Respiratory syncytial virus F envelope protein associates with lipid rafts without a requirement for other virus proteins

Abstract

Like many enveloped viruses, human respiratory syncytial virus (RSV) assembles at and buds from lipid rafts. Translocation of the envelope proteins to these membrane subdomains is essential for production of infectious virus, but the targeting mechanism is poorly understood and it is not known if other virus proteins are required. Here we demonstrate that F protein of RSV intrinsically targets to lipid rafts without a requirement for any other virus protein, including the SH and G envelope proteins. Recombinant virus deficient in SH and G but retaining F protein expression was used to demonstrate that F protein still localized in rafts in both A549 and HEp-2 cells. Expression of a recombinant F gene by use of plasmid vectors demonstrated that F contains its own targeting domain and localized to rafts in the absence of other virus proteins. The domain responsible for translocation was then mapped. Unlike most other virus envelope proteins, F is unusual since the target signal is not contained within the cytoplasmic domain nor did it involve fatty acid modified residues. Furthermore, exchange of the transmembrane domain with that of the vesicular stomatitis virus G protein, a nonraft protein, did not alter F protein raft localization. Taken together, these data suggest that domains present in the extracellular portion of the protein are responsible for lipid raft targeting of the RSV F protein.

FIG. 1.

Colocalization of F protein with a lipid raft marker in the absence of G and SH. Cells were infected with rgRSV-SGF (A) or rgRSV-F (B), viruses engineered to express GFP as a marker of infection. rgRSV-F has the G and SH coding sequences removed and does not express the G and SH proteins. Infected A549 cells grown on culture slides were stained for surface RSV F protein and GM1 at 18 h postinfection. For staining, cells were incubated with a primary antibody specific for RSV F protein and CTxB (binds to GM1) on ice, followed by incubation with secondary antibody conjugated to FITC that binds the primary F antibody for indirect staining of F protein. Fixed cells were then analyzed by confocal microscopy. For panel B, two representative fields are shown (top and bottom rows). Colocalization is shown in the overlay panels and appears pink, and the portions indicated by the boxes are magnified approximately ×5 in the insets. The F protein was not detected in samples of uninfected A549 cells (not shown). (C) RSV F protein expression from both A549 and HEp-2 cells infected with rgRSV-F was analyzed for detergent insolubility as a measure of lipid raft association. Cells were treated with 0.25% Triton X-100 at 4°C and subsequently separated on a density gradient. The lipid raft domains are shown in fractions 1 and 2 as determined by Cav-1 localization. Lanes 6, 7, and 8, are the nonraft or detergent-soluble fractions as determined by TfR localization.

FIG. 2.

Synthetic F construct design and expression. A synthetic F gene was constructed for optimal expression in mammalian cells while preserving the amino acid sequence. (A) The gene was constructed in two sections and is flanked with restriction enzyme sites and contains a cytomegalovirus promoter indicated by the arrow. S-S indicates disulfide bond. (B) The synthetic F gene product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting of HEp-2 cell lysates either infected with wild-type RSV (RSV) or transfected with the vector control (Vec), pCDNA3, or SynF gene. The F0 (uncleaved precursor) and F1 (cleaved) forms of F were detected using an anti-F1 antibody. Cell surface expression was demonstrated in HEp-2 cells by labeling cells on ice without permeabilization with an anti-RSV F antibody and analyzing by both (C) flow cytometry and (D) confocal microscopy. SynF-transfected cells (green line) were compared to RSV-infected cells (black line), and pCDNA3-transfected control cells (solid peak) were labeled with a primary antibody specific for RSV F and analyzed by flow cytometry. For each sample, 20,000 events were acquired. Surface expression of F was also seen in SynF-transfected cells that were labeled on ice with an anti-RSV F antibody (green) and analyzed by confocal microscopy. Cells were also labeled with DAPI (blue) for nuclear labeling. Images shown are optical slices from one field of cells.

FIG. 3.

RSV F colocalizes with GM1 more than with transferrin receptor in the absence of other viral proteins. Cells were transfected with the SynF gene and labeled on ice with an anti-RSV F antibody (green) and either CTxB or Tfn (red). Samples were analyzed by confocal microscopy, with colocalization shown in yellow in the overlay panel. The portions indicated by the boxes are magnified approximately ×5 in the insets.

FIG. 4.

RSV F protein membrane-proximal palmitoylation, TM domain, and CTD are not required for lipid raft association. (A) SynF sequences for TM domain and CTD mutants are listed. The final construct is a chimeric protein between the VSV G TM domain and CTD and the ectodomain of F protein. *, palmitoylation site. (B) Cells were transfected with the RSV F protein mutants, and colocalization of GM1 and the altered F proteins was analyzed by confocal microscopy. Labeling was done as described in the legend for Fig. 3, and cells were analyzed by confocal microscopy. The portions indicated by the boxes are magnified approximately ×5 in the insets.

FIG. 5.

DRM association of F protein with mutations to the palmitoylation site, TM domain, and CTD. Detergent solubility of RSV F protein was examined in cells transfected with the various F mutation constructs or with VSV G. Postnuclear supernatants were treated with 0.25% Triton X-100 at 4°C and separated on a density gradient. Subsequent blots of the fractions were probed with either an anti-RSV F antibody or an antibody specific for the CTD of VSV G (anti-VSV G), as specified to the right of the respective blots. The SynF mutant is labeled to the left of the blot. Also shown are representative blots probed with anti-Cav-1 and anti-TfR.

FIG. 6.

Transferrin receptor localization in cells expressing RSV F proteins with mutations to the TM domain and CTD. A549 cells transfected with RSV F proteins were labeled for F protein (green) and for TfR with Tfn (red) as described in the legend for Fig. 3. Samples were analyzed by confocal microscopy, and a representative image is shown for each transfection group. The portions indicated by the boxes are magnified approximately ×5 in the insets.