Proteolytic Activation of the Spike Protein at a Novel RRRR/S Motif Is Implicated in Furin-Dependent Entry, Syncytium Formation, and Infectivity of Coronavirus Infectious Bronchitis Virus in Cultured Cells

Cells and viruses.HuH-7 and Vero (ATCC CCL-81) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in the presence of penicillin (100 units/ml) and streptomycin (100 μg/ml) at 37°C in a 5% CO₂ environment. The passage 65 (designated wild-type [WT] virus here) of Vero-adapted IBV Beaudette, the recombinant IBV (rIBV [WT]), and rIBV-Luc Δ3a3b (rIBV-Luc [expressing the luciferase gene]) were described before (21, 22, 44). The recombinant vaccinia/T7 virus and IBV strains were propagated in Vero cells, and the 50% tissue culture infective doses (TCID₅₀) of viral stocks were calculated by the Reed-Muench method (42). Viral stocks were kept at −80°C until they were used.

Chemicals and antibodies.4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), leupeptin, and trypsin were purchased from Sigma Aldrich. Furin inhibitor (decanoyl-Arg-Val-Lys-Arg-chloromethylketone) was purchased from Calbiochem. The anti-IBV S polyclonal antibody (S2 Ab) was made by the immunization of rabbits with a truncated S protein (positions 387 to 687) and could recognize only the S2 subunit from amino acids 538 to 687, as confirmed by Western blotting (WB) and immunoprecipitation analysis of truncated S constructs (data not shown). Mouse monoclonal Abs against human β-tubulin and Flag epitope were purchased from Sigma Aldrich. The fluorescein isothiocyanate-conjugated anti-mouse and -rabbit immunoglobulin G (IgG) and horseradish peroxidase-conjugated anti-human, -mouse, and -rabbit IgG were purchased from Dako.

Plasmid construction and transfection.Plasmid pKT-0, which has a T7 promoter for transcription, was used for transient transfection (38). The pKT-S construct encoding the full-length S protein was previously reported (53). Epitope-tagged constructs were made by overlap PCR and cloned into pKT-0. Point mutations were made by site-directed mutagenesis using the Quikchange kit (Stratagene). Cells at about 90% confluence were infected with the recombinant vaccinia/T7 virus for 1 h, followed by transfection of plasmid DNA using the Effectene transfection reagent (Qiagen).

WB.Each sample was lysed with 1× sodium dodecyl sulfate (SDS) sample loading buffer without bromophenol blue, and the protein concentration was determined with the Bio-Rad Protein Assay kit. Equal amounts of total protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were incubated with a primary Ab and subsequently with a horseradish peroxidase-conjugated secondary Ab and detected using the ECL Advance Western Blotting Detection Kit (Amersham).

IF staining.Vero cells were cultivated in four-well chamber slides (Iwaki). IBV-infected cells or plasmid-transfected cells were washed with phosphate-buffered saline (PBS) supplemented with 10% normal goat serum, fixed with 4% paraformaldehyde in PBS for 15 min, and permeabilized with 0.2% Triton X-100 for 10 min. Immunofluorescent (IF) staining was performed by incubating the cells with the S2 Ab and subsequently with fluorescein isothiocyanate-conjugated anti-rabbit IgG. The cells were examined by fluorescence microscopy.

Quantitative real-time reverse transcription (RT)-PCR.Total RNA was extracted from virus-infected cells by using Tri Reagent (Molecular Research Center, Inc.) and treated with DNase I. The RNA concentration was measured with an ND-1000 spectrophotometer (NanoDrop Technologies), and 2 μg of RNA was reverse transcribed with Expand Reverse Transcriptase (Roche). A primer combination of IBV4527(+) (5′-TTTAGCAGAACATTTTGACGCAGAT) and IBV4805(−) (5′-TTAGTAGAACCAACAAACACGACAG) was used for either positive- or negative-strand genomic RNA (gRNA) amplification. Real-time PCR was performed using the LightCycler FastStart DNA Master SYBR green I kit according to the manufacturer's instructions (Roche). PCR was carried out for 40 cycles at 95°C for 10 s, 57°C for 10 s, 72°C for 15 s, and 75°C for 1 s. The GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA level was also quantified to normalize the IBV gRNA level with a primer combination of GAPDH(RT) (5′-TCTACATGGCAACTGTGAGGA), GAPDH(+) (5′-GACAACTTTGGTATCGTGGAA), and GAPDH(−) (5′-CCAGGAAATGAGCTTGACA). PCR for GAPDH was carried out for 40 cycles at 95°C for 10 s, 55°C for 10 s, 72°C for 15 s, and 80°C for 1 s.

In vitro transcription of full-length IBV cDNA and electroporation.The detailed procedure to create a full-length IBV cDNA clone was previously reported (21). The S gene of the partial IBV cDNA was replaced with mutant S genes and subsequently ligated into full-length IBV cDNA. Full-length transcripts were generated in vitro using the mMessage mMachine T7 kit (Ambion, Austin, TX). IBV N gene transcripts were also generated to enhance the recovery of virus. Vero cells at about 90% confluence were trypsinized, washed twice with ice-cold PBS, and resuspended in PBS. RNA transcripts were introduced into Vero cell using the Bio-Rad Gene Pulser II electroporator. The cells were cultured overnight in 1% FBS-containing DMEM in a six-well plate and further incubated in DMEM without FBS. At 48 h postelectroporation, viral-RNA replication was investigated by RT-PCR of the negative-strand gRNA. Transcription of subgenomic mRNAs was investigated by RT-PCR as described previously (21). The S genes of the recovered rIBV clones (at the second passage in Vero cells) were amplified by RT-PCR and subsequently confirmed by DNA-sequencing analysis. Characterization was carried out with the third passage of viruses in Vero cells.

Treatment of cells with protease inhibitors.Monolayers of Vero cells in 12-well plates were pretreated with furin inhibitor or protease inhibitors for 1 h at 37°C and infected with 10⁵ TCID₅₀ of rIBV for 2 h at 37°C (or 1 h at 4°C) in the presence of the agents. The cells were washed three times with PBS, and the medium was changed to fresh DMEM without agents (pretreatment). Posttreatment with furin inhibitor was carried out after virus infection (2 h at 37°C or 1 h at 4°C) by replacement of the culture media with fresh DMEM containing different concentrations of furin inhibitor, and the cells were incubated in the presence of the inhibitor. The cells were washed three times with PBS, and replication of the virus was investigated. The luciferase activity in rIBV-Luc-infected cells was measured with the Luciferase Assay System (Promega). Passive lysis buffer (Promega) was used for cell lysate preparation.

IBV S protein is cleaved by furin at both the RRFRR⁵³⁷/S and RRRR⁶⁹⁰/S positions.Prediction of a furin cleavage site on the ProP server (19) (http://www.cbs.dtu.dk/services/ProP/ ) showed the presence of an additional furin site at the ectodomain with either an RRRR₆₉₀/S or an RRKR₆₉₀/S motif in Beaudette and derivative strains of IBV (Fig. 1a). To determine if the RRRRS sequence is a furin substrate, a construct, S(1-789)Fc, covering the N-terminal 789-amino-acid region of the IBV S protein with a C-terminal Fc tag (Fig. 1a), was created and expressed in HuH-7, a cell line that cleaves the IBV S protein efficiently. In addition, two deletion constructs, Δ1 (Δ533-537) and Δ2 (Δ687-690), with deletion of the first and second furin sites, respectively, were created (Fig. 1a) and expressed. As shown in Fig. 1b, similar levels of S(1-789)Fc* (glycosylated forms are shown with asterisks) bands were detected from all constructs. The predicted C-terminal cleavage product from the first site (C terminus; cl-1C*) was detected as multiple bands in cells transfected with the WT and Δ2, but not with Δ1 (lanes 3 and 5), while the cleavage product from the second site (cl-2C*) was detected in cells expressing the WT and Δ1 (lanes 3 and 4). Interestingly, the cl-1C* species was weaker in cells expressing the WT S construct than in those expressing Δ2 (Fig. 1b, lanes 3 and 5), probably due to further cleavage of the species at the second site in the WT S protein, as described below. Treatment of samples with PNGase F confirmed that the unglycosylated bands were consistent with the predicted molecular masses of full-length [S(1-789)Fc; 111.1 kDa] and cleaved products from the S1/S2 site (cl-1C; 52.3 kDa) and the second site (cl-2C; 35.1 kDa), respectively (lane 2). The predicted dual-cleavage product could be detected in cells expressing WT, but not mutant, S constructs in a 12% gel (data not shown). To further investigate if cleavage at these two sites was furin dependent, increasing concentrations of furin inhibitor were added to cells expressing Δ1 and Δ2. Both cl-1C* and cl-2C* decreased depending on the concentration of furin inhibitor, but S(1-789)Fc* was at a constant level (Fig. 1c).

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FIG. 1.

An in vitro assay shows that RRRR₆₉₀/S of the IBV S protein is a furin substrate. (a) Schematic diagram of S protein from Vero-adapted IBV Beaudette and furin cleavage sites predicted by the ProP server. Constructs that encode full-length S and truncated S fused to the human IgG Fc domain, S(1-789)Fc, are illustrated. The positions for inserting the Flag tag, the putative signal sequence (SS), the fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), the transmembrane domain (TM), and the amino acid (aa) sequences deleted in the two deletion constructs (Δ1 and Δ2) are also shown. (b) Detection of cleavage at the two furin sites. The S(1-789)Fc constructs were expressed in HuH-7 cells for 24 h, total cell lysates were prepared, and PNGase F (New England Biotechnology) treatment was carried out for 2 h at 37°C. Cell extracts were analyzed by WB. (c) Inhibition of furin-dependent cleavage by furin inhibitor. At 6 h posttransfection of S(1-789)Fc constructs in HuH-7 cells, the culture media were replaced with fresh DMEM containing different concentrations of furin inhibitor. The cells were further incubated for 18 h, and cell extracts were analyzed by WB.

Cleavage products at two furin sites are detected in IBV-infected cells.To facilitate the detection of S protein cleavage in IBV-infected cells, two rIBV clones with a Flag-tagged S gene were created. As shown in Fig. 1a, the Flag tag (underlined) was placed either immediately after the S1/S2 cleavage site (S-Flag539; -RRFRR/SMDYKDDDDKIF-) or the C terminus of the S protein (S-Flag1163; -RPKKSVMDYKDDDDK). Infection of cells with rIBV(S-Flag539) would be expected to detect the full-length S and S2 and the N-terminal cleavage product from the second site [cl-2N; S(1-690) or S(19-690)] and the dual-cleavage fragment [cl-dual; S(538-690)]. Infection of cells with rIBV(S-Flag1163) would be expected to detect S, S2, and the C-terminal cleavage product from the second site [cl-2C; S(691-1162)]. Following electroporation of the in vitro-transcribed full-length RNA into Vero cells, infectious viruses were successfully recovered. Both S-Flag539 and S-Flag1163 viruses developed syncytia of a similar size to those of the WT (Fig. 2a). Analysis of the growth kinetics showed that the two rIBV clones propagated well in Vero cells, but the replication of S-Flag1163 was slightly delayed compared to the WT and S-Flag539 (Fig. 2b). These data indicated that insertion of Flag539 at a position immediately downstream of the S1/S2 cleavage site had minimal, if any, effect on the function of the S protein.

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FIG. 2.

Cleavage products at two furin sites are detected in IBV-infected cells. (a) Syncytium formation by rIBV was not affected by Flag-tagged S genes. Vero cells were infected with rIBV for 2 h. The cells were washed three times with PBS and incubated for 16 h with an overlay of DMEM containing 0.6% carboxymethyl cellulose. Syncytium formation was observed by IF staining with the S2 Ab. (b) Replication of S-Flag1163 was slightly delayed compared to the WT and S-Flag539. Vero cells (in a 25-cm² flask) were infected with rIBV and harvested at 0, 6, 12, 18, 24, 30, 36, 42, and 48 h postinfection. Viral stocks were prepared by freezing/thawing of the cells three times, and the TCID₅₀ was determined. The arrows indicate the time points when CPE was observed in nearly 100% of the cells. The error bars indicate standard errors of the means. (c) Cleavage at two furin sites was detected in virus-infected cells. HuH-7 cells were infected with rIBV for 18 h. Cell extracts were analyzed by WB with the anti-Flag Ab.

In cells infected with the two recombinant viruses, the glycosylated S (S*) and S2 (S2*) were detected in an SDS-6% polyacrylamide gel (Fig. 2c, top). Treatment of the samples with PNGase F efficiently removed the carbohydrate chains, leading to the detection of the unglycosylated S and S2 (Fig. 2c, top); S2 was still detected as double bands after the treatment (lanes 1 and 3). The predicted cleavage product at the second furin site ([S(691-1162)] was also detected in S-Flag1163-infected cells (lanes 3 and 4); however, its intensity was significantly reduced compared to S2. In addition, abundant bands migrating even more slowly than S and S* were detected (Fig. 2c). The identities of these bands are not clear, but they may represent higher-order oligomers/aggregates of the S protein. When samples were separated in an SDS-12% acrylamide gel, the predicted dual-cleavage fragment [S(538-690)] (lanes 1 and 2) was clearly detected. However, the putative intermediate cleavage products S(1-690)* and S(19-690)*, covering the N-terminal region up to the second furin site, were not detected in S-Flag539-infected cells (lanes 1 and 2).

Entry and syncytium formation by IBV are blocked by furin inhibitor.The effects of furin inhibitor on the attachment and entry of IBV were investigated by real-time RT-PCR optimized with specific primers on the 1a region. Pretreatment of Vero cells with different concentrations of furin inhibitor revealed that the attachment of WT virus was not affected at both 4 and 37°C, and no reduction in the positive-strand gRNAs was observed (Fig. 3a, 0 h). The effects of both pre- and posttreatment on IBV entry were evaluated at 4 h postinfection. The ratio between positive- and negative-strand gRNAs was approximately 50:1 in all samples. Interestingly, both positive- and negative-strand gRNAs were reduced by 40 to 60% by pretreatment (Fig. 3a, pretreatment at 4 h) but were only slightly decreased in posttreated cells (Fig. 3a, posttreatment at 4 h at 37°C). These data indicated that furin inhibitor blocked IBV entry with minimal effect on gRNA replication. This observation is unique for IBV and differs from MHV A59 and SARS-CoV, whose entry was not inhibited by furin inhibitor (16, 23). Posttreatment of virus-attached cells (4°C) did not block the entry, suggesting that furin may be involved in a step immediately after viral attachment to cells (Fig. 3a, posttreatment at 4 h at 4°C). At 16 h postinfection, the influence of pretreatment on viral infection was more pronounced (Fig. 3a, pretreatment at 16 h), though inhibition of viral replication was also observed in posttreated cells (Fig. 3a, posttreatment at 16 h). There was no significant difference between 5 and 10 μM treatments in this experiment.

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FIG. 3.

Entry and syncytium formation of IBV are blocked by furin inhibitor. (a) Pretreatment with furin inhibitor did not affect attachment but blocked IBV entry. The gRNA level in virus-infected cells was investigated with a quantitative real-time RT-PCR. The values are shown as relative amounts against 0 μM of furin inhibitor treatment. ND, not done. The error bars indicate standard deviations of the means. (b) The number of infected cells was reduced by pretreatment with furin inhibitor, while posttreatment blocked giant syncytium formation. Syncytium formation was observed by IF staining with the S2 Ab at 16 h postinfection. Giant syncytia caused by primary infection are indicated by arrows. (c) Inhibitory effects of pretreatment with furin inhibitor were caused by less primary infection, while those of posttreatment were due to blocking of cell-to-cell spread and secondary infection. The effects of furin inhibitor treatments on rIBV-Luc infection were investigated. Luciferase activity in virus-infected cells was measured at 7, 10, 13, and 16 h postinfection. The error bars indicate standard deviations of the means.

The effect of furin inhibitor on syncytium formation was investigated at 16 h postinfection by IF staining. The number of giant syncytia (by primary infection) was significantly decreased in a concentration-dependent manner by pretreatment, but the sizes of these syncytia were not affected (Fig. 3b, pretreatment). The reduction in tiny infected cells (by secondary infection) may have been caused by reduced viral release from the primary infection (Fig. 3b, pretreatment). In contrast, syncytium formation by primary infected cells was blocked by posttreatment in a concentration-dependent manner (Fig. 3b, posttreatment). In addition, the number of smaller syncytia was slightly reduced in posttreated cells (Fig. 3b, posttreatment). The TCID₅₀ of viruses released into the culture supernatants and the S protein expression level in virus-infected cells were also investigated at 16 h postinfection. Inhibition of WT infection by furin inhibitor was observed in both experiments (data not shown).

To investigate the kinetics of viral entry and replication in the presence of furin inhibitor, Vero cells were infected with rIBV-Luc. Following infection, the luciferase activity usually started to increase at 6 h and reached the maximum level at 16 h postinfection. As shown in Fig. 3c, analysis of the luciferase activity in virus-infected cells at 7 h postinfection revealed that pretreatment significantly reduced the viral infection in a concentration-dependent manner (Fig. 3c, pretreatment at 7 h), while posttreatment did not show any effect (Fig. 3c, posttreatment at 7 h). These results further confirmed that furin inhibitor blocked viral entry but not replication. Since the degrees of increment of the luciferase activity in pretreated cells were similar regardless of furin inhibitor concentrations (Fig. 3c, pretreatment at 7 to 16 h), less viral replication at higher concentrations of furin inhibitor may be largely due to less primary infection. In posttreated cells, a difference in the luciferase activity was observed from 13 h postinfection in a concentration-dependent manner (Fig. 3c, posttreatment at 13 and 16 h). Since formation of large and tiny syncytia was observed around this time point, less viral replication in posttreated cells was likely due to blocking of cell-to-cell spread and secondary infection.

S1/S2 cleavage mediated by furin is not essential for, but could promote, syncytium formation and infectivity of IBV.The contributions of S1/S2 cleavage by furin to IBV infection were investigated by the introduction of mutations into the constructs and the rIBV genome. The four R residues at the S1/S2 cleavage site were removed either by replacement with alanine (A; M1) or by deletion (Δ1) (Table 1), resulting in the ablation of S1/S2 cleavage with no effect on the second-site cleavage (Table 1). Furthermore, the absence of the first furin site (M1 and Δ1) reduced the induction of cell-cell fusion by the S construct (Table 1).

Two mutant viruses, rIBV(M1) and rIBV(Δ1), carrying the mutations/deletion at the S1/S2 site were successfully recovered in Vero cells (Table 1). Consistent with an in vitro cell-cell fusion assay, M1 and Δ1 mutant viruses demonstrated the formation of smaller syncytia than the WT (Fig. 4a). WB results showed that minute amounts of S2* and S2 bands were still detectable in M1- and Δ1-infected cells (Fig. 4b, lanes 3 to 6), demonstrating that the S1/S2 cleavage was not completely eliminated. Once again, multiple S2 bands were detected in WT-infected cells (Fig. 4b, lanes 1 to 6), as observed in cells infected with S-Flag539 and S-Flag1163 viruses (Fig. 2c). These results suggest that inefficient cleavage of IBV S protein in the region flanking the S1/S2 site may also occur at multiple positions, as reported for cleavage of SARS-CoV S protein by trypsin and cathepsin (8). The dual-cleavage fragment [S(538-690)* and S(538-690)] was detected only from cells infected with WT (Fig. 4b, lanes 1 and 2). Analysis of the growth kinetics of the two mutant viruses showed that the replication of M1 and Δ1 was significantly delayed compared to that of the WT, especially at early time points of infection (Fig. 4c). Cytopathic effect (CPE) was usually observed in nearly 100% of cells infected with the WT at 24 h postinfection; the infected cells were rapidly detached and died. In cells infected with M1 and Δ1, about 20% of the cells did not show CPE at 60 h postinfection. Prolonged replication of M1 and Δ1 indicated that cleavage at the S1/S2 site facilitates IBV infection and is important for efficient infection in Vero cells. The effects of pretreatment with furin inhibitor on the infectivity of the two mutant viruses were then tested, showing that, similar to the WT, infection by both M1 and Δ1 was inhibited by furin inhibitor in a concentration-dependent manner (Fig. 4d).

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FIG. 4.

S1/S2 cleavage by furin is not essential for, but could promote, syncytium formation and infectivity of IBV. (a) Cleavage deficiency at the S1/S2 site resulted in reduced size of syncytia. Syncytium formation was observed as described in the legend to Fig. 2a. (b) A minute amount of S1/S2 cleavage was detected in M1- and Δ1-infected cells, although they do not have a furin consensus motif. HuH-7 cells were infected with rIBV for 18 h. Cell extracts (20 μg protein) were analyzed by immunoblotting them with the S2 Ab. (c) Viral replication and spread were reduced in the absence of S1/S2 cleavage. Growth kinetics was investigated as described in the legend to Fig. 2b. The arrow indicates the time point when CPE was observed in nearly 100% of the cells. The error bars indicate standard deviations of the means. (d) Infection of M1 and Δ1 were inhibited by pretreatment with furin inhibitor. Vero cells were pretreated with different concentrations of furin inhibitor and infected with rIBV for 2 h. The cells were washed three times with PBS and incubated with fresh DMEM without furin inhibitor. Replication of viruses was observed by immunoblotting them with the S2 Ab at 16 h postinfection. β-tub, β-tubulin.

The XXXR₆₉₀/S motif at the second IBV furin site is a minimal sequence to support IBV infection in Vero cells.The four R residues at the second furin site were removed by either R-to-A substitutions or deletion (Table 2, M2 and Δ2). The amino acid sequence of M41 (the parental strain of Beaudette) was also reproduced at the second furin site (M2.1; PRRRS). An additional construct, M1+2.1, was made by combining the mutations of M1 and M2.1 (Table 1). Expression of M2, Δ2, and M2.1 constructs showed cleavage at the S1/S2 site, but not at the second furin site (Table 1). No cleavage at both positions was observed when M1+2.1 was expressed (Table 1). Interestingly, cell-cell fusion at neutral pH was not observed in Vero cells expressing M2, Δ2, M2.1, and M1+2.1 constructs, which lack the second furin site (Table 1). Cell-cell fusion was activated by incubation of cells expressing M2.1 and M1+2.1, but not M2 and Δ2, with 2 μg/ml trypsin for 2 h (Table 1 and Fig. 5a), suggesting that the presence of a trypsin-cleavable motif (R/S) is essential for induction of cell-cell fusion by these mutant constructs. It also indicates that these mutant S proteins could be translocated to the cell surface and that they are functional.

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FIG. 5.

Infectivity to Vero cells was abrogated by mutations of the second furin site in S protein. (a) Trypsin activation of cell-cell fusion was inefficient in the second furin site mutants, which cannot recover infectious viruses. Trypsin (2 μg/ml) was added at 10 h posttransfection of S constructs, and the cells were incubated for 2 h at 37°C. IF staining was carried out with either anti-S2 or anti-Flag Ab. (b) Replication and transcription of viral RNAs were observed in cells electroporated with mutant transcripts, although the infectious clones were not recovered. At 48 h postelectroporation of the full-length transcripts, total RNA was extracted from the cells. Negative-strand gRNA and subgenomic mRNA were detected by RT-PCR amplification.

Four additional constructs (M2.2 to M2.5) with monobasic motifs at the second furin site were made by A-to-R substitutions based on mutant construct M2 (Table 1). Sequence analysis with the peptide cutter program (http://www.expasy.ch/tools/peptidecutter/ ) showed that the probabilities of cleavage by trypsin are 91.9, 100, 100, and 100% for R₆₈₇AAAS (M2.2), AR₆₈₈AAS (M2.3), AAR₆₈₉AS (M2.4), and AAAR₆₉₀S (M2.5), respectively, although they are not cleavable by furin. Neither cleavage at the second furin site nor cell-cell fusion was observed in cells expressing the M2.2 to M2.4 S constructs (Table 1). Interestingly, a single R₆₉₀ residue (M2.5; AAARS) was sufficient to induce weak cell-cell fusion in Vero cells, even though M2.1 with the PRRRS sequence was unable to do so (Table 1). However, it was inefficient compared to the WT, M1, and Δ1, whose second site was a furin consensus motif, RRRR/S. As expected, activation of cell-cell fusion by trypsin was observed in cells expressing constructs bearing a trypsin-cleavable motif (R/A or R/S) at the second site (Table 1 and Fig. 5a). It was also noted that cell-cell fusion was less efficiently induced in cells expressing M2.2 to M2.4 than in those expressing M2.1 and M2.5, probably due to reduced cleavage efficiencies by trypsin at the second site.

These mutations were then introduced into the IBV genome, and infectious viruses were successfully recovered in Vero cells electroporated with M2.1, M1+2.1, and M2.5 constructs, but not with M2, Δ2, M2.2, M2.3, and M2.4 (Table 1), although the 64-terminal-residue sequence which mediates S protein incorporation into the CoV particle (25) is maintained. As no infectious virus was recovered with M2, Δ2, M2.2, M2.3, and M2.4 mutant transcripts, RT-PCR amplification of the negative-strand gRNA and the subgenomic mRNAs was performed at 48 h postelectroporation of transcripts. As shown in Fig. 5b, both negative-strand gRNA and subgenomic mRNAs (mRNA3 and -4) were detected in cells electroporated with these transcripts (lanes 2 and 3). These data indicated that the inability to recover infectious viruses from these constructs was due to S protein modification and that R residues at the second furin site may play a role in the function and conformation of S protein. Therefore, the XXXR₆₉₀/S motif at the second furin site was likely the minimal sequence required to support IBV infectivity in Vero cells.

An additional construct, S(Δ538-690), with deletion of the region between the two furin sites, was also made; however, no induction of cell-cell fusion and recovery of the infectious virus was observed (Fig. 5a and Table 1). Amplification by RT-PCR detected both negative-strand gRNA and subgenomic RNAs in Vero cells electroporated with S(Δ538-690) transcripts (Fig. 5b, lane 8), confirming that the mutation did not affect the replication and transcription of viral RNA.

Proteolytic activation at the second IBV furin site is mediated by furin and other serine protease(s).The recovered mutant viruses M2.1, M1+2.1, and M2.5 were then characterized. M2.1 and M1+2.1 did not induce syncytium formation in Vero cells (Fig. 6a) . Instead, rounded single cells were noticed from 24 h postinfection in M2.1- and M1+2.1-infected cells (Fig. 6a) (only M2.1 is shown). Delayed syncytium formation was observed in M2.5-infected cells compared to the WT, M1, and Δ1 viruses (Fig. 6a). The smaller syncytia induced by M2.5 were usually seen only at 24 to 30 h postinfection in Vero cells.

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FIG. 6.

Proteolytic activation at the second IBV furin site is mediated by furin and other serine protease(s). (a) Giant syncytium formation by rIBV was mediated by proteolytic activation at the second furin site. Syncytium formation was observed as described in the legend to Fig. 2a. For observation of CPE, cells were incubated with DMEM instead of overlay medium. (b) S1/S2 cleavage occurred efficiently in M2.1- and M2.5-infected cells, but not in M1+2.1-infected cells. HuH-7 cells were infected with rIBV for 18 h. Cell extracts (20 μg protein) were analyzed by immunoblotting them with the S2 Ab. (c) M2.1 and M1+2.1, which lack induction of syncytia, demonstrated prolonged replication with high productivity of virions. The growth kinetics was investigated as described in the legend to Fig. 2b. The error bars indicate standard deviations of the means. (d) Infection of M2.1, M1+2.1, and M2.5 was not affected by pretreatment with furin inhibitor. The effect of pretreatment with furin inhibitor was investigated as described in the legend to Fig. 4d. (e) Infection of the WT, M2.1, and M2.5 was inhibited by pretreatment with AEBSF, but not leupeptin. The effects of protease inhibitors were investigated as described in the legend to Fig. 4d, except that the cells were pretreated with leupeptin or AEBSF.

WB results confirmed that cleavage at the S1/S2 site occurred efficiently in M2.1 and M2.5 but not in M1+2.1 (Fig. 6b). The dual-cleavage fragment [S(538-690)* and S(538-690)] was detected only in cells infected with WT (Fig. 6b). Analysis of the growth kinetics showed that the replication of all mutant viruses was delayed, reaching peak titer at 24, 30, and 60 h postinfection for M2.1, M2.5, and M1+2.1, respectively (Fig. 6c). Interestingly, both M2.1 and M1+2.1 mutant viruses produced higher titers, with M2.1 reaching a peak titer of nearly 10⁷, more than 1 log unit higher than the WT (Fig. 6c). Without formation of syncytia, rounded cells remained scattered at 60 h postinfection, with prolonged replication and production of high titers of virus (Fig. 6c). The much delayed replication of M1+2.1 compared to M2.1 further supported the conclusion that S1/S2 cleavage by furin facilitates infection by IBV in Vero cells. The growth kinetics of M2.5 that induces syncytium formation was similar to that of M1 virus (Fig. 6c, M2.5, and 4c, M1).

Pretreatment of M2.1, M2.5, and M1+2.1 with furin inhibitor showed that infection by the three mutant viruses was not inhibited, suggesting that furin dependence of IBV entry was mediated by the second furin site (Fig. 6d). Taken together, the evidence shows that proteolytic activation of IBV S protein by furin occurs at the second furin site for both virus-cell and cell-cell fusion events, but not S1/S2 cleavage.

To test whether proteolytic activation of M2.1 and M2.5 S proteins is mediated by a trypsin-like protease(s), the effects of two broad-range protease inhibitors, leupeptin (a cysteine and serine protease inhibitor) and AEBSF (a serine protease inhibitor), at concentrations from 0 to 200 μM were investigated. In the presence of AEBSF, but not leupeptin, inhibition of WT infection was observed in a concentration-dependent manner (Fig. 6e), consistent with the inhibitory effects of those agents on furin activity. Pretreatment with leupeptin did not inhibit either M2.1 or M2.5 infection (Fig. 6e). In contrast, treatment with AEBSF reduced infection by M2.1 and M2.5 in a concentration-dependent manner (Fig. 6e). These data demonstrate that the entry of M2.1 and M2.5 is also mediated by a yet-to-be-identified serine protease(s). As the infectivity of M2.1 and M2.5 mutant viruses was not inhibited by leupeptin, this rules out the possibility that the mutant S proteins may be activated in Vero cells by type II transmembrane serine proteases, which cleave monobasic motifs, as shown with influenza A virus hemagglutinin (10, 11, 28). Differences in the infectivity and syncytium formation of these mutant viruses may be due to the expression level and cellular localization of the associated protease(s) and the cleavage efficiency of the substrate sequence.

The minimal XXXR/S motif, cleavable by furin, general PCs, or trypsin, is strictly conserved at the equivalent position of the second IBV furin site in most CoV S proteins.As demonstrated above, XXXR₆₉₀/S is likely the minimal motif of IBV S protein at the second furin site, and the associated proteases may vary depending on the first 3 amino acids. Analysis of 52 published IBV S protein sequences by multiple alignments, the ProP server, and the peptide cutter program showed that only the Beaudette-related strains (15/52) are cleavable by furin at the second furin site. However, most S proteins (36/52) could be cleaved by general PCs, including PC1, PC2, furin, PC4, PC5, PACE4, and PC7, and all (52/52) by trypsin at this position, confirming that the XXXR/S motif is strictly conserved. The same analysis also showed that all S proteins (52/52) are furin cleavable at the S1/S2 site.

Similar analysis was then extended to other CoVs. As expected, similarity analysis of CoV S proteins showed that the S2 domain, but not the S1 domain, is conserved (Fig. 7). The low similarity (gaps) in the S1 domain was largely due to amino acid insertions in group 1 (at positions 1 to 200) and group 2 (at 730 to 880) (Fig. 7). The S1/S2 cleavage site was mapped to the variable region, and the second IBV furin site straddled the variable and consensus regions (Fig. 7). The multiple alignments and furin site prediction showed the presence of a furin site at a position equivalent to the second IBV furin site in group 1 CoVs, including canine CoV, FECV 1683, porcine epidemic diarrhea virus, transmissible gastroenteritis virus (TGEV), and bat CoV 1B, and group 2 bat CoV HKU5-1 (Table 2). However, the low probability score and the lack of a preferred motif, RXR(K)R, indicate that cleavage may be inefficient. Analysis of the predicted cleavage by general PCs showed the presence of a cleavable site in feline CoV (NTU2 and C1Je), group 2 CoVs (MHV BHK, SARS-CoV, and bat CoV), and IBV M41 (Table 2). The minimal XXXR/S motif is strictly conserved in most examined CoVs, with the exception of FIPV strains 1146 and DF-2, in which the R residue was replaced by a G (NSKRKYG_{961 or 963}S) (Table 2). The equivalent positions in other feline CoV S proteins are an XXXR/S motif and a furin site (underlined) (NSKRKYR₉₆₃/S), found in FECV 1683, as well as canine CoV and TGEV (Table 2). Cathepsin B, but not cathepsin L, was shown to mediate cleavage of the FIPV 1146 S protein at a position further downstream from the S1/S2 cleavage site (43). Cleavage of the FECV 1683 S protein occurs at the same position but is mediated by both cathepsins B and L (43). The S protein of munia CoV has an R-to-K substitution (underlined) (SNKIGEK₆₆₉/SV), which is cleavable by trypsin (Table 2).

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FIG. 7.

A novel XXXR/S motif, cleavable by furin, general PCs, or trypsin, is strictly conserved in CoV. The similarity alignments of CoV S were obtained with the Vector NTI Align X software (Informax). The fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), and the transmembrane domain (TM) are also shown. The gaps in the HR1 and HR2 regions were due to amino acid insertion in a group 1 CoV. Receptor-binding domains (RBD) are shown with dashed lines. The S1/S2 cleavage and the second IBV furin site are indicated by arrows. The asterisk indicates the site of S1/S2 cleavage by furin in group 1 (feline CoV Black and NTU2), group 2 (MHV, bovine CoV, human CoV OC43 and HKU-1, porcine hemagglutinating encephalomyelitis virus, and equine CoV), and group 3 (IBV and turkey CoV). S1/S2 cleavage in SARS-CoV by trypsin (empty arrow) and cathepsin (solid arrow) is also shown.

Replacement of the second IBV furin site with FIPV 1146 (NSKRKYGS) and FECV 1683 (NSKRKYR/S) sequences showed induction of mild cell-cell fusion in Vero cells expressing the FECV sequence, but not the FIPV sequence (Table 2). However, cell-cell fusion could be activated upon trypsin treatment in cells expressing the FIPV sequence, suggesting that the FIPV sequence is trypsin cleavable at the NSKRKYGS position (underlined). The probabilities of cleavage by trypsin were 65, 89.7, and 84.3% for NSK₉₅₇R₉₅₈K₉₅₉YGS at positions K_957, R₉₅₈, and K₉₅₉, respectively. Furthermore, cleavage by furin at the second site was detectable in cells expressing the chimeric S(1-789) construct containing the FECV, but not FIPV, sequence (Table 2), suggesting that the RKYR/S motif in FECV, TGEV, canine CoV, and bat CoV HKU5-1 is furin cleavable. These data indicated that the RXXR motif is a minimal furin-cleavable sequence, as in the hepatitis B virus e antigen (30).

Similarly, replacement of the second IBV furin site with the equivalent sequences from human CoV 229E (GSRVAGR/S) and NL63 (SSRIAGR/S), MHV (PSAIRGR/S), MHV-2 (MAAQTGR/S), and SARS-CoV (PLKPTKR/S) showed induction of either mild or weak cell-cell fusion in cells expressing MHV, MHV-2, human CoV 229E, and human CoV NL63, but not SARS-CoV, sequences (Table 2). It suggests that induction of cell-cell fusion by these S constructs may be activated by a yet-to-be-identified trypsin-like protease(s), as in M2.5. Upon trypsin treatment, more extensive cell-cell fusion was observed in cells expressing the SARS-CoV sequence (Table 2) than in cells expressing the FIPV sequence, suggesting that XXXR₆₉₀S may be a preferred motif in the context of the IBV S protein. Nevertheless, these results confirm that the equivalent motifs in other CoV S proteins can be efficiently activated by trypisn-like proteases in the context of the IBV S protein.