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. 2003 Dec;77(23):12807-18.
doi: 10.1128/jvi.77.23.12807-12818.2003.

The membrane-proximal region of vesicular stomatitis virus glycoprotein G ectodomain is critical for fusion and virus infectivity

E Jeetendra  1 , Kakoli Ghosh, Derek Odell, Jin Li, Hara P Ghosh, Michael A Whitt
Affiliations

The membrane-proximal region of vesicular stomatitis virus glycoprotein G ectodomain is critical for fusion and virus infectivity

E Jeetendra et al. J Virol. 2003 Dec.

Abstract

The glycoprotein (G) of vesicular stomatitis virus (VSV) is responsible for binding of virus to cells and for mediating virus entry following endocytosis by inducing fusion of the viral envelope with the endosomal membrane. The fusion peptide of G is internal (residues 116 to 137) and exhibits characteristics similar to those of other internal fusion peptides, but recent studies have implicated the region adjacent to the transmembrane domain as also being important for G-mediated membrane fusion. Sequence alignment of the membrane-proximal region of G from several different vesiculoviruses revealed that this domain is highly conserved, suggesting that it is important for G function. Mutational analysis was used to show that this region is not essential for G protein oligomerization, transport to the cell surface, or incorporation into virus particles but that it is essential for acid-induced membrane fusion activity and for virus infectivity. Deletion of the 13 membrane-proximal amino acids (N449 to W461) dramatically reduced cell-cell fusion activity and reduced virus infectivity approximately 100-fold, but mutation of conserved aromatic residues (W457, F458, and W461) either singly or together had only modest effects on cell-cell fusion activity; recombinant virus encoding these mutants replicated as efficiently as wild-type (WT) VSV. Insertion of heterologous sequences in the juxtamembrane region completely abolished membrane fusion activity and virus infectivity, as did deletion of residues F440 to N449. The insertion mutants showed some changes in pH-dependent conformational changes and in virus binding, which could partially explain the defects in membrane fusion activity, but all the other mutants were similar to WT G with respect to conformational changes and virus binding. These data support the hypothesis that the membrane-proximal domain contributes to G-mediated membrane fusion activity, yet the conserved aromatic residues are not essential for membrane fusion or virus infectivity.

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Figures

FIG. 1.
FIG. 1.
Sequence alignment of the membrane-proximal domains of vesiculovirus glycoproteins. The sequences shown are from San Juan (accession number J02428) (37) and Orsay strains; a VSV Indiana strain (accession number M11048) (19); a VSV New Jersey strain (accession number J02433) (18); a Cocal virus strain (accession number AF045556) (2); a Chandipura virus strain (accession number J04350) (28); a Piry virus strain (accession number D26175) (4); and a spring viremia of carp virus strain (accession number U18101) (3). Residues represented by black characters with a light-gray background were conserved among all the vesiculoviruses. Residues represented by white characters with a black background were identical in the virus sequences examined. Black characters with a dark-gray background indicate residues with similar properties. Stars at the bottom of the sequences represent residues that were invariant across the sequences examined.
FIG. 2.
FIG. 2.
Schematic representation of mutations in the membrane-proximal stem region of VSV G. A linear diagram of the full-length G protein (with the ectodomain, juxtamembrane GS region, TM, and cytoplasmic domains demarcated) is shown at the top of the panel. The sequence of the 42-aa stem region is also shown. The numbers at the beginning and end of the sequence indicate the positions of the amino acid residues from the N terminus of VSV GIND (San Juan strain). Amino acid K462 is believed to be the boundary between the TM domain and ectodomain (37). Mutations in the conserved tryptophan (W) (positions 457 and 461), the glutamic acid (E452), the glycine (G456), and the phenylalanine (F458) residues are shown. The sequences of the insertion, deletion, and inverted sequence mutants are also shown. The protein G(AXB) introduces two additional serines at the ectodomain TM junction.
FIG. 3.
FIG. 3.
Expression and stability of the mutant proteins. COS-1 cells were transfected with plasmids encoding the indicated G proteins, and the proteins were labeled with [35S]Met and then analyzed by immunoprecipitation with a polyclonal anti-G antibody followed by SDS-PAGE. (A) Substitution mutants and WT G protein (WT-G). (B) Deletion and insertion mutants. The lanes labeled VSV represent immunoprecipitated proteins from cells that were infected with WT VSV and labeled with [35S]Met. The positions of the G and N proteins are indicated.
FIG. 4.
FIG. 4.
Transport kinetics of WT and mutant G proteins. BHK-21 cells expressing WT G or the mutant proteins were labeled with [35S]Met for 15 min. The medium was removed, and medium containing excess unlabeled Met was added for 0, 10, 30, or 60 min. The G proteins were immunoprecipitated from cell lysates using an anti-G tail peptide antibody. One-half of the immunoprecipitates was digested with endo H. Proteins were resolved on an SDS-10% PAGE gel and visualized by fluorography. The amounts of endo H-resistant and -sensitive forms of the proteins were quantified using ImageQuant software (Molecular Dynamics). (A) Results of an experiment examining WT, GΔ13, and Gsrev11 proteins. (B) Results from a separate experiment comparing WT, G10DAF, ΔF440-N449, and GΔ9-10DAF proteins.
FIG. 5.
FIG. 5.
Syncytium formation assays. Approximately 5 × 105 BHK-21 cells were infected at a multiplicity of infection of 10 for 1 h at 37°C. At 6 h postinfection, the cells were treated with fusion medium buffered to pH 5.9, 5.5, or 5.2 for 1 min at room temperature. The medium was replaced with DMEM-5% FBS, and the cultures were incubated at 37°C for 20 min to 1 h. Cells were then fixed and processed for indirect immunofluorescence using a G-specific MAb (I1). Rhodamine-conjugated goat anti-mouse antibody was used as the secondary antibody. Fluorescence and phase-contrast images were digitally captured using a Zeiss Axiocam fitted on a Zeiss Axiophot microscope with a 10× water-immersible ceramic objective. The images were then processed using Adobe Photoshop to adjust for brightness and contrast. (A) Syncytium formation induced in cells infected with rVSV-WT, -GΔ9, -GΔ13, and -GΔ9-10DAF strains after treatment with fusion medium buffered to pH 5.9. The arrows point to small syncytia in the mutant-infected cells. (B) Cells infected with rVSV-ΔF440-N449, -G10DAF, and -G(+9)gBG after treatment at pH 5.2. Upper and lower panels correspond to fluorescence and phase-contrast images, respectively.
FIG. 6.
FIG. 6.
Virus infectivity assay. BHK-21 cells were infected with either WT or G-complemented mutant viruses at a multiplicity of infection of 10 for 1 h; the cells were then washed three times with growth medium. At 16 h postinfection, an aliquot of the supernatant was taken and plaque assays on BHK-21 cells were used to determine virus titers. At 36 h postinfection, the number of plaques were counted and averaged between at least two dilutions to determine the titers. Virus titers shown are averages from three independent experiments. Standard deviations are also shown.
FIG. 7.
FIG. 7.
Incorporation of WT and mutant G proteins in virions. BHK-21 cells were infected with viruses encoding the WT or mutant proteins at a multiplicity of infection of 10 as described in the legend to Fig. 5. At 16 h postinfection, virus released into the supernatant was pelleted through a 20% sucrose cushion. The viral pellets were resuspended in sample buffer, and the proteins from one-fifth of the viral pellets were resolved by SDS-PAGE. The proteins were visualized by staining with Coomassie blue. Digital images of the gels were obtained using a Nikon camera with a 35- to 80-mm-focal-length Nikkor lens. Protein amounts were quantified by densitometry using Image Quant software (Molecular Dynamics). Relative amounts of G protein incorporated into virions were determined by calculating the ratio of G protein to N protein. The results are expressed as percentages relative to the G-to-N ratio found in the WT VSV control.
FIG. 8.
FIG. 8.
Virus binding assay. Radiolabeled virions (∼80,000 cpm) were resuspended in binding medium buffered to pH 7.0 or 5.9 and incubated at room temperature for 30 min. The suspensions were cooled on ice for 10 min and then added to prechilled confluent monolayers of BHK-21 cells. Virus binding was done for 3 h on ice. The medium was removed, and the amount of radioactivity was determined. This represented the unbound virus fraction. The cells were then washed three times with ice-cold binding buffer at the same pH used for binding, and the washes were collected for quantitation. Cells were lysed in PBS containing 1% Triton X-100, and the amount of radioactivity in the lysates (bound fraction) was determined. Virus binding was expressed as the percentage of the bound virus compared to the total input virus.
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