INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Rubella virus (RV), the etiological agent of German measles, is a small enveloped RNA virus that belongs to the togavirus family (31) and bears similarities to the prototype alphaviruses in terms of its genomic organization and strategy for viral gene expression (11). RV virions contain two glycosylated envelope proteins (E1 [58 kDa] and E2 [42 to 47 kDa]), located on the virion surface (32), that exist as heterodimers in the viral spike complexes (3, 49). E1, the major target antigen of RV, contains virus-neutralizing and hemagglutinin epitopes (6). The biological role of E2 is not well defined, although it has been reported to possess strain-specific epitopes and possibly a neutralizing domain (9). The RV structural proteins are synthesized from a subgenomic RNA as a polyprotein precursor that is cleaved to form the viral polypeptides C, E2, and E1 (33). The coordinate and individual expression of the C, E2, and E1 proteins shows that E2 is necessary for the cell surface expression of E1 and that E1 is not required for exit of E2 from the endoplasmic reticulum (ER) and its passage through the Golgi complex to the cell surface (16). However, E1 seems to increase the rate of transport of E2 to the cell surface (16). In the absence of E2, E1 is arrested in a post-ER, pre-Golgi complex compartment (18) and is only transported to the cell surface in the presence of E2 (16). Since the association of E1 and E2 is a hydrophobic interaction (3, 16), it is likely that E2 interacts specifically with an E1 hydrophobic domain to form the E1-E2 complexes that are transport competent.

Although RV appears to be similar to alphaviruses in terms of its structure as well as its structural-protein expression, its replication cycle kinetics are different. Cells infected with alphaviruses generally reach maximum rates of virus production 4 to 8 h after infection (22). RV, in contrast, has a latent period of more than 12 h, and peak virus production is reached between 24 and 48 h postinfection (13). The maturation and intracellular transport of alphavirus structural proteins have been extensively studied (27, 47, 48, 51). Compared to that of alphaviruses, RV glycoprotein processing occurs relatively slowly and the transport of glycoproteins E2 and E1 to the plasma membrane is inefficient (16). Like Semliki Forest virus (SFV), RV infects cells via endocytosis and an acid-triggered fusion step (1, 3, 26, 45). The reported acid-induced conformational changes in RV E1 and E2 and the removal of E2 by proteolytic digestion of RV particles, resulting in E1 particles that can still bind to liposomes (23), suggest that RV E1 plays a dominant role in membrane fusion in the acidic endosomal compartment, similar to that of SFV E1 (24, 25, 30), and may contain a noncleavable fusion peptide in the internal region of E1.

Inspection of the amino acid sequence of RV E1 revealed that there is an internal hydrophobic domain within E1, 29 amino acids long and 82 residues from the E1 NH₂ (Fig. 1). We speculated that this internal hydrophobic domain is involved in E1-E2 interaction as well as in cell fusion. We initiated experiments to determine the role of this E1 hydrophobic region in the E1-E2 interaction and fusogenic activity of BHK cells expressing RV antigens. In vitro mutagenesis was used to introduce four mutations into the hydrophobic region of E1. BHK cells were transfected with recombinant plasmids, and stable transformed BHK cell lines expressing wild-type or mutant spike proteins were isolated via methotrexate selection (38). Here we present evidence that the internal hydrophobic domain of RV E1 plays a major role in E1-E2 interaction and membrane fusion of RV.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Schematic diagram of RV cDNAs and the hydrophobic domain of the RV E1 glycoprotein. Respective portions of the C, E2, and E1 genes are indicated above the constructs. Intergene borders are marked by vertical lines extending through the cDNAs. The translation start site of the capsid protein is utilized in all constructs. Signal peptides are indicated by open boxes, and the transmembrane regions are shown as closed boxes. The hydrophobic domain of E1 is depicted as an open box 81 amino acid residues from the N terminus of E1. Restriction endonuclease sites are abbreviated as follows: E, EcoRI; H, HindIII; B, BamHI; and N, NcoI. The bar represents approximately 500 nucleotides. Beneath the arrows are the single-amino-acid changes and the deletion (dt) mutation. 24S, the cDNA containing all three structural protein genes of RV; E2E1, the cDNA containing the E1 and E2 genes.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Plasmid construction and mutagenesis. RV cDNAs encoding the polyprotein precursor for all three structural proteins (24S) (7) and the E2E1 polyprotein precursor (E2E1) (16) are shown in Fig. 1. The cDNAs were subcloned into the SmaI site of the transfer vector pNUT (34) under the control of the metallothionein 1 promoter. The construction of plasmids pNUT-24S and pNUT-E2E1 has been previously described (38). Plasmid pNUT-E2 was constructed by insertion of a blunted EcoRI-HindIII fragment from plasmid pCMV5-E2 (15) into the SmaI site of the pNUT vector.

Generation of stable transformed BHK cell lines. BHKtk cells (46) grown in Dulbecco modified Eagle medium (DMEM) with 5% fetal bovine serum were transfected with recombinant plasmids by the calcium phosphate method (12). After 24 h, the medium was changed to DMEM containing 500 µM methotrexate. The presence of dihydrofolate reductase cDNA driven by the simian virus 40 early promoter in the pNUT vector (34) permits the selection of transfected BHK cells in the presence of high concentration of methotrexate (38). After 10 days in the selection medium, methotrexate-resistant colonies were picked and screened for the expression of RV structural proteins by Western blot analysis (44). The resultant cell lines were named BHK-E2E1(wt), BHK-E2E1(C82S), BHK-E2E1(dt), BHK-E2E1(G93D), and BHK-E2E1(P104G).

Metabolic labelling, biotinylation, immunoprecipitation, and endoglycosidase digestion. Transformed BHK cells, grown as monolayers (in 35-mm-diameter petri dishes) in growth medium (DMEM containing 2.5% fetal bovine serum), were induced with zinc sulfate (40 µM) for 16 h. Induced BHK cells were labelled with 100 µCi of [³⁵S]methionine for 30 min and chased with 1 mM unlabelled methionine for various time periods. Cell surface biotinylation was carried out as described by Duffus et al. (10). Briefly, labelled cells were placed on ice, washed twice with phosphate-buffered saline (PBS), and incubated with 0.5 ml of sulfosuccinimidobiotin (0.5 mg/ml in PBS) for 15 min on ice. The biotinylation reaction was repeated once with fresh reagent. The reaction was stopped by washing with cold 10 mM glycine containing 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl. The cells were lysed with lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 2 mM EDTA, and 1% Triton X-100), and spike proteins were immunoprecipitated with human anti-RV serum. The biotinylated spike proteins were recovered from streptavidin-agarose and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (29). Immunoprecipitation of cellular spike proteins was performed as described by Wahlberg et al. (47). Briefly, at the end of the pulse or chase period, the monolayers were washed twice with PBS and lysed with buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40 (NP-40), and 74 µM antipain dihydrochloride (Boehringer Mannheim). RV antiserum-coated protein A-Sepharose was washed with lysis buffer and incubated with ³⁵S-labelled cellular lysate overnight at 4°C. The beads were washed twice with washing buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, and 0.2% NP-40 and once with 10 mM Tris-HCl (pH 7.4). RV antigen was eluted into 100 mM sodium citrate (pH 5.5) containing 0.15% SDS and 1 mM phenylmethylsulfonyl fluoride at 100°C for 4 min and analyzed by SDS-PAGE (29). Some immunoprecipitates were digested with endoglycosidase H (5 mU/50 µl) as described previously (16).

Fusion assay. BHK cells expressing either wild-type or mutant RV spike proteins were cultured in six-well plates. After ZnSO₄ induction for 16 h, the cells were washed once with fusion medium (DMEM without bicarbonate, containing 0.2% bovine serum albumin, 10 mM HEPES, and 10 mM morpholinoethanesulfonic acid; pH 7.0) and then incubated for 20 min at 37°C with fusion medium adjusted to a pH of 4.0 to 7.0 as required. After exposure to the desired pH, the fusion medium was replaced with growth medium of pH 7.0 and the cells were incubated for an additional 4 h at 37°C to allow both maximal RV spike protein expression in the newly fused cells and morphological reorganization of the cell nuclei. Polykaryons containing more than five nuclei were counted under a phase-contrast microscope. Fusion activity was presented as the percentage of fused cells, which is the ratio of the mean total number of polykaryons counted in five random fields to the mean total number of unfused cells and polykaryons.

Localization of spike protein by immunofluorescence. Transformed BHK cells were seeded onto poly-L-lysine-coated coverslips. After ZnSO₄ induction, the cells were washed three times with PBS containing 0.7 mM CaCl₂ and 0.3 mM MgCl₂, fixed for 20 min at room temperature in 2% formaldehyde-PBS, and then washed with PBS. Some cells were permeabilized with 0.075% NP-40-PBS for 30 min prior to being blocked with bovine serum albumin (1% in PBS) for localization of intracellular RV antigen (16). The cells were then treated with mouse monoclonal antibodies against E1 (1:50) or E2 (1:50) followed by incubation with fluorescein- or rhodamine-conjugated secondary antibodies (Kirkegaard and Perry Laboratories).

Sucrose velocity gradients. Samples (500 µl) were layered onto 11.5-ml linear gradients of 5 to 20% sucrose in 50 mM Tris-HCl (pH 7.5)-150 mM NaCl-2 mM EDTA-0.1% NP-40 and centrifuged for 30 h at 40,000 rpm in a Beckman SW41 rotor. Fractions (0.5 ml) were collected from the bottom of the tube.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Expression of mutant proteins. To determine whether the internal hydrophobic region (residues 81 to 109) is involved in E1-E2 interaction and cell-cell fusion, four mutants (C82S, dt, G93D, and P104G) were constructed by oligonucleotide-directed mutagenesis. Since the antigenic structure of E1 is dependent on N-linked glycosylation and intramolecular disulfide bonding (37), the Cys82 residue in the hydrophobic region was selected as a target for studying E1-E2 interaction. The substitution of aspartic acid at Gly93 was based on the assumption that if the E1 internal hydrophobic region interacts directly with the host cell membrane as a prerequisite of or during the fusion event, the introduction of a charged residue may inhibit the ability of E1 to induce membrane fusion. The internal fusion domains have been postulated to contain helix-breaking residues near their centers (50); therefore, proline 104 was changed to glycine.

View larger version (58K): [in this window] [in a new window]   FIG. 2.   Expression of mutant proteins. Induced BHK cells were labelled with [35S]methionine for 30 min and chased with 1 mM unlabelled methionine for 2 h. Intracellular RV antigens were isolated by immunoprecipitation with human anti-RV serum (A) or with anti-E1 monoclonal antibody (B), followed by SDS-PAGE and fluorography. Portions of the immunoprecipitates were (+) or were not () digested with endo-H. The positions of apparent molecular mass markers are shown at the right (in kilodaltons). BHK, BHK cells; Wt, BHK-E2E1(wt); Cys, BHK-E2E1(C82S); dt, BHK-E2E1(dt); Gly, BHK-E2E1(G93D); Pro, BHK-E2E1)(P104G); Human, human anti-RV serum; Mab-E1, monoclonal antibody against RV E1.

View larger version (81K): [in this window] [in a new window]   FIG. 3.   Localization of RV spike proteins by immunofluorescence. Induced BHK cells were fixed with 2% formaldehyde-PBS, incubated with monoclonal antibodies against E1 (1:50) or E2 (1:50), and then incubated with fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G. Wt, BHK-E2E1; Gly, BHK-E2E1 (G93D); Pro, BHK-E2E1(P104G); Cys, BHK-E2E1(C82S); dt, BHK-E2E1(dt). Magnifications, ×400.

View larger version (44K): [in this window] [in a new window]   FIG. 4.   Cell surface expression of wild-type and mutant proteins. Induced BHK cells were labelled with [35S]methionine for 30 min and chased with 1 mM unlabelled methionine for 2 or 8 h. Cell surface RV antigens were derivatized with sulfosuccinimidobiotin and isolated as described in Materials and Methods. The virus spike proteins were precipitated and separated into internal (A) and surface (B) proteins by streptavidin binding. Portions of the immunoprecipitates were (+) or were not () digested with endo-H. The positions of apparent molecular mass markers are shown at the right (in kilodaltons). Internal, intracellular antigens; surface, cell surface antigens; medium, RV antigens released into the culture medium; Wt, W, BHK-E2E1; Gly, G, BHK-E2E1(G93D); Pro, P, BHK-E2E1(P104G).

Glycan processing and intracellular stability of mutant proteins. To study the processing and stability of the C82S and dt mutant proteins, pulse-chase experiments were carried out. In the wild type, after 30 min of pulse-labelling, E2 was found predominantly as a 39-kDa form, and removal of high-mannose glycans by digestion with endo-H reduced the molecular size to 31 kDa (Fig. 5, top panel). Approximately 20, 40, and 60% of E2 was further processed to complex-type glycans (16) (indicated by asterisks in Fig. 5, top panel). In contrast, E2 from the mutants was not processed to complex-type glycans and was not detected after the 8-h chase period (Fig. 5, middle and bottom panels). It is likely that the unassociated E2 in the C82S and dt mutants was either degraded in the ER or transported to the cell surface by itself.

View larger version (47K): [in this window] [in a new window]   FIG. 5.   Time course of glycan processing of wild-type and mutant proteins. Induced transformed BHK cells were pulse-labelled with [35S]methionine for 30 min and chased for various periods of time as indicated. Some immunoprecipitates were digested with endo-H for 8 h (+); others were not (). E1 and E2 arrowheads indicate the E1 and E2 protein species in the absence or presence of endo-H treatment. The processing of E2 is indicated by the asterisks. The positions of molecular size markers are shown on the left (in kilodaltons). WT, BHK-E2E1; CYS, BHK-E2E1(C82S); DT, BHK-E2E1(dt).

Effect of mutation on E1 conformation. RV E1 and E2 are rich in cysteine residues and contain intramolecular disulfide bridges that are important in maintenance of the proper protein folding (49). RV E1 and E2 show increased mobility in SDS-PAGE gels if disulfide bond reduction is omitted (49). This difference in mobility is probably due to the presence of intrachain disulfide bonds in E1 and E2. To determine whether a mutation would affect the conformation of E1, Western blot analysis was used to detect the binding of E1 to human anti-RV serum and monoclonal antibodies against E1 or E2 under reducing and nonreducing conditions. Under reducing conditions, the binding of antibodies to the wild-type E1 and their binding to mutant E1 proteins were similar (Fig. 6). In contrast, under nonreducing conditions, E1 proteins from the wild type and the G93D and P104G mutants retained their antibody binding activities, whereas the E1 proteins from the C82S and dt mutants lost most of their ability to bind the human anti-RV serum and anti-E1 monoclonal antibody (Fig. 6) and no E1-E2 heterodimer was detected (note asterisks in Fig. 6). As expected, E2 binding activity was not affected under nonreducing conditions in either the wild type or the mutants (Fig. 6). This decrease in E1 antibody binding activity of the C82S and dt mutants is not due to the formation of E1-E1 or E1-E2 oligomers under nonreducing conditions, since no higher-molecular-weight protein species were observed (Fig. 6). Thus, the antibodies appear to detect conformational changes with respect to intrachain disulfide bonding in the C82S and dt mutants. This result is consistent with our data on the immunoprecipitation of RV antigen with a conformation-specific anti-E1 monoclonal antibody (Fig. 2B).

View larger version (49K): [in this window] [in a new window]   FIG. 6.   Immunoblot analysis of E1 mutant proteins. Monolayers of induced BHK cells were lysed in lysis buffer containing 10 mM iodoacetamide to prevent the formation of disulfide bonds. The protein samples were denatured in the presence (+-Me) or absence (-Me) of 2-mercaptoethanol, separated by SDS-PAGE, and transferred to nitrocellulose membranes for immunoblot analysis. RV antigens were detected by using human anti-RV serum (Human), anti-E1 monoclonal antibody exhibiting hemagglutination-inhibiting activity (Mab-E1), and anti-E2 monoclonal antibody (Mab-E2). The positions of the apparent molecular mass standards are indicated on the right (in kilodaltons). E1-E2 heterodimer is indicated by an asterisk. Wt, BHK-E2E1; Cys, BHK-E2E1(C82S); dt, BHK-E2E1(dt); Gly, BHK-E2E1(G93D); Pro, BHK-E2E1(P104G).

E1-E2 heterodimer interaction. Since correct E1-E2 oligomerization appears to be required for efficient transport of these two proteins through the Golgi apparatus and to the plasma membrane (3), alteration of the E1 conformation may affect the E1-E2 heterodimer interaction. Biochemical studies have demonstrated that RV particles can be solubilized by nonionic detergent into an E1-E2 heterodimer structure and nucleocapsid (32). This E1-E2 noncovalent protein complex is not dissociated by NP-40 lysis buffer. Therefore, the formation of intracellular E1-E2 dimers can be detected by sedimentation analysis in sucrose gradients. To determine whether the mutations in E1 affect the interaction between E1 and E2, induced BHK cells were solubilized in lysis buffer containing 1% NP-40 and cellular lysates were fractionated on isokinetic gradients (3). Gradient fractions were analyzed by SDS-PAGE under nonreducing conditions. RV antigens transferred to membranes were detected with human anti-RV serum. Two distinct E1 peaks were observed in the wild type and in both the G93D and P104G mutants (Fig. 7, top three panels). The slower-migrating peak (fractions 15 to 19) was the monomeric E1, and the faster-sedimenting peak (fractions 7 to 10) contained E1-E2 heterodimers as well as E1 oligomers. Baron and Forsell (3) have reported that RV E1 migrates as two peaks in sucrose velocity gradients. The lighter peak is the E1 monomer, while the heavier peak is the E1 oligomer, which is larger than the E1-E2 heterodimer. Therefore, it is likely that the fractions between 11 and 14 contained mostly E1-E2 heterodimer. This interpretation is further supported by the band intensity of undissociated E1-E2 in the Western blots (Fig. 7, top three panels). In the Cys and dt mutants, only a small fraction of E1 and E2 was found as a heterodimer. Most of E2 was detected as E2 oligomers (fractions 7 to 13), and no oligomeric E1 was observed (Fig. 7, bottom two panels). It appears that alteration of the conformation of E1 in the C82S and dt mutants resulted in prevention of E1-E2 interaction.

View larger version (51K): [in this window] [in a new window]   FIG. 7.   Sucrose velocity analysis of E2 and E1 from cellular lysates. Induced BHK cells were lysed in lysis buffer, and the lysates were spun to remove the nuclei. Each lysate was analyzed on a 5 to 20% linear sucrose gradient as described in Materials and Methods. Fractions from each gradient were analyzed by immunoblotting. E2 and E1 were detected with human anti-RV serum (1:100 dilution). Sedimentation is from right to left. The positions of the apparent molecular mass standards are indicated on the right (in kilodaltons). Wt, BHK-E2E1; Gly, BHK-E2E1(G93D); Pro, BHK-E2E1(P104G); Cys, BHK-E2E1(C82S); dt, BHK-E2E1(dt).

Cell-cell fusion activities of mutants. Katow and Sugiura (23) suggested that RV E1 plays a vital role in membrane fusion in the acidic endosomal compartment. Bernasconi et al. (5) reported that RV E1 containing a glycosylphosphatidylinositol (GPI) anchor was transported to the cell surface, where it retained the hemadsorption activity characteristic of the wild-type E1-E2 heterodimer. To examine whether E1 is the fusogenic protein of RV, transformed BHK cell lines expressing RV E2 (BHK-E2) were isolated and used in the fusion assay in parallel with the wild type, BHK-E2E1. Induced BHK cells were treated with fusion medium at pH 5.0, 6.0, or 7.0 for 20 min at 37°C. The cells were washed with growth medium (pH 7.0) and incubated with the same medium at 37°C for an additional 3 to 4 h. The polykaryons formed were viewed under a phase-contrast microscope. The wild type, BHK-E2E1, showed extensive polykaryon formation at both pH 5.0 and 6.0 but not at pH 7.0 (Fig. 8A). The majority of polykaryons contained 20 to 50 nuclei at pH 5.0 and 5 to 20 nuclei at pH 6.0 (Fig. 8A). No syncytium formation was observed in the induced BHK-E2 cells at any pH tested (data not shown). The lack of detectable cell fusion observed in BHK-E2 cells is not due to limited cell surface expression, since we have shown previously that RV E2 can reach the cell surface by itself (16) and the expression level in transformed BHK cells is five times higher than that of the transfected COS cells (data not shown). Taken together, these results indicate that E1 contains the fusogenic domain of RV, consistent with the finding that RV E1 particles can bind to liposomes in the absence of E2 (23).

View larger version (167K): [in this window] [in a new window]   View larger version (113K): [in this window] [in a new window]   FIG. 8.   Syncytium formation in cells expressing wild-type or mutant proteins. (A) pH dependence of polykaryon formation. BHK-E2E1 (WT) and BHK cells were induced with ZnSO4 for 16 h, exposed to fusion medium at pH 5.0, 6.0, or 7.0 for 20 min, incubated in regular growth medium for 4 h, and photographed. (B) Polykaryon formation by mutant proteins. Induced BHK cells were exposed to fusion medium at pH 5.0 for 20 min, incubated in regular growth medium for 4 h, fixed, and photographed. WT, BHK-E2E1; Gly, BHK-E2E1(G93D); Pro, BHK-E2E1(P104G).

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

RV envelope glycoproteins E1 and E2 are targeted to the Golgi complex as heterodimers. It has been suggested that heterodimerization of E1 with E2 is required for the former protein to be correctly folded and transported to the Golgi complex (4, 19), where the complex accumulates due to a Golgi retention signal in the membrane-spanning domain of E2 (21). However, RV E1 can be rendered transport competent in the absence of E2 by the addition of a GPI anchor to the C terminus of the E1 ectodomain (5). Addition of a GPI anchor has been used for studying the role of individual domains of several membrane proteins in oligomerization and transport (8, 41) and has been shown to influence folding (2). Thus, it is conceivable that E1 retention is mediated by the binding of E1 to another protein, perhaps via a sequence(s) in the ectodomain, and this interaction may be prevented by oligomerization with E2 or fusion to a GPI anchor.

Our results showed that the internal hydrophobic domain (residues 81 to 109) in RV E1 plays a major role in the formation of the E1-E2 heterodimer and in the low-pH-induced cell fusion. We have shown previously that the antigenic structure of E1 is dependent on both N-linked glycans and intramolecular disulfide bonding (37). It is likely that substitution of serine at Cys82 disturbed the intramolecular disulfide bridges in E1 that are critical in maintaining the conformation of E1 for E1-E2 interaction and cell surface transport. The defect in dimerization and transport in the deletion mutant may imply that the E1 hydrophobic domain indirectly interacts with E2. However, we believe that the transport defect is more likely due to an overall disruption of the conformation of E1 in the absence of this hydrophobic domain. It is possible that the E1 hydrophobic domain is normally masked in the ER by association of E1 and E2, in order for transport to proceed further. In the absence of this hydrophobic moiety, E1 became misfolded or bound to ER proteins, such as the resident ER protein Bip (35).

Our results with G93D and P104G mutants demonstrated that the hydrophobic domain of E1 is closely involved in RV membrane fusion. This interpretation is further supported by our recent experiments using virus-like particles and an infectious clone containing a G93D or P104G mutation. We have shown previously that C protein of RV mediates the assembly of RV spike glycoproteins into virus-like particles (38). We found that virus-like particles containing either G93D or P104G mutations were produced as efficiently as the wild type (unpublished results). In the G93D mutant, the release of spike proteins into the medium was not observed in the presence of C protein. We have also carried out the fusion assay using BHK cells producing G93D or P104G virus-like particles; the results obtained are in agreement with those reported here. The other evidence supporting our interpretation lies in experiments in which defective G93D virus was produced from an RV infectious RNA transcript containing the G93D mutation. Further studies on G93D and P104G viruses to define the steps in fusion processes that are affected by these mutations are in progress.

A variety of mutations affecting the fusion activity of SFV have been reported (30). Although mutations at many positions affect the pH dependence of fusion, only a mutation (Gly91 to Asp) in the hydrophobic domain of SFV E1 was found to completely block fusion (30). It is interesting that in RV E1, substitution of aspartic acid at Gly93 also resulted in complete blockage of cell fusion.

The binding and fusion process mediated by the spike proteins in the envelope of the virus particle usually involve a series of conformational changes in these proteins. In the case of SFV, conformational changes in E1 and E2 on exposure to a pH that induces fusion have been investigated by using protease digestion and monoclonal antibody assays (24, 25). Under the acidic conditions of the endosomes, the E1-E2 heterodimer of SFV dissociates and an NP-40-resistant E1 trimer is formed (48). Due to the lack of conformation-specific monoclonal antibodies available for RV, little is known about the low-pH-mediated fusion process of RV structural proteins. Although RV E2 is transported to the plasma membrane in the absence of E1 (16) and does not have fusion activity, the involvement of RV E2 in the fusion process cannot be ruled out. It is possible that conformational changes in RV E2 are necessary for activation of E1 acid sensitivity. Unfortunately, the E2 and E1 subunits are correctly folded and transported only as a heterodimer. No direct evidence for this mechanism is yet available. It is also possible that a maturation step occurring late in the exocytic pathway is required to confer acid sensitivity on newly synthesized E1.