Cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion

Abstract

Preparations of purified full-length fusion (F) protein of human respiratory syncytial virus (HRSV) expressed in recombinant vaccinia-F infected cells, or of an anchorless mutant (F(TM(-))) lacking the C-terminal 50 amino acids secreted from vaccinia-F(TM(-))-infected cells contain a minor polypeptide that is an intermediate product of proteolytic processing of the F protein precursor F0. N-terminal sequencing of the intermediate demonstrated that it is generated by cleavage at a furin-motif, residues 106-109 of the F sequence. By contrast, the F1 N terminus derives from cleavage at residue 137 of F0 which is also C-terminal to a furin recognition site at residues 131-136. Site-directed mutagenesis indicates that processing of F0 protein involves independent cleavage at both sites. Both cleavages are required for the F protein to be active in membrane fusion as judged by syncytia formation, and they allow changes in F structure from cone- to lollipop-shaped spikes and the formation of rosettes by anchorless F.

Figure 1

Identification of two cleavage sites in anchorless F protein. (A) Diagram of the F protein primary structure denoting cysteine residues (●), potential glycosylation sites (▴), hybrophobic regions (■), heptad repeat sequences (▨) and cleavage sites by furin-like proteases (↓). Inserted is a partial amino acid sequence of the F protein where the furin-sequence motifs (boldface) and the cleavage sites I and II are indicated. (B) Western blot of purified F_TM⁻ developed with anti-F_255–275 and anti-F_104–117 sera. The location of bands is shown at right (see text for explanation). (C) Coomassie blue-stained poly(vinylidene difluoride) membrane of purified F_TM⁻ after SDS/PAGE. N-terminal sequences of F_Δ1–109 and F1 bands, determined by Edman degradation (see Materials and Methods), are shown at right. The band with the asterisk was identified as a contaminant of bovine apolipoprotein I by N-terminal sequencing. The F0 and F2 bands yielded no sequence. (D) The mass spectrum of tryptic peptides of the F2 band denotes peaks with masses matching the expected values of peptides from F2 (arrows). (Insert) Magnified spectrum of peptides N88-R106 and R88-A107 denoting by-products with oxidized methionines (+16 m/z) and metastable degradation products (Δ56.3 m/z) of these peptides. The location of the F2 peptides in the F primary structure is shown in A, below the protein diagram.

Figure 2

Immunoblots of full-length and anchorless F protein preparations. (A) Purified HRSV virus or F protein purified from extracts of HEp-2 cells infected with either HRSV or VAC-F or F_TM⁻ protein purified from the supernatant of HEp-2 cells infected with VAC-F_TM⁻ were blotted with the anti-F_255–275 serum. The amount of each sample was standardized to yield F1 bands of similar intensity. (B) The same protein samples from A were blotted with the anti-F2_104–117 serum. The amount of each sample loaded in the gel relative to the blot of part A was as follows: purified virus, 10:1; F(HRSV), 2:1; F(VAC-F), 1:1; F(VAC-F_TM⁻), 1:1.

Figure 3

Analysis of F-protein mutants with changes at sites I or II. (A) HEp-2 cells were infected with vaccinia recombinants (multiplicity of infection, 5 plaque-forming units per cell) expressing the F_TM⁻ protein with the wild-type sequence or with the mutations R109N, R108N, or R108N/R109N. Cells were labeled from 4 to 24 h after infection with [³⁵S]methionine (100 μCi/ml). Cell supernatants were harvested at the end of the labeling period and aliquots were immunoprecipitated with a monoclonal antibody (47F) specific for the F1 chain (17). Proteins were resolved by SDS/PAGE and visualized by fluorography. (B) Immunoprecipitate of cell culture supernatants from cells infected as indicated in A, but labeled with [³H]glucosamine (100 μCi/ml) from 4 to 24 h after infection. (C) HEp-2 cells were infected with vaccinia recombinants expressing either wild-type F or the same mutants from A but in the full-length protein. Cells were labeled from 4 to 24 h after infections with [³⁵S]methionine, and extracts were immunoprecipitated as in A. (D) HEp-2 cells were infected with the vaccinia virus MVA-T7 (26) and transfected with pTM1 plasmids carrying either the full-length F gene or mutants K131Q or Δ131–134, by using Lipofectin (23). Extracts were made 48 h after infection and analyzed by SDS/PAGE and Western blotting with monoclonal antibody 47F.

Figure 4

Changes associated with trypsin digestion of purified F protein. Aliquots of purified F_TM⁻ (1 μg) were incubated with the indicated amounts of trypsin for 60 min at 37°C. Subsequently, a large excess of sample buffer was added and the proteins were resolved by SDS/PAGE and blotted with the anti-F_255–275 (A) or anti-F_104–117 (B) sera. (C) Samples of F_TM⁻ (≈100 μg) were digested with trypsin in PBS (equivalent to the conditions of the 250-ng lane of A), or mock-digested. Then, one-fifth volume of a trypsin inhibitor (complete miniEDTA, Roche Diagnostics) was added and the samples were loaded on top of a 10–25% sucrose gradient and centrifuged as indicated in Materials and Methods. Fractions (1-ml) were analyzed by Western blotting with the anti-F_255–275 serum. Pools of fractions from the gradients shown at the top (fractions 4–7) or at the bottom (fractions 6–9) of C were observed under the electron microscope by negative staining (D and E, respectively). A diagram of the changes observed in F_TM⁻ molecules is shown at right.

Figure 5

Syncytia formation in BSR-T7/5 cells transfected with pTM1 plasmids carrying wild type or mutant full-length F genes. Cells were transfected with the indicated plasmids and processed for immunofluorescence 40 h later with an antibody specific for the F1 chain, as indicated in Materials and Methods. (Magnification, ×200.)