624/1169 produced smaller plaques. Consistently, removal of both N-glycans on Gc resulted in poorer growth after a low MOI and delayed shutoff of host protein synthesis. The single mutation of Gc glycosylation sites, especially at N624, had less impact, although virus growth was delayed at the lower MOI. At an MOI of 1 PFU/cell, the growth curves of all three mutant viruses were similar. This indicates that the two Gc glycans, although dispensable of virus replication, are important for efficient virus infection. The different patterns of growth kinetics at the two MOIs suggested that the glycans on Gc are likely involved in early stages of infection, such as virus entry into or spread between neighboring cells, rather than in virus morphogenesis.

In summary, our study demonstrated that N-glycan on Gn is crucial for protein folding, heterodimerization, and intracellular trafficking of both Gn and Gc proteins. The mutation at Gn glycosylation site (N60) is lethal to BUN viability. Two sites on Gc, although dispensable for in vitro infectivity, likely play a role in early infection, and the elimination of both sites led to significant attenuation of the mutant virus vQ624/1169 in vitro infection. The availability of N-glycosylation-site-deficient viruses will be a valuable tool to enable us to investigate the role of N glycosylation in infectivity, virulence, and tissue tropism in vivo.


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ACKNOWLEDGMENTS
 
We thank Klaus Conzelmann, Martin Lowe, and Bernard Moss for providing reagents used in this study.

This study was supported by grant 065121 from the Wellcome Trust to R.M.E.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Virology, University of Glasgow, Church St., Glasgow G11 5JR, Scotland, United Kingdom. Phone: 44-141-330-4024. Fax: 44-141-337-2236. E-mail: elliott{at}vir.gla.ac.uk. Back


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Journal of Virology, November 2005, p. 13725-13734, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13725-13734.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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