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. 2004 May;78(10):5079-87.
doi: 10.1128/jvi.78.10.5079-5087.2004.

Prediction and identification of a permissive epitope insertion site in the vesicular stomatitis virus glycoprotein

Lisa D Schlehuber  1 , John K Rose
Affiliations

Prediction and identification of a permissive epitope insertion site in the vesicular stomatitis virus glycoprotein

Lisa D Schlehuber et al. J Virol. 2004 May.

Abstract

We developed a rational approach to identify a site in the vesicular stomatitis virus (VSV) glycoprotein (G) that is exposed on the protein surface and tolerant of foreign epitope insertion. The foreign epitope inserted was the six-amino-acid sequence ELDKWA, a sequence in a neutralizing epitope from human immunodeficiency virus type 1. This sequence was inserted into six sites within the VSV G protein (Indiana serotype). Four sites were selected based on hydrophilicity and high sequence variability identified by sequence comparison with other vesiculovirus G proteins. The site showing the highest variability was fully tolerant of the foreign peptide insertion. G protein containing the insertion at this site folded correctly, was transported normally to the cell surface, had normal membrane fusion activity, and could reconstitute fully infectious VSV. The virus was neutralized by the human 2F5 monoclonal antibody that binds the ELDKWA epitope. Additional studies showed that this site in G protein tolerated insertion of at least 16 amino acids while retaining full infectivity. The three other insertions in somewhat less variable sequences interfered with VSV G folding and transport to the cell surface. Two additional insertions were made in a conserved sequence adjacent to a glycosylation site and near the transmembrane domain. The former blocked G-protein transport, while the latter allowed transport to the cell surface but blocked membrane fusion activity of G protein. Identification of an insertion-tolerant site in VSV G could be important in future vaccine and targeting studies, and the general principle might also be useful in other systems.

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Figures

FIG. 1.
FIG. 1.
Diagram of insertion sites in VSV G. VSV G is shown as a linear molecule inserted into the lipid bilayer. The relative positions of the six insertion sites are shown and numbered by the amino acid after which ELDKWA was inserted. The two N-linked glycosylation sites are indicated by the Ψ symbol. The computer-aligned (DNAstar; Megalign program) sequences of five serotypes of vesiculovirus G proteins around the permissive 191 site (Indiana [IND], New Jersey [NJ], Chandipura [Chandi], Cocal, and Piry) are shown. Gaps introduced into the sequences to maximize sequence alignment are indicated by dashes. The single conserved cysteine in the region is shown boxed. Abbreviations: SS, signal sequence; ECTO, ectodomain; CYT, cytoplasmic domain.
FIG. 2.
FIG. 2.
Immunoprecipitation of G-ELDKWA mutants expressed from transfected cells. BHK cells were infected with vTF7-3 and then transfected with the indicated constructs or not transfected (−). After 4 h, proteins were labeled with [35S]methionine for 1 h, and cell lysates were immunoprecipitated with polyclonal anti-VSV G antibody or with VSV G MAb I1 or I14 as indicated. Proteins were then separated by SDS-PAGE. The positions of the unprocessed form (G0) and processed form (G1) of the glycoprotein are shown to the right of the gel.
FIG. 3.
FIG. 3.
Acquisition of endoglycosidase H resistance by wt and mutant proteins. BHK cells transfected with plasmids encoding wt or mutant G proteins were labeled with [35S]methionine and then incubated in chase medium with nonradioactive methionine for the indicated number of minutes (A and B). Cell lysates were immunoprecipitated with polyclonal anti-VSV G, and samples were incubated in the presence (+) or absence (−) of endoglycosidase H (Endo H). The kinetics of acquisition of endoglycosidase H resistance of the processed glycoproteins (A) were quantitated using ImageQuant software (C). The percentage of resistance is expressed as the percent of processed G protein relative to the total G protein detected.
FIG. 4.
FIG. 4.
Indirect immunofluorescence to detect intracellular or cell surface G proteins. HeLa cells were infected with vTF7-3 and transfected with the indicated mutants. After 6 h, cells were fixed and stained with rabbit polyclonal anti-VSV serum and then with a Texas red-conjugated anti-rabbit secondary antibody to detect surface G expression. The samples were then permeabilized and incubated with guinea pig anti-VSV G serum and then with a FITC-conjugated anti-guinea pig secondary antibody to show internal G expression. Cells were visualized by indirect immunofluorescence with a Nikon Microphot FX microscope and photographed with a SPOT digital camera.
FIG. 5.
FIG. 5.
Detection of G-ELDKWA mutants by indirect immunofluorescence with MAb 2F5. BHK cells were infected with vTF7-3 and transfected with plasmids encoding the indicated proteins. After 6 h, cells were fixed and incubated with the human MAb 2F5 and then with a FITC-conjugated anti-human secondary antibody. Cells were photographed as described in the legend to Fig. 4.
FIG. 6.
FIG. 6.
Fusion activity of 191 and 464 G mutants. BHK cells were infected with vTF7-3 and then transfected with plasmid encoding either wild-type G, 191, or 464 protein. After 6 h, cells were incubated for 1 min in fusion medium at pH 5.2 and again 1 h later as described in Materials and Methods. Large syncytia (multinucleated cells) are indicated (arrows).
FIG. 7.
FIG. 7.
Indirect immunofluorescence microscopy of cells infected with VSV, VSV 191 G-ELDKWA, or VSV G-2F5e. BHK cells were infected with wt VSV or VSV 191 G-ELDKWA (A), or cells were infected with wt VSV or VSV G-2F5e (B). Cells were fixed and then incubated with mouse MAbs I1 and I14 to VSV G (α-G MAbs) and with the human MAb 2F5 as indicated. Cells were then incubated with a Texas red-conjugated anti-mouse antibody and a FITC-conjugated anti-human antibody. Cells were photographed as described in the legend to Fig. 4.
FIG. 8.
FIG. 8.
Neutralization of VSV-ELDKWA by MAb 2F5. Approximately 100 PFU of wt VSV or VSV-ELDKWA were incubated with the indicated concentrations of MAb 2F5 for 1 h at 37°C before addition to BHK cells. Cells were then overlaid with DMEM containing methylcellulose. After 48 h, cells were stained with crystal violet, and plaques were counted. Results are expressed as a percentage of the titer obtained with no antibody.
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