key: cord-0004889-i0a3igc7
authors: Nagata, S.; Okamoto, Y.; Inoue, T.; Ueno, Y.; Kurata, T.; Chiba, J.
title: Identification of epitopes associated with different biological activities on the glycoprotein of vesicular stomatitis virus by use of monoclonal antibodies
date: 1992
journal: Arch Virol
DOI: 10.1007/bf01309581
sha: 8b491859966fa9484f29dea1a33b0cd2abe1bfe2
doc_id: 4889
cord_uid: i0a3igc7

Thirteen monoclonal antibodies (MAbs) to the glycoprotein (G) of vesicular stomatitis virus (VSV) serotype Indiana were prepared and examined for their effects on various biological activities of VSV, including in vitro infection, hemagglutination, adsorption to cells, and mediation of cell fusion. Competitive binding assays with these MAbs revealed the presence of at least seven distinct antigenic determinants (epitopes) on the G protein. In some cases, overlappings among epitopes to various degrees were observed as partial inhibition or binding enhancement. The MAbs to all the epitopes but one (epitopes 1–6) reacted with the denatured G protein in a Western immunoblot analysis. Four of the epitopes (epitopes 2, 4, 5, and 7) were involved in neutralization and two (epitopes 1 and 2) in hemagglutination inhibition. None of the MAbs inhibited the adsorption of radiolabeled VSV to BHK-21 cells; the MAbs to epitope 2 slightly enhanced the virus adsorption. All neutralization epitopes except epitope 2 (epitopes 4, 5, and 7) were associated with inhibition of VSV-mediated cell fusion. These results show a direct spatial relationship between the epitopes recognized by the MAbs and functional sites on G protein and further insights into the structure and function of G protein.

Many enveloped viruses including vesicular stomatitis virus (VSV), family Rhabdoviridae, genus Vesiculovirus, transfer their nucleocapsids to the cytoplasm of host cells by the adsorption and receptor-mediated endocytosis, followed by fusion with the endosomal membrane [20, 21] . The glycoprotein (G) of VSV is the sole protein anchored in the viral envelope and plays a critical role in this early stage of virus infection. Many biological properties of G protein are associated with the virus entry [37] , which include adsorption to host cells [3, 6] , hemagglutination (HA) [9, 23] , and mediation of in vitro cell-cell fusion [7, 38] . The cell-cell fusion occurs only at low pH, which mimics the acidic environment of the endosomal lumen [7, 38] . As expected from its central role in infection, the G protein also gives rise to and reacts with neutralizing antibodies [12] . In recent years, much effort has been made to reveal the structurefunction relationships of the G protein, especially regarding its role in fusion [2, 10, 27, 29, 39, 40] , but the underlying molecular mechanisms are still poorly understood.

One approach to the structure-function relationship of surface glycoproteins of viruses is to analyze for the sites and effects of the monoclonal antibody (MAb) binding [4, 13, 31, 33] . Production of MAbs against G proteins of two major serotypes of VSV (Indiana and New Jersey) has been reported by two research groups [5, 14, 15, 36] . These MAbs have mainly been used to map neutralization and non-neutralization epitopes on G protein and to analyze the mutation leading to antigenic variations of G protein [8, 11, [16] [17] [18] 35] . The effects of the MAbs specifically reacting with G protein on biological functions other than neutralization have not been reported.

In the present study, we prepared thirteen MAbs specific for seven distinct epitopes on G protein of VSV-Indiana and examined for their effects on various biological activities of VSV including in vitro infection, HA, adsorption to the cells, and mediation of cell-cell fusion. Our findings defined the spatial relationship between the epitopes recognized by the MAbs and the functions of G protein.

The San Juan strain of VSV-Indiana originally provided by Dr. R. R. Wagner, University of Virginia, was obtained from Dr. K. Yamamoto, National Institute of Health, Tokyo. The virus stock was prepared by infecting BHK-21 cells (Japanese Cancer Research Resources Bank) at a multiplicity of 0.1 PFU/cell. Virus harvested at 22 h postinfection was concentrated by ultrafiltration and ultracentrifugation [22, 25] , and purified by sucrose density gradient centrifugation [22] . This preparation containing 1.1 mg/ml of viral protein (2.3 x 1011PFU/ml) was stored at -80 °C. The protein content of the preparation was determined with BCA protein assay reagent (Pierce Chemical Co., Rockford, IL, U.S.A.) with bovine serum albumin (BSA) as a standard. Virus infectivity was determined by plaquing on monolayer cultures of BHK-21 cells [41] .

The G protein was extracted from the purified virus with 30 mM octyl-13-D-glucopyranoside (Sigma Chemical Co., St. Louis, MO, U.S.A.) as described by Petri and Wagner [30] . After removal of the nucleocapsids by ultracentrifugation at 150,000 x g, the supernatant containing G protein was dialyzed against 10 mM HEPES (pH 7.4) containing 0.15 M NaCI. G protein thus obtained was free from any other virus protein in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie brilliant blue.

For immunization, female BALB/c mice were subcutaneously injected twice each with 25 gg of the purified G protein emulsified in the same quantity of Freund's complete and Freund's incomplete adjuvants, respectively. Then, additional two intraperitoneal injections with 40 gg of G protein were given. Three days before fusion for hybridoma production, the final 40 gg of G protein was injected intravenously.

The spleen cells of the immunized mice and SP 2/O-Ag 14, BALB/c mouse non-secretory plasmacytoma cells, were fused with polyethylene glycol according to Oi and Herzenberg [28] or by the novel VSV-mediated cell fusion method described previously [25, 26] . Media preparation and HAT selection of hybridomas were described previously [25] . In 2 weeks, the hybridomas were screened for production of anti-G protein antibody by enzyme-linked immunosorbent assay (ELISA) with purified G protein as the antigen (see below). The positive cultures were cloned several times by the limiting dilution method. The isotypes of the specific antibodies were determined with a mouse monoclonal antibody isotyping kit (Amersham International, Buckinghamshire, England).

Thirteen hybridomas were established and each was over-grown in about 11 of a serumfree medium (Iscove's modified Dulbecco's medium, Sigma) containing 1 mg/ml of BSA, 1 mM sodium pyruvate (Gibco, Grand Island, NY, U.S.A.), 8 ~tg/ml of bovine insulin (Sigma), 5 gg/ml of iron-saturated human transferrin (Miles Scientific, Naperville, IL, U.S.A.), 50 ~tM 2-mercaptoethanol, 20 gM ethanolamine, 2.5 ~tg/mt of linoleic acid, 2.5 ~tg/ ml of oleic acid, 2.5 gg/ml of palmitic acid, and 10 gg/ml of gentamicin. MAb in the culture supernatant was precipitated with ammonium sulfate at 40% saturation and the precipitate was further purified by high performance liquid chromatography on hydroxyapatite beads [42] or by affinity chromatography on protein G-Sepharose (Pharmacia, Uppsala, Sweden). The eluate was concentrated to 4 ml by membrane ultrafiltration (30,000 tool. wt. cut-off Centriprep; Amicon, Danvers, MA, U.S.A.), and dialyzed against phosphate-buffered saline (PBS). The antibody concentration was determined from absorbance at 280 nm with an extinction coefficient of 1.4 per mg of protein. For use as negative controls in various assays, two MAbs prepared at National Institute of Health were purified by the same procedure. One of them was Ig G1 specific for the core antigen of feline immunodeficiency virus (unpubl.) and the other was Ig G2a specific for sheep red blood cells (not crossreactive with goose erythrocytes) [25] .

Production of anti-G protein antibody in hybridoma culture supernatants was examined by ELISA. Wells of microtiter plates (Costar 3690; Costar, Cambridge, MA, U.S.A.) were coated with the purified G protein (1 gg/ml) in 50mM sodium carbonate buffer (pH9.6) for 2h at room temperature. The wells were washed with PBS containing 0.05% (v/v) Tween 20 (PBS-Tween) and blocked overnight at 4°C with 0.5% (w/v) gelatin in PBS. After washing, each culture supernatant was added to the wells and the plates were incubated for t h at room temperature. The antibody bound was detected by incubation for 1 h at room temperature with alkaline phosphatase-conjugated goat anti-mouse IgG + M (Tago 6553; Tago Inc., Burlingame, CA, U.S.A.) diluted 5,000-fold in PBS-Tween. The enzyme reaction was started by adding 1 mg/ml of p-nitrophenylphosphate (Wako Pure Cemical Ind., Osaka, Japan) in 1% (v/v) diethanolamine (pH 9.8) containing 0.5 mM MgC12. The 156 S. Nagata et al.

absorbance at 410 nm was measured with an EIA autoreader (Sanko Junyaku Co., Tokyo, Japan). As a positive control, a 2,000-fold dilution of mouse immune serum was used. This control usually showed an absorbance of approximately 1.0 after incubation for 20 rain at room temperature. The well with absorbance higher than 0.2 was regarded as positive.

Purified MAbs were titrated by the same ELISA to compare the relative reactivities, in which the wells of the plates were coated with VSV virions (2 gg/ml) instead of G protein.

The relative reactivity was defined as the concentration of MAb needed to attain 50% of the absorbance value of the positive control.

Purified VSV was separated by SDS-PAGE in 10% polyacrylamide gel under reduced conditions. The proteins separated were then blotted onto immobilone membrane (Millipore Corp., Bedford, MA, U.S.A.) according to Towbin et al. [34] . The blots were allowed to react with culture supernatants of established hybridomas, and specific bands were visualized with alkaline phosphatase-conjugated goat anti-mouse IgG + M (Tago 6553) and the BCIP/ NBT phosphatase substrate system (Kirkegaard & Perry Lab. Inc., Gaithersburg, MD, U.S.A.).

A 500-gg portion of each purified MAb was mixed with 100gg of biotinyt N-hydroxysuccinimide ester (NHS-LC-Biotin, Pierce) in 0.5 ml of 0.1 M sodium bicarbonate (pH 8.4). After incubation for 4 h at room temperature, the mixtures were dialyzed extensively against PBS at 4 °C; BSA was then added to a final concentration of 5 mg/ml.

For competitive binding assay, the wells of plates were coated with purified VSV (2 ~tg/ ml) and blocked with 5% (w/v) unfatted bovine milk in PBS. Serial 10-fold dilutions of unlabeled competitor MAb (2 x 10-1 to 2 x 10 -5 mg/ml) were added to the wells (40 ~tl/ well) and the plates were incubated for 3 h at room temperature. Subsequently, 40 gl of biotinylated MAb was mixed with competitor MAb. The concentrations of biotinylated MAbs were 1 gg/ml for 2B9 and 3B1, 0.5 gg/ml for V20 B12, and 0.25 gg/ml for the other MAbs. These concentrations were about half-maximal in their titration curve and were within the range where the binding was linear. After incubation overnight at 4 °C, biotinylated MAb bound was detected with a 5,000-fold dilution of alkaline phosphataseconjugated streptavidin (Bethesda Research Lab., Gaithersburg, MD, U.S.A.). Dilution was made in PBS-Tween containing 5% (w/v) unfatted bovine milk. The enzyme reaction and absorbance determination were carried out as described above. All assays were performed in duplicate and the results were expressed as the percentage of binding calculated with the formula: average ofabsorbance in the presence of competitor Binding(%) =

x 100 average ofabsorbance in the absence of competitor

For the neutralization assay, the stock of VSV was diluted to a final concentration of approximately 1,000 PFU/ml with bicarbonate-free Eagle's minimum essential medium (MEM, Nissui Pharmachemical Co., Tokyo, Japan) containing 0.2 mg/ml of BSA and 20 mM HEPES (pH 7.2). The virus was mixed with an equal volume of each of serial twofold dilutions of each purified MAb (from 250 gg/ml) in the same medium. The mixtures were incubated for 1 h at 37 °C and then plated in duplicate on monolayers of BHK-21 cells in 6-well culture plates for plaque assay (100 gl/well). The neutralization antibody titer was defined as the reciprocal of the highest dilution reducing more than 50% of the plaques of the control without MAb.

Hemagglutination inhibition (HI) was assayed with 4 or 8 hemagglutinating units of VSV in V-bottom microtiter plates as described by Halonen et al. [9] , except that goose erythrocytes were used after the treatment with trypsin (Sigma) at 100 l~g/ml for 30 rain at 37 °C. This pretreatment of the erythrocytes enhanced HA, thus increasing the sensitivity of HI [19; unpubt, data] . The reciprocal of the highest dilution of purified MAb causing complete inhibition of hemagglutination was taken as the HI titer.

For adsorption assays, 35S-labeled VSV was prepared by the addition of 20 ~ Ci/ml of L-[35Slmethionine (Amersham) to the infection medium as described by Bailey et al. [1] . The radiolabeled virions were concentrated and purified as described for the unlabeled virus. The final preparation was free of contaminating labeled materials as judged by SDS-PAGE and autoradiography. The final preparation contained 3.4 x 10 l° PFU/ml (1.8 mg/ml viral protein) with a specific activity of 1.5 x 104 cpm/~tg.

The radiolabeled virus absorbed to cells was quantified essentially as described by Matlin etal. [21] . BHK-21 cells grown to confluency in 12-well culture plates (about 10 6 cells/well) were washed twice with the binding medium, bicarbonate-free MEM buffered with 20 mM HEPES (pH 7.2) containing 2 mg/ml of BSA, and cooled for 5 rain on ice. The radiolabeled purified virus (33,500cpm) was mixed with each purified MAb at various concentrations in the binding medium. The mixtures were incubated for 1 h at 37 °C, chilled, and then plated in duplicate on the BHK-21 cell (100 gl/well). After incubation for 1 h on ice, unbound virus was removed. The cells were washed four times with the binding medium. The cells bound with the virus were solubilized in 0.4 ml of Solubable (NEN Research Product, Boston, MA, U.S.A.), and its radioactivity was measured with a liquid scintillation counter. The average radioactivities bound to the cells in the presence of MAb were expressed as the percentage of the radioactivity bound in the absence of MAb. Nonspecific interaction of the virus with the ceils and the surface of the plates was minimized by adding BSA to the binding medium. The addition of BSA reduced the nonspecific binding of labeled VSV to the surface of the plates from 9.3% to less than 1.1% of the input radioactivity. The amount of virus used was within the range where the radioactivity bound to cells increased proportionally with the amount of the input virus.

The effect of MAbs on VSV-mediated cell fusion was assayed by inhibition of polykaryon formation of BHK-21 cells [24, 38] . The cells in 24-well culture plates (about 1.5 x 105 celts/well) for 18-24 h were washed twice with the ice-cold binding medium (bicarbonatefree MEM buffered with 20 mM HEPES, pH 7.2, containing 2 mg/ml of BSA). The purified virus (25 gg) in 200 gl of the cold binding medium was applied onto the cells. After incubation for 1 h on ice to allow viral adsorption, free virus was removed, and the cells were treated on ice for 45 min with 25 ~g/ml of each MAb in 200 ~.1 of the binding medium. After removing the MAb solutions, 0.5 ml of prewarmed (37 °C) acidic medium, bicarbonate-free MEM buffered with 10 mM MES (pH 5.5), was added for triggering fusion and the plates were incubated for 2 min at 37 °C. The medium was replaced with 0.5 ml of the prewarmed binding medium and the cells were incubated for an additional hour at 37 °C. After fixation with 20% formalin in PBS and staining with hematoxylin, inhibition of polykaryon for-marion was examined under a phase-contrast microscopy. The amount of the virus used in this assay was enough to induce fusion in about 80% of the cells.

Two fusion experiments yielded 13 stable hybridomas secreting M A b s specifically reacting with G protein of VSV. Their characteristics are listed in Table 1 . Their relative reactivities varied over a 40-fold range, but were still within a relatively high range c o m p a r e d with those of other M A b s to different antigens determined by us. All the M A b s except for P2F3 reacted with G protein in Western blotting analysis.

Competitive binding E L I S A was carried out a m o n g these M A b s to classify the epitopes of G protein recognized by them. Typical results o f the competitive binding assay are shown in Fig. 1 , in which the binding o f biotinylated 1A7 M A b to G protein was challenged by several unlabeled MAbs. We observed four types of competition. In addition to homologous M A b (1A7), P2F9 completely inhibited the binding. 5C6 partially inhibited the binding. 3F4, P2 E11 or P2F3 a All MAbs had ~: light chain b Relative reactivity was defined in ELISA as the MAb concentration needed to attain an absorbance of 0.6 when a positive control (immunized mouse serum diluted 2,000-fold) had an absorbance value of 1.2 c Reactivity to G protein in Western blotting analysis d Epitopes (antigenic determinants) were identified by competitive binding assay among MAbs as described in Table 2 Table 2 ). The 13 MAbs were assigned to seven distinct epitopes on G protein based on the complete inhibition. When an unlabeled MAb inhibited the binding of a biotinylated MAb at 100gg/ml (at least 100-fold excess of biotinylated MAbs) to less than 10% and when this inhibition was observed in pair-wise assays, both MAbs were considered to share the same (or a closely adjacent) epitope. These seven epitopes were designated as Ep 1 to Ep 7. The MAbs to the same epitope showed similar patterns of partial inhibition or enhancement of binding against MAbs to different epitopes (Table 2) . Only MAbs to Ep 3 were subgrouped into two based on the pattern; MAbs to Ep 3a had no effect on the binding of MAbs to Ep 2, whereas MAb to Ep 3b enhanced the binding of MAbs to Ep 2.

The 13 MAbs were assayed for the neutralizing activity by the plaque reduction test ( Table 3 ). The MAbs assigned to four (Ep 2, Ep 4, Ep 5 and Ep 7) of the seven epitopes had neutralizing activities. The neutralization titers of the MAbs to Ep 5 and Ep 7 were about 10-times higher than the others. Inhibition of hemagglutination by the MAbs was also examined ( Table 3 ). The MAbs assigned to two epitopes (Ep 1 and Ep 2) had higher HI activity than the others. Goose erythrocytes used in this experiment were pretreated with trypsin to enhance the sensitivity of HI. Untreated erythrocytes gave similar results, although higher MAb concentrations were required (data not shown). Titers for neutralization represent the reciprocal of the highest twofold dilution of purified MAb (125 gg/ml initial) causing more than 50% reduction in the plaque number b Titers for HI represent the reciprocal of the highest twofold dilution of purified MAb (250 gg/ml initial) inhibiting HA caused by 4 or 8 HAU of VSV. Control Ig G1 and Ig G2a MAbs and the serum-free medium for growing hybridomas showed no HI activity (< 1)

We examined MAbs for the influence on the adsorption of radiolabeled VSV to BHK-21 cells. MAbs to Ep 1 and Ep 3 had very little, if any, effects on the virus adsorption (Fig. 2 a and c) similar to the control MAbs (Fig. 2 h) . MAbs to Ep 4, Ep 5, Ep 6 and Ep 7 slightly reduced the virus binding (Fig. 2 d, e, f and g). Even at the highest concentration (125 gg/ml), they exerted partial inhibition (65-75% of the binding of control). On the other hand, MAbs to Ep2 slightly enhanced the VSV adsorption only at certain concentrations (Fig. 2 b) . In another experiment with a different preparation of radiolabeled VSV with a higher specific activity, similar results were obtained: no MAb completely inhibited the virus binding and MAbs to Ep 2 enhanced the virus binding (data not shown).

We examined MAbs for the effects on VSV-mediated polykaryon formation of BHK-21 cells to test whether the MAbs inhibit the fusion induced by VSV. Extensive cell fusion was induced by acid treatment of the virus-bound cells (Fig. 3 o) but not by the same treatment of unbound cells (Fig. 3 p) . MAbs reacting with Ep 4 (Fig. 3 g and h) , Ep 5 ( Fig. 3 i) , and Ep 7 (Fig. 31) completely inhibited polykaryocyte formation. The other MAbs (Fig. 3 a-f, j, and k) P2F3 + + --+ a + +, +, and -Neutralization titers of 1> 1,000, >f 50 and > 1, respectively for 50% reduction of plaques, shown in the third column of Table 3 b + and -HI titers against 4 H A U of >t 32 and ~< 16, respectively, given in the 4th column of Table 3 c _ No inhibition or slight inhibition of adsorption (65-125%); e enhancement of adsorption (~> 135%) in the virus binding assay shown in Fig. 2 d + Inhibition of fusion; -no inhibition e Not tested to the cell fusion activity. W e reported here for the first time identification of the G protein epitopes associated with H A and fusion activities.

In the competitive binding assay, the m u t u a l complete competition o f paired M A b s revealed the presence of at least seven distinct epitopes on G protein of VSV-Indiana. In addition, some topographical relationships a m o n g some o f these epitopes were suggested by partial inhibition or enhancement o f the binding o f M A b s ( Table 2 ). In particular, association a m o n g Ep 1, Ep 2, and E p 3 w o u l d be quite possible since the m u t u a l binding enhancement o f the respective M A b s was observed. Such enhancement is p r o b a b l y due to an a d v a n t a g e o u s allosteric alteration of G protein after binding with the first M A b , thereby resulting in increased binding o f the second M A b . Similar competitive binding assays with anti-G protein M A b s were reported by Volk etal. [36] and Le-Francois and Lyles [14, 15] , w h o d e m o n s t r a t e d 11 and 10 epitopes on G protein of VSV-Indiana, respectively. The enhancement of binding was f o u n d also by LeFrancois and Lyles [14, 15] .

O f the seven epitopes identified on G protein, all b u t one (Ep 1-6) reacted with respective M A b s even in Western blot analysis. These are p r e s u m a b l y linear epitopes not dependent on the secondary structure. The M A b s assigned to four epitopes (Ep 2, Ep 4, Ep 5, and Ep 7) had VSV-neutralizing activity, although of varying efficiency. Previous reports also demonstrated the same number of neutralizing epitopes on G protein of VSV-Indiana [14, 36] .

MAbs to Ep 1 and Ep 2 showed HI activity. The proximity between these HI epitopes were suggested by the competitive binding assay and it is likely that these epitopes concurrently form the functional domain for the HA activity. These HI epitopes were not always neutralization epitopes and were different from the fusion-inhibition epitopes. This indicates that the sites involved in HA activity are different from those involved on the other functions. In general, viral HA is equivalent to the viral attachment to cells. However, the HI MAbs did not inhibit the VSV binding to BHK-21 cells. The little correlation between these two activities is likely ascribed to the difference of the target cells and/or conditions in these assays. "l;he result of another experiment showed that the HI MAbs markedly inhibit the binding of radiolabeled VSV to goose erythrocytes, in which the binding is measured under the same condition as the HI assay (data not shown).

Attempts to identify the cell-binding domain of G protein were unsuccessful in this study. In the binding assay, no MAb completely inhibited the VSV adsorption (Fig. 2) . MAbs to Ep 4, Ep 5, Ep 6, and Ep 7 at high concentrations slightly inhibited VSV adsorption, but such low inhibitory effects were not related to the efficient neutralization or HI. The lack of the complete inhibition suggests that all neutralizing MAbs prepared in this study block the virus infection at a step subsequent to adsorption. On the other hand, a certain concentration of MAbs to Ep2, one of the neutralization epitopes, rather enhanced VSV adsorption. Although it is difficult to explain the biological significance of this enhancing effect, a similar enhancement of the VSV adsorption by immune serum was reported by Schlegel and Wade [32] . They suggested that the VSV-antibody complex binds to a different or an additional cell binding site, thus altering the adsorption efficiency. The same explanation may be given to the enhancing effect of MAbs to Ep 2.

In the fusion inhibition assay, MAbs reacting with three epitopes (Ep 4, Ep 5, and Ep 7) inhibited the VSV-mediated cell fusion. These epitopes are probably located on or close to the fusogenic domain of G protein. Although hydrophobic domains involved in fusion have been identified in several viral fusion proteins [10, 27] , such a domain has not been identified in G protein of VSV [27] . A most recent finding that introduction of a glycosylation site into residue 117 of G protein resulted in fusion-defective mutant suggests that the residues 118 to 136 are involved in the fusion activity [39] . Some of our fusion-inhibiting MAbs may recognize these residues and the location of the fusion-inhibition epitopes will be required. We found the presence of multiple epitopes related to the fusion activity. This suggests that these different regions of G protein contribute to the fusion activity in partnership and might explain the lack of highly hydrophobic fusion sequence in the G protein of VSV. All fusion-inhibiting MAbs had the neutralizing activity. In the VSV infection process, after endocytosis of the b o u n d virion, the fusion of viral envelope with the endosomal m e m b r a n e is necessary for the entry of the nucleocapsids into the cytoplasm [20, 21] . Fusion-inhibiting MAbs neutralize VSV probably by blocking the fusion stage of the infection process.

To analyze further the structure-function relationship of G protein, our studies are currently aimed at locating these epitopes on the G protein by testing the reactivity of the MAbs to G protein fragments expressed in Escherichia coli.

Effects of DEAE-dextran on infection and hemolysis by VSV. Evidence that nonspecific electrostatic interactions mediate effective binding of VSV to cells

Glycosylation is not required for the fusion activity of the G protein of vesicular stomatitis virus in insect cells

Restitution of infectivity to spikeless vesicular stomatitis virus by solubilized viral components

Identification of distinct antigenic determinants on Semliki Forest virus by using monoclonal antibodies with different antiviral activities

Monoclonal antibodies to the glycoprotein of vesicular stomatitis virus (New Jersey serotype): a method for preliminary mapping of epitopes

Surface structure of vesicular stomatitis virus

A cell line expressing vesicular stomatitis virus glycoprotein fuses at low pH

Effect of glycosylation on the conformational epitopes on the glycoprotein of vesicular stomatitis virus (New Jersey serotype)

Hemagglutinin of rabies and some other bullet-shaped viruses

Membrane fusion of envelope viruses: especially a matter ofproteins

Epitope mapping by deletion mutants and chimeras of two vesicular stomatitis virus glycoprotein genes expressed by a vaccinia virus vector

The glycoprotein of vesicular stomatitis virus is the antigen that gives rise to and reacts with neutralizing antibody

Biological activities of monoclonal antibodies reactive with antigenic sites mapped on the G1 glycoprotein of La Crosse virus

The interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus I. Analysis of neutralizing epitopes with monoclonal antibodies

The interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus II. Monoclonal antibodies to nonneutralizing and cross-reactive epitope of Indiana and New Jersey serotypes

Antigenic determinants of vesicular stomatitis virus: analysis with antigenic variants

Point mutations in glycoprotein gene of vesicular stomatitis virus (New Jersey serotype) selected by resistance to neutralization by epitope-specific monoclonal antibodies

Spontaneous mutations leading to antigenic variations in the glycoproteins of vesicular stomatitis virus field isolates

pH-dependent hemolysis and cell fusion of rhabdoviruses

Virus entry into animal cells

Pathway of vesicular stomatitis virus entry leading to infection

Transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of Madin-Darby canine kidney cells. I. Morphological evidence

Biological properties of VSV glycoprotein. II. Effects of the host cell and of the glycoprotein carbohydrate composition on hemagglutination

Vesicular stomatitis virus-mediated cell fusion subsequent to virus adsorption at different pH values

Production of monoclonal antibodies by the use of pH-dependent vesicular stomatitis virus-mediated cell fusion

Preferential generation ofmonoclonal IgG-producing hybridomas by use of vesicular stomatitis Virus-mediated cell fusion

Membrane fusion in fertilization, cellular transport, and viral infection

Immunoglobulin-producing hybrid cell lines

Vesicular stomatitis virus membrane proteins and their interactions with lipid bilayers

Functional epitopes on glycoprotein of VSV

Reconstitution into liposomes of the glycoprotein of vesicular stomatitis virus by detergent dialysis

Analysis ofmurine coronavirus surface glycoprotein functions by using monoclonal antibodies

Neutralized vesicular stomatitis virus binds to host cells by a different "receptor

Topographical mapping of epitopes on the glycoproteins of murine hepatitis virus-4 (strain JHM): correlation with biological activities

Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications

Sequences of the major antibody binding epitopes of the Indiana serotype of vesicular stomatitis virus

Monoclonal antibodies to the glycoprotein of vesicular stomatitis virus: comparative neutralizing activity

Rhabdovirus biology and infection: an overview

Cell fusion by Semliki Forest, influenza, and vesicular stomatitis viruses

A fusion-defective mutant of the vesicular stomatitis virus glycoprotein

Fatty acid acylation is not required for membrane fusion activity or glycoprotein assembly into VSV virions

Growth, purification and titration of rhabdoviruses. In: Mahy BWJ (ed) Virology: a practical approach

High performance liquid chromatography of mouse monoclonal antibodies on spherical hydroxyapatite beads

We thank Dr. Kiichi Yamamoto for the gift of VSV-Indiana and Drs. Shudo Yamazaki and Robert R. Wagner for their useful information on this strain. We thank also Dr. Yoshio Yamakawa for his help with HPLC and Dr. Akiko Taniguchi for her assistance in some assays. Several helpful discussions with Dr. Michiyuki Matsuda are gratefully acknowledged. We thank also Mr. John Meissner and Mr. Russell Nash for their critical reading of the manuscript. We thank also the members of Department of Pathology, National Institute of Health, for their encouragement.