Molecular Architecture of the Bipartite Fusion Loops of Vesicular Stomatitis Virus Glycoprotein G, a Class III Viral Fusion Protein

The glycoprotein of vesicular stomatitis virus (VSV G) mediates fusion of the viral envelope with the host cell, with the conformational changes that mediate VSV G fusion activation occurring in a reversible, low pH-dependent manner. Based on its novel structure, VSV G has been classified as class III viral fusion protein, having a predicted bipartite fusion domain comprising residues Trp-72, Tyr-73, Tyr-116, and Ala-117 that interacts with the host cell membrane to initiate the fusion reaction. Here, we carried out a systematic mutagenesis study of the predicted VSV G fusion loops, to investigate the functional role of the fusion domain. Using assays of low pH-induced cell-cell fusion and infection studies of mutant VSV G incorporated into viral particles, we show a fundamental role for the bipartite fusion domain. We show that Trp-72 is a critical residue for VSV G-mediated membrane fusion. Trp-72 could only tolerate mutation to a phenylalanine residue, which allowed only limited fusion. Tyr-73 and Tyr-116 could be mutated to other aromatic residues without major effect but could not tolerate any other substitution. Ala-117 was a less critical residue, with only charged residues unable to allow fusion activation. These data represent a functional analysis of predicted bipartite fusion loops of VSV G, a founder member of the class III family of viral fusion proteins. The glycoprotein of vesicular stomatitis virus (VSV G) mediates fusion of the viral envelope with the host cell, with the conformational changes that mediate VSV G fusion activation occurring in a reversible, low pH-dependent manner. Based on its novel structure, VSV G has been classified as class III viral fusion protein, having a predicted bipartite fusion domain comprising residues Trp-72, Tyr-73, Tyr-116, and Ala-117 that interacts with the host cell membrane to initiate the fusion reaction. Here, we carried out a systematic mutagenesis study of the predicted VSV G fusion loops, to investigate the functional role of the fusion domain. Using assays of low pH-induced cell-cell fusion and infection studies of mutant VSV G incorporated into viral particles, we show a fundamental role for the bipartite fusion domain. We show that Trp-72 is a critical residue for VSV G-mediated membrane fusion. Trp-72 could only tolerate mutation to a phenylalanine residue, which allowed only limited fusion. Tyr-73 and Tyr-116 could be mutated to other aromatic residues without major effect but could not tolerate any other substitution. Ala-117 was a less critical residue, with only charged residues unable to allow fusion activation. These data represent a functional analysis of predicted bipartite fusion loops of VSV G, a founder member of the class III family of viral fusion proteins. Vesicular stomatitis virus (VSV) 2The abbreviations used are:VSV Gglycoprotein of vesicular stomatitis virusPBSphosphate-buffered salineMES4-morpholineethanesulfonic acidMLVmurine leukemia virusgBglycoprotein BHVS-1herpes simplex virus 1. is a prototypic virus in the Rhabdoviridae, which includes many important human, animal, and plant pathogens, including Rabies virus (1Lyles D.S. Rupprecht C.E. Knipe D.M. Howley P.M. 5th Ed. eds). Fields Virology. Lippincott Williams & Wilkins, Philadelphia2007: 1363-1408Google Scholar). VSV is an enveloped virus that is well known to infect cells via a low pH-dependent fusion reaction within endosomes (2Le Blanc I. Luyet P.P. Pons V. Ferguson C. Emans N. Petiot A. Mayran N. Demaurex N. Faure J. Sadoul R. Parton R.G. Gruenberg J. Nat. Cell Biol. 2005; 7: 653-664Crossref PubMed Scopus (262) Google Scholar, 3Regan A.D. Whittaker G.R. Pohlmann S. Simmons G. eds. Viral Entry into Host Cells. Landes Bioscience, Austin, TX, in press2008Google Scholar, 4Sun X. Yau V.K. Briggs B.J. Whittaker G.R. Virology. 2005; 338: 53-60Crossref PubMed Scopus (191) Google Scholar). The virus contains a single envelope protein, termed the glycoprotein (G), which mediates both attachment to host cells and fusion between the virus envelope and the host cell membrane. This fusion event delivers the VSV genome into the host cell for viral replication. In addition to being a critical determinant of viral pathogenesis, VSV G has also served as an important model for protein folding and transport through the secretory pathway of cells (5Hammond C. Helenius A. Curr. Opin. Cell Biol. 1995; 7: 523-529Crossref PubMed Scopus (589) Google Scholar). VSV G is used extensively in pseudotyped virus systems and as a delivery system for gene therapy applications (6Cronin J. Zhang X.Y. Reiser J. Curr. Gene Ther. 2005; 5: 387-398Crossref PubMed Scopus (386) Google Scholar), and, due to the fact that VSV preferentially replicates and destroys immortalized or tumorigenic cells, the virus has been of great interest as an oncolytic agent in anticancer treatment (7Duntsch C.D. Zhou Q. Jayakar H.R. Weimar J.D. Robertson J.H. Pfeffer L.M. Wang L. Xiang Z. Whitt M.A. J. Neurosurg. 2004; 100: 1049-1059Crossref PubMed Scopus (27) Google Scholar). glycoprotein of vesicular stomatitis virus phosphate-buffered saline 4-morpholineethanesulfonic acid murine leukemia virus glycoprotein B herpes simplex virus 1. The mature VSV G protein is an ∼65-kDa type I transmembrane protein containing 511 amino acids that oligomerizes into a homotrimer during transport to the cell surface, where the trimer is then assembled into the viral particle (8Jayakar H.R. Jeetendra E. Whitt M.A. Virus Res. 2004; 106: 117-132Crossref PubMed Scopus (102) Google Scholar). Unlike many other viral glycoproteins (9Klenk H.-D. Garten W. Wimmer E. Cellular Receptors for Animal Viruses. Cold Spring Harbor Press, Cold Spring Harbor, NY1994: 241-280Google Scholar), VSV G is not subject to proteolytic priming for fusion activation. The fusogenic ability of VSV G has been of significant interest, because, unlike the proposed "spring-loaded" and essentially irreversible metastable state for the pre-fusion state of other fusion proteins such as influenza HA strain X-31 (10Carr C.M. Kim P.S. Cell. 1993; 73: 823-832Abstract Full Text PDF PubMed Scopus (791) Google Scholar), VSV G is apparently fully reversible for fusion activation; it exists in a dynamic equilibrium between the pre-fusion and post-fusion states (11Gaudin Y. Subcell. Biochem. 2000; 34: 379-408Crossref PubMed Google Scholar). In addition, whereas most viral fusion proteins can be categorized into an obvious structural group, either class I or class II (12Colman P.M. Lawrence M.C. Nat. Rev. Mol. Cell. Biol. 2003; 4: 309-319Crossref PubMed Scopus (387) Google Scholar, 13Kielian M. Virology. 2006; 344: 38-47Crossref PubMed Scopus (156) Google Scholar), the distinct structural features of VSV G (14Roche S. Bressanelli S. Rey F.A. Gaudin Y. Science. 2006; 313: 187-191Crossref PubMed Scopus (354) Google Scholar, 15Roche S. Rey F.A. Gaudin Y. Bressanelli S. Science. 2007; 315: 843-848Crossref PubMed Scopus (286) Google Scholar) have resulted in it being considered a novel "class III" fusion protein (16Weissenhorn W. Hinz A. Gaudin Y. FEBS Lett. 2007; 581: 2150-2155Crossref PubMed Scopus (182) Google Scholar). A critical feature of any viral fusion protein is the so-called "fusion peptide" (17Earp L.J. Delos S.E. Park H.E. White J.M. Curr. Top. Microbiol. Immunol. 2005; 285: 25-66PubMed Google Scholar), which inserts into the target membrane and is instrumental in initiating the merging of the two lipid bilayers (18Durell S.R. Martin I. Ruysschaert J.M. Shai Y. Blumenthal R. Mol. Membr. Biol. 1997; 14: 97-112Crossref PubMed Scopus (191) Google Scholar). In class I fusion proteins (e.g. influenza hemagglutinin and paramyxovirus F), the fusion peptide is a linear sequence that, in the case of influenza HA, is externalized by proteolytic cleavage and comprises a short kinked α-helix (19Han X. Bushweller J.H. Cafiso D.S. Tamm L.K. Nat. Struct. Biol. 2001; 8: 715-720Crossref PubMed Scopus (406) Google Scholar). In class II fusion proteins, e.g. Semliki Forest virus and tick-borne encephalitis virus, the fusion peptide comprises an internal loop at one end of the fusion domain (20Allison S.L. Schalich J. Stiasny K. Mandl C.W. Heinz F.X. J. Virol. 2001; 75: 4268-4275Crossref PubMed Scopus (275) Google Scholar). Prior to determination of its x-ray structure, the identification of the VSV G fusion peptide was challenging. Initial attempts to understand membrane fusion utilized hydrophobic photolabeling and demonstrated that VSV was able to interact with the host cell membrane in response to low pH, and that residues 59-221 of the G protein were in close proximity to the membrane during this process (21Durrer P. Gaudin Y. Ruigrok R.W. Graf R. Brunner J. J. Biol. Chem. 1995; 270: 17575-17581Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Mutational analysis of VSV G demonstrated that modification of a highly conserved region (residues 118-139) abolished fusion activity or modified the pH of fusion activation (22Fredericksen B.L. Whitt M.A. J. Virol. 1995; 69: 1435-1443Crossref PubMed Google Scholar, 23Li Y. Drone C. Sat E. Ghosh H.P. J. Virol. 1993; 67: 4070-4077Crossref PubMed Google Scholar, 24Zhang L. Ghosh H.P. J. Virol. 1994; 68: 2186-2193Crossref PubMed Google Scholar); in particular, the mutations G124A, P127G/L, and A133K dramatically decreased cell-cell fusion activity. Other mutations at this region, such as F125Y and D137N, shifted the optimum pH for G protein-mediated cell-cell fusion. Overall, the region between amino acids 118 and 139 was generally considered to represent an internal fusion peptide for VSV G (17Earp L.J. Delos S.E. Park H.E. White J.M. Curr. Top. Microbiol. Immunol. 2005; 285: 25-66PubMed Google Scholar). However, other studies demonstrated that amino acids 395-418 (the membrane-proximal region) have an significant influence on fusion (25Shokralla S. He Y. Wanas E. Ghosh H. Virology. 1998; 75: 39-50Crossref Scopus (33) Google Scholar), and additional studies identified region 145-164, termed the p2-like peptide, as being a pivotal domain in facilitating glycoprotein G-mediated membrane fusion (26Carneiro F.A. Lapido-Loureiro P.A. Cordo S.M. Stauffer F. Weissmuller G. Bianconi M.L. Juliano M.A. Juliano L. Bisch P.M. Poian A.T. Eur. Biophys. J. 2006; 35: 145-154Crossref PubMed Scopus (39) Google Scholar, 27Carneiro F.A. Stauffer F. Lima C.S. Juliano M.A. Juliano L. Poian Da A.T. J. Biol. Chem. 2003; 278: 13789-13794Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). A synthetic p2 peptide was shown to mediate liposome-liposome fusion in a low pH- and phosphatidylserine-dependent manner (27Carneiro F.A. Stauffer F. Lima C.S. Juliano M.A. Juliano L. Poian Da A.T. J. Biol. Chem. 2003; 278: 13789-13794Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Further studies revealed that the action of the p2-like peptide was specifically facilitated by electrostatic interactions between phosphatidylserine and two histidine residues within this region (26Carneiro F.A. Lapido-Loureiro P.A. Cordo S.M. Stauffer F. Weissmuller G. Bianconi M.L. Juliano M.A. Juliano L. Bisch P.M. Poian A.T. Eur. Biophys. J. 2006; 35: 145-154Crossref PubMed Scopus (39) Google Scholar, 28Da Poian A.T. Carneiro F.A. Stauffer F. Braz. J. Med. Biol. Res. 2005; 38: 813-823Crossref PubMed Scopus (30) Google Scholar). Unlike for some other viral fusion proteins, notably Semliki Forest virus E1 (29Ahn A. Gibbons D.L. Kielian M. J. Virol. 2002; 76: 3267-3275Crossref PubMed Scopus (109) Google Scholar), there is no apparent requirement for interaction of the VSV G fusion loops with specific lipids (30Teissier E. Pecheur E.I. Eur. Biophys. J. 2007; 36: 887-899Crossref PubMed Scopus (81) Google Scholar), although VSV G-mediated binding to and fusion with liposomes is enhanced by the presence of acidic phospholipid head groups (e.g. phosphatidylserine) and cholesterol (31Yamada S. Ohnishi S. Biochemistry. 1986; 25: 3703-3708Crossref PubMed Scopus (54) Google Scholar). With the availability of high resolution x-ray structures for both the pre- and post-fusion forms of the VSV G ectodomain (amino acids 1-410), it was subsequently suggested that domain IV of VSV G comprises the fusion domain (14Roche S. Bressanelli S. Rey F.A. Gaudin Y. Science. 2006; 313: 187-191Crossref PubMed Scopus (354) Google Scholar, 15Roche S. Rey F.A. Gaudin Y. Bressanelli S. Science. 2007; 315: 843-848Crossref PubMed Scopus (286) Google Scholar), because it has considerable structural similarity to the fusion domains of class II fusion proteins. Domain IV (residues 51-180) of VSV G is composed of an extended β-sheet with two obvious loops at one end and has a conserved set of four hydrophobic residues exposed at the end of the loops (Trp-72, Tyr-73, Tyr-116, and Ala-117) (Fig. 1). The hydrophobic nature of this bipartite fusion loop is conserved across a wide range of divergent rhabdoviruses (Fig. 1). This arrangement of hydrophobic loops is highly reminiscent of the fusion peptide (or fusion loop) of a class II fusion protein (20Allison S.L. Schalich J. Stiasny K. Mandl C.W. Heinz F.X. J. Virol. 2001; 75: 4268-4275Crossref PubMed Scopus (275) Google Scholar). However, a significant difference for VSV G is that the hydrophobic amino acids are shared over two non-contiguous loops, whereas for class II fusion proteins the key amino acids are on a contiguous stretch of primary sequence. In support of the suggestion that this bipartite loop comprises a critical fusion domain, selection of fusion-defective mutants previously identified one of these amino acids (Ala-117 in the second loop region) as having a critical function in VSV G-mediated membrane fusion (22Fredericksen B.L. Whitt M.A. J. Virol. 1995; 69: 1435-1443Crossref PubMed Google Scholar). This mutation was previously referred to as an A133K substitution due to different numbering of amino acids in the G protein sequence. Based on the available x-ray structure of VSV G, here we systematically mutated the proposed bipartite fusion loop of VSV G (residues Trp-72, Tyr-73, Tyr-116, and Ala-117) and characterized how these amino acids modulate membrane fusion activity. These data represent an analysis of the molecular architecture of the fusion-active loops of VSV G, a founder member of the class III family of viral fusion proteins. Cell Culture—Vero E6 cells and 293T cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium (Cellgro) containing 10% fetal bovine serum, 100 units/ml penicillin, and 10 μg/ml streptomycin. Plasmids and Site-directed Mutagenesis—Plasmid phCMV-VSV G encoding the vesicular stomatitis virus Indiana glycoprotein (VSV G) (NCBI accession number CAC47944) (32Beyer W.R. Westphal M. Ostertag W. Laer von D. J. Virol. 2002; 76: 1488-1495Crossref PubMed Scopus (151) Google Scholar), was kindly provided by Dr. Jean Dubuisson (Institut Pasteur de Lille, Lille Cedex, France). Site-directed mutagenesis using phCMV-VSV G as a template was performed using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). According to the manufacturer's protocol, pairs of complementary oligonucleotides were designed to introduce the desired mutations. Mutations were then confirmed by sequencing using an Applied Biosystems Automated 3730 DNA Analyzer at the Cornell University Life Sciences Core Laboratories Center. Biotinylation of Surface Protein and Immunoprecipitation—Vero E6 cells grown on 6-well plates were transfected with 1 μg of wild-type or mutant VSV G-expressing plasmid, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, for 24 h at 37 °C, or for 36 h at 32 °C. For cell-surface biotinylation, the transfected cells were washed twice with ice-cold phosphate-buffered saline (PBS), and then cells were labeled with 250 μg/ml Sulfo-NHS-SS-biotin (Pierce) for 30 min on ice. The biotin-labeled cells were then added to 150 mm ice-cold glycine solution for 20 min to quench unlabeled free biotin followed by an ice-cold PBS wash. The cells were then lysed in 500 μl of radioimmune precipitation assay buffer (100 mm Tris-HCl, 150 mm NaCl, 0.1% SDS, 1% Triton X-100, 1% deoxylcholic acid, pH 7.4), including complete protease inhibitor mixture (Roche Applied Science), and the cell lysates were affinity-purified using immobilized NeutrAvidin beads (Pierce) overnight at 4 °C. Finally, the NeutrAvidin beads were washed with radioimmune precipitation assay buffer followed by the addition of SDS-PAGE Laemmli sample loading buffer containing 50 mm dithiothreitol. The surface-biotinylated VSV G protein was analyzed by Western blot using the anti-VSV G monoclonal antibody P5D4 (kindly provided by Dr. Ari Helenius, ETH-Zurich), and images were obtained from LAS-3000 mini Fujifilm imaging system (Fuji Photo Film Co., Ltd). The biotinylation assay was repeated three times, and the results obtained in the Western blot were quantified using IP Lab software (Scanalytics) and plotted in Sigma Plot 9.0 (Systat Software). Syncytium Formation Assay and Luciferase Reporter Gene Fusion Assay—Subconfluent Vero cells in 24-well plates were transfected with phCMV-VSV G plasmids encoding either wild-type or mutant G protein, using Lipofectamine 2000, for 24 h at 37 °C or 32 °C before induction of syncytium formation. To examine low pH-induced syncytia formation, the transfected cells were rinsed once with PBS and then incubated with HMSS fusion buffer (5 mm HEPES, 5 mm MES, 5 mm sodium succinate 150 mm NaCl, adjusted to the indicated pH with HCl, stored at room temperature) for 1 min. The cells were then washed with PBS and incubated with fresh culture medium for another 3 h after fusion induction. Finally, the cells were fixed with 3% paraformaldehyde, and without being permeabilized, the cells were processed for indirect immunofluorescence microscopy using the anti-VSV G ectodomain-specific monoclonal antibody I 14 (clone 1E9F9) (33Lefrancois L. Lyles D.S. J. Immunol. 1983; 130: 394-398PubMed Google Scholar) (a kind gift from Dr. Ari Helenius, ETH-Zurich) and Alexa 488-labeled anti-mouse secondary antibody (Invitrogen-Molecular Probes). Using a 20× Plan Apo objective (numerical aperture, 0.75). Images were captured with a Sensicam EM camera (Cooke Corp.) using IP Lab software (Scanalytics). For quantification of syncytium formation, the percentage of cells involved in syncytia was determined by counting the ratio of cells in syncytia containing three or more nuclei to total cells in the field. The experiments were repeated three times, and >500 cells from at least 5 different fields were counted in each experiment. Cell-cell fusion mediated by G protein was also quantified by a luciferase reporter gene assay, with plasmids kindly provided by Dr. Tom Gallagher, Loyola University, Chicago, IL. In brief, 293T cells in each well of a 24-well plate were co-transfected with 125 ng of plasmid encoding luciferase cDNA under control of T7 promoter and 125 ng of VSV G wild-type or mutant plasmids, using 1 μl of Exgen 500 (Fermentas, Ontario, Canada) at either 37 °C or 32 °C depending on the mutants used for transfection. 24 h post-transfection, transfected 293T cells were overlaid at a 1:3 ratio with 293T cells, which had been previously transfected with a plasmid encoding T7 polymerase. The 293T cell mixtures were cultured for 1 h to allow cells to adhere to the plate. Cell-cell fusion was induced with low pH buffer as described above. The cells were lysed 4 h post-fusion, and the supernatants were measured for luciferase activity using the Luciferase Assay System (Promega, Madison WI), according to the manufacturer's instructions. Light emission was measured using a Glomax 20/20 luminometer (Promega, Madison, WI). Production and Transduction of VSV G-pseudotyped Virions—VSV G protein-pseudotyped particles were generated from a murine leukemia virus (MLV)-based transfer vector system as described previously (34Bartosch B. Dubuisson J. Cosset F.L. J. Exp. Med. 2003; 197: 633-642Crossref PubMed Scopus (957) Google Scholar, 35Blanchard E. Belouzard S. Goueslain L. Wakita T. Dubuisson J. Wychowski C. Rouille Y. J. Virol. 2006; 80: 6964-6972Crossref PubMed Scopus (442) Google Scholar) with plasmid-encoding luciferase flanked by retroviral packaging sequences and an MLV Gag-Pol construct were kindly provided by Dr. Jean Dubuisson (Institut Pasteur de Lille, Lille Cedex, France). The phCMV-VSV G wild-type or mutant plasmids, together with the plasmids encoding luciferase, murine leukemia virus Gag-Pol, were transfected into 293T cells using Exgen 500 (Fermentas) according to the manufacturer's instructions. The transfected cells were then cultured at 37 °C for 48 h or 32 °C for 72 h depending on the VSV G mutants used in the transfection. The supernatants containing pseudotyped particles were harvested 48 h post-transfection and filtered through 0.45-μm-pore-sized membranes before used for infection assay. To analyze G protein incorporation into pseudotyped particles, the viral particles were concentrated by ultracentrifugation. Briefly, 700 μl of viral supernatants was layered on the top of 300 μl of 30% sucrose cushion and subject to ultracentrifugation at 50,000 rpm for 2 h in a TLA55 rotor (Beckman), before the samples were analyzed by Western blot. The monoclonal antibodies P5D4 and R187 (American Type Culture Collection), recognizing the G protein and the MLV Gag protein, respectively, were used to detect VSV G and the retroviral Gag protein in the Western blot. Transduction of pseudoparticles was performed using Vero E6 cells. For a typical infection assay, 100 μl of supernatant containing either wild-type or mutant G protein-pseudotyped MLV particles was used to infect Vero E6 cells for 72 h. The cells were lysed 72 h post-infection, and luciferase activity in the lysates was measured using the same method as described above for the luciferase-based cell-cell fusion assay. Mutagenesis of the VSV G Fusion Loops and Cell Surface Expression—To determine the functional properties underlying the molecular architecture of the VSV G fusion loops, we performed a systematic mutagenesis study of the four residues predicted to comprise the bipartite fusion loop (Trp-72, Tyr-73, Tyr-116, and Ala-117). In each case we substituted a variety of amino acids with different R groups; comprising bulky aromatic residues (e.g. Phe, Tyr, and Trp), polar uncharged residues (e.g. Asn), non-polar aliphatic residues (e.g. Val and Ala), and both positively and negatively charged residues (e.g. Arg and Asp). These mutants are shown in Table 1, along with a qualitative measure of their surface expression, using immunofluorescence microscopy of the G protein in non-permeabilized Vero cells. Mutants were qualitatively scored as being surface-expressed at medium or high levels (at least half of the level of expression of wild type), or at low or very low levels (less than half of the level of expression of wild type) (see Table 1). At 37 °C, many mutants showed low, or very low, levels of surface expression. However, in these cases surface expression could often be rescued by lowering the temperature to 32 °C. All mutants showed high expression at both 37 °C and 32 °C when total G protein was detected by immunofluorescence microscopy of permeabilized Vero cells (data not shown). Mutants with low or very low surface expression had extensive localization to an intracellular compartment (data not shown). Only those mutants that showed a qualitatively medium or high level of cell surface expression at 32 °C were considered suitable for subsequent fusion and entry assays. These mutants were tested further by quantitative assays, using a combination of cell surface biotinylation and streptavidin pull down of total cell surface material, followed by Western blotting for the G protein. These results are shown in supplemental Fig. S1. All the mutants selected for further analysis had surface expression of >50% of the wild-type at 32 °C.TABLE 1Surface expressions for amino acid substitutionsTarget amino acidAmino acid substitutionSurface expression at 37 °CSurface expression at 32 °CTarget amino acidAmino acid substitutionSurface expression at 37 °CSurface expression at 32 °CTrp-72F+++++++Tyr-116F++++++++Y++++++W++++++++V+++++V+++++A+++++A+++++T+++T+++K+++K+++Tyr-73F++++++++Ala-117K+++++++W++++++++N++++NTaNT, not tested.V++++F++++NTA++++H++++NTT+++D++++NTN+++R++++NTa NT, not tested. Open table in a new tab Cell-Cell Fusion and Luciferase Reporter Gene Assays of Wild-type and Mutant VSV G—To examine membrane fusion mediated by wild-type and mutant VSV G, we first performed cell-cell fusion assays on Vero cells followed by indirect immunofluorescence microscopy to visualize syncytia formation. Plasmids encoding wild-type or mutant G protein were transfected into Vero cells for 24 h, and then the transfected cells were treated briefly with fusion buffer at pH 6.6, pH 6.1, or pH 5.7. These pH values were chosen based on previous studies showing that, although G is optimally fusogenic at pH 5.7 and does not fuse at pH 6.6, some mutants only show changes in syncytia formation under suboptimal conditions (i.e. pH 6.1) (36Shokralla S. He Y. Wanas E. Ghosh H.P. Virology. 1998; 242: 39-50Crossref PubMed Scopus (33) Google Scholar). Using visual scoring of syncytia as a read-out of membrane fusion, the wild-type G protein did not fuse at pH 6.6, fused extensively at pH 5.7, and caused limited syncytia formation at pH 6.1 (Fig. 2). In the case of substitution of Trp-72, only small syncytia could be detected for the mutant W72F at pH 5.7, with syncytia barely detectable at pH 6.1. Essentially no syncytia formation occurred for any other substitution of Trp-72, at either pH 6.1 or 5.7. In contrast, substitutions of Tyr-73 and Tyr-116 to Trp or Phe did not dramatically affect cell-cell fusion at pH 5.7, however much more limited fusion than wild-type was apparent at pH 6.1. Substitutions of Tyr-73 and Tyr-116 to Val or Ala completely abolished fusion at both pH 6.1 and 5.7. In general, mutations at position 117 allowed efficient fusion at pH 5.7 when Ala-117 was mutated to uncharged residue (Asn or Phe), but allowed much less fusion when Ala-117 was mutated to a charged residues (Asp, Lys, His, or Arg). These data are quantified in Fig. 3.FIGURE 3Quantification of syncytium formation in cells expressing wild-type or mutant VSV G protein. The percentage of cells involved in syncytia at pH 5.7 (A) or pH 6.1 (B) was determined by counting the ratio of cells in syncytia containing three or more nuclei to total cells in the field. The experiments were repeated three times, and greater than 500 cells from at least five different fields were counted in each experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next employed luciferase as a reporter gene, to quantify the VSV G-based membrane fusion activity. In this assay, we expressed the VSV G protein in 293T cells because of their better transfection efficiency. In all cases, the expression level of the wild type and mutants was comparable to that in Vero E6 cells (data not shown). In this luciferase reporter gene cell-cell fusion assay, induction of luciferase gene expression occurs upon the fusion of cells expressing the T7 polymerase gene and cells expressing both a T7 polymerase driven-luciferase gene and VSV G. Luciferase activity after induction of fusion is used as an indicator of the fusogenic activity of G protein. Co-cultures of 293T cells transfected with wild-type or mutant G protein and T7 polymerase driven-luciferase, and 293T cells transfected with T7 polymerase, were treated with buffer at several pH values (pH 5.1, 5.4, 5.7, 5.9, 6.1, and 6.6), and luciferase activities were then measured after 4 h. These data are shown in Fig. 4, with luciferase activity at pH 6.6 normalized to 1.0 and activities at the lower pH values expressed as the -fold enhancement compared with pH 6.6. Mutation of tryptophan 72 to other residues had a marked effect on membrane fusion (Fig. 4A). At pH 5.7, only substitution of Trp-72 with phenylalanine (W72F) resulted in fusion activity (27% of the wild-type fusion level). In this case, fusion was rescued by lowering the pH, with fusion at pH 5.1 being close to the wild-type level. The substitution of tryptophan for tyrosine (Trp-72Y), also gave a marked pH shift in fusion activation, however in this case fusion at pH 5.1 was only 54% of the wild-type level. For the other mutants tested (W72V and W72A), membrane fusion was at background levels for all pH values tested. Mutation of tyrosine 73 also markedly affected membrane fusion (Fig. 4B). Both phenylalanine and tryptophan substitutions gave a pH-shifted fusion phenotype; at pH 5.1, Y73F and Y73W allowed efficient membrane fusion, although fusion activity at pH 5.7 was only 31 and 29%, respectively, of the level of the wild type. For the other mutants tested (Y73V and Y73A), membrane fusion was at background levels for all pH values tested. Although in this case we cannot exclude the possibility that the lack of fusion was due to limited cell surface expression (see supplemental Fig. S1 and Table 1). Mutation of tyrosine 116 gave essentially similar results to Tyr-73 (Fig. 4C). The Y116V and Y116A mutations resulted in background fusion, however substitution with aromatic residues was generally better tolerated; the Y116W mutation behaved essentially as wild-type, and the Y116F mutation had 66% of the fusion activity of wild type at pH 5.7. Interestingly, fusion of the Y116F mutant could not be rescued to wild-type levels by lowering the pH;