Contribution of cysteine residues in the extracellular domain of the F protein of human respiratory syncytial virus to its function
© Day et al; licensee BioMed Central Ltd. 2006
Received: 01 November 2005
Accepted: 24 May 2006
Published: 24 May 2006
The mature F protein of all known isolates of human respiratory syncytial virus (HRSV) contains fifteen absolutely conserved cysteine (C) residues that are highly conserved among the F proteins of other pneumoviruses as well as the paramyxoviruses. To explore the contribution of the cysteines in the extracellular domain to the fusion activity of HRSV F protein, each cysteine was changed to serine. Mutation of cysteines 37, 313, 322, 333, 343, 358, 367, 393, 416, and 439 abolished or greatly reduced cell surface expression suggesting these residues are critical for proper protein folding and transport to the cell surface. As expected, the fusion activity of these mutations was greatly reduced or abolished. Mutation of cysteine residues 212, 382, and 422 had little to no effect upon cell surface expression or fusion activity at 32°C, 37°C, or 39.5°C. Mutation of C37 and C69 in the F2 subunit either abolished or reduced cell surface expression by 75% respectively. None of the mutations displayed a temperature sensitive phenotype.
To determine the contribution of the individual cysteine residues in the extracellular domain (ECD) to its functions, a panel of mutations in which each cysteine residue in the ECD of the HRSV F protein (residues 37, 69, 212, 313, 322, 333, 343, 358, 367, 382, 393, 416, 422, 439) was individually changed to a serine, and the effect of these mutations upon the function of the HRSV F protein was determined.
Summary of results for HRSV F cysteine mutants. Processing is defined as relative amounts of F0, F1, and F2, and is described as being equivalent to wild-type HRSV F protein (complete) or reduced. Cell surface and total expression were measured by ELISA under permeabilizing (total F protein) or non-permeabilizing (cell surface F protein) conditions using palivizumab as described in methods and reported as percent relative to wild-type HRSV F protein. Reactivity with neutralizing mAbs (palivizumab, Mab19, 47F, and 101F) as determined by flow cytometry is shown and reported as percent relative to wild-type HRSV F protein. Cell fusion activity is reported as luciferase activity measured at 32°C, 37°C, and 39.5°C as described in . All values are expressed as % relative to wild-type at the respective temperatures.
Cell surface expression (Flow cytometry)
Cell fusion (% of WT)
Cell surface protein (Non-permeabilized)
Total protein (permeabilized)
To extend these results, the effect of the cysteine mutations upon the level of cell surface expression was examined by flow cytometry using four different antibodies, 47F , 101F (a monoclonal which recognizes the site IV, V, VI region), palivizumab  or mAb19  directed against one of two major antigenic sites (II or IV, V, VI) in the F protein. Consistent with results obtained using ELISA under non-permeabilizing conditions, flow cytometry analysis demonstrated that mutation of cysteine residues 37, 313, 322, 333, 343, 358, 367, 393, 416, and 439 reduced binding of all four antibodies, while mutation of cysteine mutants C382S, and C422S retained similar levels of antibody binding as the wild-type F protein (Table 1). As the same set of cysteine mutations that reduced or abolished F0 protein cleavage and cell surface expression, also reduced or abolished cell surface binding of the four mAbs tested here, we conclude that cysteine residues 37, 313, 322, 333, 343, 358, 367, 393, 416, and 439 play a key role in the proper folding, processing, and cell surface transport of the HRSV F protein. Again, as the epitopes of these antibodies are directed against two different antigenic regions of F protein and have been shown to be largely non-conformational [43, 45], we suggest that it is unlikely that the inability to detect these cysteine mutation F proteins on the cell surface is attributable to protein misfolding which would simultaneously block the epitopes recognized by these four different antibodies, but rather reflects a true defect in cell surface transport caused by these mutations. Interestingly, mutation of residue C212, which had wild-type levels of protein expression as determined by ELISA, appeared to have somewhat reduced levels of cell surface protein (37–47% of wild-type) as determined by flow cytometry. Although the exact reason for this is not clear, it could reflect a sensitivity of this particular mutant (folding, reactivity to fixation agent, etc.) to the differences in the experimental conditions used for ELISA and flow cytometry.
Limited direct structure-function data exists for the HRSV F protein. This study utilizes a genetic approach to analyze the contribution of the individual cysteine residues in the extracellular domain in protein expression and cell fusion of the HRSV F protein and represents the first analysis of the contribution of the cysteine residues of the HRSV F protein ECD to its function. Generally, cysteine residues are critical for folding and provide structural stability to a protein via the formation of disulfide bonds. Mutation of cysteine residues 37, 313, 322, 333, 343, 358, 367, 393, 416, and 439 abolished or reduced cell surface expression to less than 7% of wild type HRSV F protein. This suggests that these residues play a key role in the proper folding and subsequent transport through the Golgi to the cell surface. Identification of the stages at which these specific cysteine mutations block the folding, maturation, and transport of the HRSV F protein is currently ongoing. Mutation of cysteine residues can often lead to a temperature sensitive (ts) phenotype such as that observed for the herpes simplex type 1 gD glycoprotein . The lack of an observable ts phenotype in this study is supported by the high thermostability of the HRSV F protein among paramyxoviruses .
From direct mapping of disulfide bonds in Sendai virus , and based upon the positional conservation of the cysteine 69 residue in the HRSV F proteins with that of Sendai virus F protein and the F proteins from other of the Paramyxoviridae, it is likely that cysteine residues 69 and 212 participate in the disulfide linkage between the F1 and F2 subunits. The Pneumovirinae members have a positionally conserved second cysteine residue in the F2 subunit (corresponds to residue 37 in HRSV F protein) (Figure 2) not found in the other Paramyxovirinae. In the model of the HRSV F ECD, this cysteine residue is predicted to make a disulfide bond with cysteine residue 439, which is also only conserved in the F proteins of the Pneumovirinae members and not found in the F proteins of the other Paramyxovirinae members. This would suggest that two disulfide bonds are formed between the F1 and F2 subunits. We are currently performing direct biochemical mapping of the disulfide linkages to formally demonstrate this. This could explain, in part, the unique thermostability described for the HRSV F protein ECD .
HRSV is a significant human pathogen, and the F protein has been identified as the target of multiple neutralizing antibodies [47, 52, 53] as well as small molecule inhibitors [54–58]. As such, the HRSV F protein represents a critical viral target for the development of new and improved preventions and treatments for HRSV induced disease. A greater understanding of its structure-function relationships would greatly facilitate the development of these new agents. The results of this study provide further support that the highly conserved HRSV F protein cysteine residues play a critical role in the structure and function of this protein. As disulfide bonds have been shown to play roles beyond proper protein folding and stabilization of protein structure , it is tempting to speculate that, similar to HIV , the disulfide bonds of the Pneumovirus F proteins may have a direct role in fusion. Our modeling and analysis suggest the presence of two disulfide bonds which join the F1 and F2 subunits of the HRSV F protein. If formally demonstrated, this would highlight a distinct structural feature of the F proteins of the Pneumovirinae not described for the F proteins of the Paramyxovirinae.
Cells, plasmids and transfections
293T cells were grown at 37°C in a humidified atmosphere of 5% CO2 and maintained in Dulbecco's modified Eagle media (DMEM) with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose and 10% FBS. Cells were tested and confirmed to be free of mycoplasma contamination. Plasmid pHRSVFoptA2, which expresses the HRSV F protein of the A2 strain whose sequence was codon optimized and derived from a known infectious HRSV cDNA , has been previously described  and served as the template for the generation of the panel of cysteine mutations by site directed mutagenesis using the QuikChange® Site-Directed Mutagenesis kit (Stratagene®, La Jolla, CA). Cells were transiently transfected using FuGENE 6 reagent (Roche Applied Science, IN) as previously described .
Metabolic labeling and immunoprecipitation
[35S]-methionine/cysteine radiolabeled cell lysates were prepared and immunoprecipitated with a cocktail of four anti-HRSV F mAbs (palivizumab, 47F, Mab19, and 101F) directed against the two major antigenic sites II and IV, V, VI  as previously described .
The binding of neutralizing monoclonal antibodies (mAbs) to HRSV F protein was assayed by ELISA using 293T cells transiently transfected with plasmids expressing either wild-type F protein, the panel of cysteine mutants, or a vector only control. 293T cells (2.0 × 104 cells/well) were plated the day before transfection in 96-well plates in DMEM, supplemented with 1.5 gms./liter sodium bicarbonate and 10% FBS. A total of 50 ngs of plasmid DNA was complexed with 0.15 μl of FuGENE 6 reagent and incubated 20 minutes room temperature in OptiMEM reduced-serum medium prior to addition to cells in serum containing medium. At 20–24 hours post-transfection, cells were assayed for binding of palivizumab under permeabilizing or non-permeabilizing conditions. Cells were fixed by the addition of 0.05% glutaraldehyde (Sigma) in 1X PBS for 15 minutes at room temperature. Cells were then either washed under conditions which permeabilizing (0.1% Triton-X100 in PBS) or non- permeabilizing (0.05% Tween-20 in PBS) conditions. These conditions were verified using an anti-RSV N protein mAb (clone # M291207, Fitzgerald Industries International, Concord, MA) and HRSV infected cells. HRSV N protein is only produced within the cytoplasm of HRSV infected cells. The anti-N mAb yields a strong positive signal on infected cells when the wash buffer containing 0.1% Triton-X100 is used, but not when wash buffer containing 0.05% Tween 20 is used (data not shown). Cells were blocked for one hour with SuperblockTM (Pierce Biotechnology, Inc., Rockford, IL) followed by incubation with either 1 μg/ml chimeric 101F IgG, 1 μg/ml palivizumab or a 1:600 dilution of mAb19 hybridoma supernatant for one hour at room temperature. Samples were then incubated with an anti-human IgG-HRP or an anti-mouse IgG-HRP as appropriate (Amersham Biosciences, Inc.) at 1:800 for one hour at room temperature followed by detection with TMB substrate (Sigma, Inc.). The reaction was stopped with the addition of 2N sulfuric acid, and the optical density at 450 nm was read. Values were calculated as percents relative to wild-type HRSV F after adjusting for background signal from the vector only control.
To confirm cell surface expression, 293T cells were transfected with plasmids expressing either wild-type F protein, the panel of cysteine mutants or a vector only control in either 6-well or 96-well formats as described above. Cells were fixed with 2% paraformaldehyde in PBS for 15 minutes at 4°C. Cells were washed with PBS containing 2% FBS and then stained with either a chimerized human version of 101F (murine V region grafted onto human IgG1κ framework) or palivizumab (IgG1κ) at 1 μg/ml with an anti-human IgG-Alexa-Fluor-488 conjugated secondary (Molecular Probes, Eugene, OR) for analysis with the FACSCalibur (BD Bisociences) and determining the mean fluorescence intensity. Data analysis was performed with Cell Quest and FloJo Analysis Software. Values were calculated as percents relative to wild-type HRSV F after adjusting for background signal from the vector only control.
Cell fusion assays
Cell fusion assays were conducted as previously described . Briefly, one population of 293T cells was co-transfected with pHRSVFOptA2 and pBD-NFκB (effectors cells), and another population of 293T cells was transfected with the pFR-Luc luciferase reporter plasmid (reporter cells). At 24 hours post transfection, effector cells were mixed with an equal amount of reporter cells in a 96-well plate and incubated an additional 24 hours prior to measurement of luciferase activity using the Steady Glo Luciferase reporter system (Promega, Inc.).
The molecular structure of HRSV F protein ECD was modeled using the human parainfluenza virus 3 virus F protein ECD structure as template [pdb code 1ztm], essentially in the same way as previously described  with a small adjustment of the residues between 331 and 346, thus allowing all pairs of cysteine residues to be positioned close enough to form disulfide bonds. Sequence alignment was carried out in ICM (Molsoft, CA) and manually adjusted. The monomer molecular model was first generated in ICM and then the trimer was assembled.
Sequence alignment was performed using the CLUSTAL W method in MegAlign program (version 5.05) from DNASTAR, Inc. (Madison, WI). Genbank accession numbers for the sequences of the viral F proteins used for the alignment are: HRSV , BRSV (NC_001989), PVM (AY729016), HMPV (NC_004148), APV (AY590688), hPIV3 (NC_001796), Sendai virus (NC_001552), Mumps virus (NC_002200), NDV (AF309418), Simian parainfluenza virus 5 (SV5) (NC_006430), Measles virus (P69353), Rinderpest virus (NC_006296), Nipah virus (NC_002728), Hendra virus (NC_001906).
We thank Geraldine Taylor for generously providing mAb19 hybridoma supernatant as well as helpful discussions and comments. We thank William Glass, Jarrat Jordan, and Lamine Mbow for critical review of this manuscript.
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