INTRODUCTION

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Varicella-zoster virus (VZV) is a highly fusogenic virus, but the degree of fusion is dependent on the cell substrate in which the virus is propagated (20). In human fibroblast cells, fusion formation is limited to a small number of nuclei per syncytium. In contrast, in human melanoma cells, all VZV strains examined to date exhibit fusion formation in which the entire monolayer is eventually involved. Polykaryon formation also occurs during primary VZV infection in human epidermal cells. Therefore, fusion formation appears to be related to cells of ectodermal origin (20). The question of which glycoproteins are involved in VZV-induced fusion was addressed in two earlier reports in which transfection studies were carried out with the VZV gH and gL genes (13, 14). Transfection with gH alone caused little or no syncytium formation. In contrast, cotransfection with gH and gL genes led to multiple foci of fusion within the monolayer, where syncytia from 6 to 25 nuclei were easily detected. This set of experiments also documented the utility of confocal microscopy as an instrument to detect syncytium formation and glycoprotein expression within a polykaryon.

Of interest, the VZV results differed markedly from the herpes simplex virus type 1 (HSV-1) data, which showed that cotransfection with four glycoprotein genes, namely those of gH, gL, gB, and gD, was required for syncytium formation (42, 55). In the latter case, each syncytium often included 10 to 20 nuclei but rarely larger foci. While VZV gH is easily detected in the plasma membrane, VZV gL is not detected on the surface of the transfected cell monolayer. The above data suggest that VZV gH is a more fusogenic molecule than HSV-1 gH; however, the precise fusion domains have not been mapped in either VZV gH or HSV-1 gH. The degree of genetic identity between VZV gH and HSV gH is 25% (31).

VZV has the smallest genome (125 kbp) among the human alphaherpesviruses. Because no gD homologous open reading frame (ORF) is present in the VZV Us segment (9), gD cannot play a role in VZV-induced fusion. It has been postulated that some functions of gD may be transferred to another VZV glycoprotein, but no obvious regions of homology between HSV gD and a VZV glycoprotein have been identified (7). Given the results obtained with VZV gH and gL, the unanswered question is the role of VZV gB in fusion. Prior studies have demonstrated that a murine monoclonal antibody (MAb) to VZV gH inhibits fusion formation in cell culture (33, 45). In a similar analysis, antibody to VZV gB was shown to reduce fusion formation (32). By analogy, therefore, the hypothesis was set forward that VZV gB was involved in fusion. The gB molecule is one of the most highly conserved genes in the herpesvirus genome. VZV gB, which is encoded by ORF 31, was shown to have a 49% degree of genetic identity with HSV gB (9, 16). The mature VZV gB is a highly glycosylated disulfide-linked heterodimer (19). Trafficking domains from the endoplasmic reticulum to the Golgi bodies within the cytoplasmic tail have been defined (21).

Our preliminary studies with an individually expressed VZV gB gene failed to demonstrate a strong fusogenic property. The possibility was entertained that the gB gene in the particular laboratory strain may have undergone mutation and thereby lost its fusogenic domain (44). However, sequencing of 10 VZV strains, including the most commonly used laboratory strains, did not detect an obvious mutation which could account for the loss of fusogenic potential (17, 47). Finally, a more detailed investigation involving individual VZV glycoprotein genes came to the surprising discovery that gB required the coexpression of a second VZV protein in order for fusion to occur.

	MATERIALS AND METHODS

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Construction of recombinant VVs. Vaccinia virus (VV) strain Praha was used for the construction of all VV-VZV recombinants. A schematic representation of single and double recombinant viruses containing the VZV genes, those of gB (ORF 31), gE (ORF 68), gH (ORF 37), and gL (ORF 60), which were inserted into thymidine kinase or hemagglutinin genes of the VV, was described recently (30). Expression of the extrinsic genes was controlled by either the early-late VV p7.5 promoter or the late VV 11k promoter. The recombination and thymidine kinase selection were performed as described by Perkus et al. (41). The recombinant viruses with foreign genes inserted in the hemagglutinin gene were isolated from plaques that were negative in hemadsorption. The Escherichia coli xanthine-guanidine phosphoribosyl transferase (gpt) gene under the control of the VV I₃ promoter served as a selection marker for recombinant viruses (5). The construction of the single recombinant viruses expressing glycoproteins gB (VV-gB), gE (VV-gE), gH (VV-gH), and gL (VV-gL), as well as double recombinant viruses VV-gH+gL and VV-gE+gB, was described in detail previously (26, 29, 35). Likewise, VZV cloning strategies in the VV T7-pTM1 expression system were previously published (59). The source of VZV DNA for all VZV glycoprotein genes was the VZV strain 80-2 genomic library (15). The sequences of the glycoprotein genes in this library have been published (17). DNA sequencing of all cloned VZV genes was performed by the DNA Core Facility at the University of Iowa.

Antibodies. Human anti-gH specific MAb V₃ recognizes a conformation-dependent virus neutralization epitope of mature gH (52). Murine MAb 258 binds to an epitope present on both mature gH and its glycosylated precursor form but does not recognize the unglycosylated gH precursor (13). Human anti-gB specific MAb V₁ (52) and murine MAb 158 (34) were used to detect VZV gB. Murine MAb 3B3 recognizes a linear epitope in the ectodomain of VZV gE (47, 48).

Analysis of infected cells by laser confocal scanning microscopy. HeLa cells (ATCC CCL2) were obtained from the American Type Culture Collection. HeLa cells were seeded onto coverslips in six-well culture dishes, grown in Eagle minimal essential medium with 10% fetal bovine serum to confluency, and infected at a multiplicity of infection (MOI) of 0.6 to 1.8 for 22 h by appropriate VV recombinants. The infected cells were fixed with 2% paraformaldehyde in 0.2 M Na₂HPO₄ for 1 h and then washed five times with phosphate-buffered saline (PBS) (pH 7.4). If the cells were to be permeabilized, 0.05% Triton X-100 was included in the fixative. The monolayers were blocked with 5% milk for 30 min at room temperature. Primary antibodies were diluted in PBS containing 1% milk: MAbs V₁ and V₃ were diluted 1:1,000, MAb 3B3 was diluted 1:750, and MAb 158 as well as MAb 258 were diluted 1:500. After incubation for 1 h and washing with PBS, secondary antibodies along with DNA marker TOTO-3 (Molecular Probes, Inc.) were added simultaneously for 1 h. Secondary antibodies, including Texas Red-conjugated goat anti-mouse antibody and Alexa 488-conjugated goat anti-human antibody as well as Alexa 488-conjugated goat anti-mouse antibody (Molecular Probes, Inc.), were diluted 1:1,000 in PBS. For multiple labeling experiments, staining with all secondary antibodies was done simultaneously. Samples were analyzed by confocal laser scanning microscopy (LSM 510; Zeiss, Göttingen, Germany). Images were stored in the Carl Zeiss Laser Scanning software system. Quantitative analysis of confocal images was carried out by previously described procedures (47).

Quantitative analysis of fusion. HeLa cells were seeded onto coverslips in six-well culture dishes, grown to confluency, and infected at an MOI of 0.6 to 1.8 by appropriate recombinant VVs. The infected cells were fixed and permeabilized with 2% paraformaldehyde and 0.05% Triton X-100 in 0.2 M Na₂HPO₄ for 30 min. After being washed with PBS, the cells were stained with hematoxylin and eosin and analyzed by light microscopy (Leitz Diaplan).

Pulse-chase labeling protocol. Cultured HeLa cells (6 × 10⁵) were infected for 3 h with a recombinant VV and incubated for 16 h. Afterward, the monolayers were starved for 3 h in methionine- and cysteine-deficient Dulbecco's modified Eagle medium (Sigma) (labeling medium) supplemented with 10% fetal bovine serum, 10% methionine-cysteine, 1% L-glutamine, 1% nonessential amino acids, and 1% penicillin-streptomycin and pulse labeled with 250 µCi of [³⁵S]methionine-cysteine (Pro-Mix; Amersham) in 0.2 ml of labeling medium for 45 min. The radioactive medium was removed, and the monolayers were washed once with complete medium and chased in complete medium for increasing time intervals prior to harvesting. The cell cultures were harvested as previously described (59). MAb 3B3 was added, and the samples were incubated overnight at 4°C. After addition of either anti-gE MAb or anti-gB MAb, precipitates were collected on protein A-Sepharose beads. After standard elution procedures, the proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% gel under reducing conditions (27). Protein mobility was calibrated by using ¹⁴C-radiolabeled molecular mass standards (Amersham).

	RESULTS

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Syncytium formation mediated by coexpression of VZV gH and gL. In an earlier study, VZV fusion mechanisms had been assessed in a VV T7-pTM1 transient-expression system. Even though this system demonstrated gH-gL-mediated syncytium formation, we wanted to determine whether an even higher level of expression of both glycoproteins led to even more syncytium formation. We also wanted to verify our initial results because of the obvious differences between the glycoproteins sufficient to mediate VZV- and HSV-1-mediated fusion (36). To investigate this issue in a new series of experiments, infection was performed with a double recombinant VV-gH+gL virus. The coexpression of both gH and gL in one vector led to processing of gH to its fully mature form, as demonstrated by detection by conformation-dependent MAb V₃. A large syncytium contained over 60 nuclei; colocalization, demonstrated by a yellow color of the two merged antibody probes, was most intense near the clusters of nuclei (Fig. 1A), sites known to contain Golgi bodies (56). Of particular importance, these results confirmed earlier VZV studies using the VV T7-pTM1 transfection system, which demonstrated syncytium formation after cotransfection with individual expression plasmids containing gH and gL in the absence of any other VZV glycoproteins (14). As in the latter expression system, infection with VV-gH alone was not a highly fusogenic event (Fig. 1B).

View larger version (102K): [in this window] [in a new window]   FIG. 1.   Confocal microscopic imaging following infection with VZV recombinant VVs. (A) HeLa cells were infected by VV-gH+gL double recombinant virus and labeled with anti-gH MAb 258 plus Texas Red-conjugated secondary antibody as well as anti-gH MAb V3 plus Alexa 488-conjugated secondary antibody. Yellow represents colocalization of the two fluoroprobes within syncytia. Blue represents TOTO-3 staining of nuclei. (B) HeLa cells were infected by VV-gH and labeled as described above; no antibody attached to gH. (C) HeLa cells were infected by VV-gB and labeled with anti-gB MAb 158 plus Texas Red-conjugated secondary antibody as well as anti-gB MAb V1 plus Alexa 488-conjugated secondary antibody. Yellow indicates colocalization of gB forms within syncytia. (D) HeLa cells were infected by VV-gE and labeled with anti-gE MAb 3B3 plus Alexa 488-conjugated secondary antibody.

Effect of VZV gB on cell fusion. In the HSV-1 system, gB is one of four glycoproteins required for fusion formation (55). Based on the extensive homology between the gB molecules in the alphaherpesviruses, a set of experiments was performed to determine the fusogenic potential of VZV gB. After infection with VV-gB, the cells were probed with both human MAb V₁ to gB and mouse MAb 158 to gB. The gB staining was concentrated near the clustered nuclei but was also detectable throughout the cytoplasm. Small syncytia, often with about 10 nuclei, were formed after expression of gB alone and demonstrated a modest fusogenic potential of VZV gB (Fig. 1C). As a control experiment, cells were infected with VV-gE and probed with a mouse MAb 3B3 to gE. As expected from earlier transfection experiments, no syncytia formed after expression of gE alone in a recombinant VV (Fig. 1D). The gE immunoreactivity was found throughout the cytoplasm and localized near nuclei. This experiment demonstrated that gE alone did not mediate cell membrane fusion and confirmed similar negative results in the VV T7-pTM1 system (14).

Enhancement of VZV gB fusion by VZV gE. In earlier experiments, VZV gH when expressed by itself was not able to exit the Golgi apparatus and reach the cell surface. When gH and gE were coexpressed, gE facilitated the trafficking of gH to the cell surface (13). Because of this observation, the gB experiment was repeated in a second set of experiments. Cells were infected with a VV carrying gB (VV-gB) and another VV with gE (VV-gE). In contrast with the results with gB alone (Fig. 1C), larger syncytia were formed after fusion induced by coexpression of both VZV gE and VZV gB. A yellow polykaryon (expressing both gB and gE) is shown in Fig. 2.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Confocal microscopic imaging of VZV gB and gE expressed by single recombinant VVs. HeLa cells were coinfected by single recombinant viruses VV-gE and VV-gB and labeled with anti-gB MAb V1 plus Alexa 488-conjugated secondary antibody as well as anti-gE MAb 3B3 plus Texas Red-conjugated secondary antibody. (A) VZV gB-specific staining. (B) VZV gE-specific staining. (C) Colocalization of VZV gB and gE (yellow) produced by merging the fluoroprobes in panels A and B.

View larger version (72K): [in this window] [in a new window]   FIG. 3.   Confocal microscopic imaging of a polykaryon induced by VV-gE+gB. HeLa cells were infected by VV-gE+gB double recombinant virus, and the two glycoproteins were labeled as described in the legend to Fig. 2. Colocalization of VZV gE together with gB is indicated by the orange pseudocolor produced from merging of the two fluoroprobes. Cell nuclei were pseudocolored blue with TOTO-3. Note that this micrograph represents one horizontal plane; more nuclei would be detectable below and above that represented in the figure.

Comparative analysis of gE and gB biosynthesis and cell surface expression. Because the above results demonstrated an apparent interaction between gE and gB, pulse-chase labeling experiments were carried out to assess the rate of maturation of gB and gE alone and in combination. Prior published studies have defined the maturation of both gE and gB in cell culture (19, 32). Of note, mature gE appeared 60 min more quickly in the double recombinant system than with a single gE recombinant; however, maturation of gB was less affected when gB alone and gB-gE coexpression were compared (Fig. 4A to D). Additional experiments were performed to measure the surface expression of the two glycoproteins when expressed singly and in combination. Quantification was carried by a previously described method using Adobe Photoshop software (47). When the pixels of gE were compared between gE alone and gE coexpressed with gB, the number of fluorescent gE pixels increased 3.3-fold in the gE+gB construct (Fig. 4E and F). When the pixels of gB were compared between gB alone and gB expressed with gE, there was only a 1.4-fold increment in the fluorescent pixel count in the gE+gB construct (Fig. 4G and H). The difference between gE alone and gE+gB was statistically significant (P < 0.001).

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Analysis of VZV gE as well as gB biosynthesis and cell surface expression. HeLa cells were infected with either VV-gE (A and E) or VV-gB (C and G) recombinant virus alone or double recombinant virus VV-gE+gB (B, D, F, and H). The cultures were pulse-labeled with [35S]methionine-cysteine for 45 min (A to D), after which the radioactive medium was replaced with regular minimum essential medium-fetal bovine serum for increasing time intervals indicated in the figure. Cell lysates were immunoprecipitated with either MAb 3B3 (A and B) or MAb V1 (C, D, and A and B, Pulse). After elution, the samples were analyzed by SDS-PAGE. Molecular mass markers are on the right. The infected cultures (E to H) were examined for surface expression of gE and gB by laser scanning microscopy. Unpermeabilized cell cultures were labeled with anti-gE MAb 3B3 and Alexa 488-conjugated secondary antibody (E and F) as well as anti-gB MAb 158 and Alexa 488-conjugated secondary antibody (G and H). In the micrographs (20× magnification), the green color is represented by white; quantification of fluorescent pixels was carried out in an Adobe Photoshop software program.

Fusion after gB and gE transfection in pTM1 expression system. The marked fusogenic properties of VV-gE+gB were striking. Because the initial VZV gH-gL fusion studies were performed in the VV T7-pTM1 system (14), the gE and gB genes were also cloned into individual pTM1 plasmids and subsequently coexpressed in a HeLa cell monolayer. The monolayers were labeled with both anti-gB and anti-gE antibodies to verify that both glycoproteins were expressed. The amount of fusion was similar to that seen in the previously mentioned gH-gL experiments; namely, syncytia with 10 nuclei were easily detected (Fig. 5). The sizes of these syncytia corresponded to those previously seen with VZV gH-gL in the same expression system and also to those described following transfection with HSV-1 gH-gL, gB, and gD (55).

View larger version (122K): [in this window] [in a new window]   FIG. 5.   Confocal microscopic imaging of VZV gB and gE expressed by the VV T7-pTM1 transfection system. HeLa cells were cotransfected by plasmids containing the VZV gB and gE genes, and the two glycoproteins were labeled as described in the legend to Fig. 2 to verify that both VZV products were coexpressed. In this micrograph, the yellow color demonstrating colocalization within a syncytium is represented by white.

Quantification of VZV glycoprotein-induced fusion. To quantify the degree of fusion in the recombinant VV system, HeLa cells were infected by the following viruses: VV-gB, VV-gH, VV-gE+gB, VV-gE+VV-gH, VV-gH+gL, and VV-gE. The infected monolayers, after being stained with hematoxylin and eosin, were examined for multinucleated cells by light microscopy, and the images were captured with a digital camera and analyzed in Adobe Photoshop (Fig. 6). Examination of the micrographs demonstrated a dramatic difference between fusion induced by coexpression with either gE+gB or gH+gL and fusion induced with gB or gH expressed alone as well as gE and gH coexpressed together (compare Fig. 6C and F to other panels).

View larger version (192K): [in this window] [in a new window]   FIG. 6.   Polykaryocyte formation induced by different VZV glycoproteins. HeLa cells were infected by the following recombinant VVs: VV-gE (A), VV-gB (B), VV-gE+gB (C), VV-gH (D), VV-gE and VV-gH (E), and VV-gH+gL (F). The infected cell monolayers were processed as described in Materials and Methods. Bar, 100 µm.

Comparison with conditions used in HSV glycoprotein fusion experiments. The positive VZV fusion results in the recombinant VV system contrasted with the negative results found by others following infection with recombinant VVs expressing the HSV glycoproteins (10). When the experimental protocols were compared, one difference emerged as an explanation; namely, much larger inocula of HSV recombinant VVs were used: for VZV, from 0.6 to 1.8 PFU, and for HSV, from 5 to 20 PFU. To test the effect of increasing inocula of VV on the VZV fusion assay, cultures were infected with VZV recombinant VVs at an MOI of 5 PFU as in the HSV fusion assay. Some of these cultures were simultaneously coinfected with wild-type VV in an amount which approximated the total PFU of VV used in the HSV fusion assays (Fig. 7). Of interest, as the PFU of wild-type virus was increased in the presence of a constant amount of recombinant VZV, the amount of fusion diminished. For example, when the PFU of VV strain Praha was increased to 15 PFU, less fusion was observed (Fig. 7B). As an additional control, the same experiment was repeated with the WR strain of VV, which was used in the HSV fusion assays (10). Of note, the inhibition of VZV fusion was even more pronounced when the same number of PFU was added (Fig. 7D). In short, increasing the total PFU of VV decreased the amount of fusion. Even increasing the PFU of VV-gE+gB from 5 to 15 PFU appeared to increase the lytic component of VV while decreasing the fusogenic component of the expressed VZV glycoproteins (Fig. 7F). Therefore, for recombinant VV-based fusion assays to be successful, a balance must be established between the fusogenic potential of the expressed herpesvirus glycoprotein and the fusion-inhibitory effect of the VV infection itself.

View larger version (176K): [in this window] [in a new window]   FIG. 7.   Inhibition of VZV gE+gB-mediated fusion by VV. HeLa cell monolayers were infected with a recombinant VV as described in the text; in addition, some monolayers were coinfected with wild-type VV. (A) VV-gE+gB at an MOI of 5 PFU per cell; (B) VV-gE+gB at an MOI of 5 as well as VV strain Praha at an MOI of 15; (C) VV strain Praha at an MOI of 15; (D) VV-gE+gB at an MOI of 5 as well as VV strain WR at an MOI of 15; (E) VV strain WR at an MOI of 15; (F) VV-gE+gB double recombinant virus at an MOI of 15. The infected cell monolayers were processed as described in Materials and Methods. Bar, 100 µm.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Virus-cell fusion events mediated by viral membrane glycoproteins constitute a crucial primary step in the infectious cycle of all enveloped viruses. In spite of dissimilarities between viruses, viral fusion glycoproteins share several common features (23, 39, 43). Those that have been studied are composed of one or two type 1 integral membrane glycoproteins, contain extended ectodomains carrying N-linked carbohydrates, and form higher-order oligomers which are present on the viral membrane at a high surface density (8). A key feature is the involvement of a common motif, a fusion peptide in a membrane-anchored polypeptide chain (12, 58). Many viral fusion proteins are tight complexes of two glycoprotein subunits that confer binding as well as fusion activity, and many are made as larger precursors which require proteolytic activation of their fusogenic potential (57). Generally, for viruses that fuse at neutral pH, it is suggested that an interaction between fusion proteins themselves, or between a fusion protein and a second protein of either viral or cellular origin, can trigger an activating conformational change in the fusion protein (2, 22, 28, 37, 38, 46, 50, 51). Exposure of a previously cryptic fusion peptide enables it to insert into the lipid bilayer and initiate a fusion reaction by disturbing membrane integrity (6, 11).

A successful assay to measure alphaherpesvirus-induced membrane fusion by individually expressed glycoprotein genes was reported for the VZV gH-gL complex (13, 14). These VZV experiments were carried out in a VV T7-pTM1 transfection system, with appropriate control experiments. The same transient-transfection system has been used extensively by investigators of parainfluenza and influenza virus-induced fusion over the last decade. In many control experiments, all these investigators have demonstrated the absence of fusion activity by VV containing T7 polymerase alone (25, 49, 53).

In addition, other virologists have investigated the potential of recombinant VVs to analyze fusion. Davis-Poynter et al. (10) performed a detailed analysis of the fusogenic potential of individual HSV-1 glycoproteins within a recombinant VV system; they did not detect fusion and further concluded that VV itself was not a fusogen. Subsequently, in a Cos cell transfection system with a pSMH3 expression vector, HSV-polykaryocyte formation was observed only if gB, gD, gH, and gL components of HSV-1 were coexpressed (55). Each syncytium often included 11 to 20 nuclei but rarely larger foci. They mentioned further that not every transfected COS cell expressed the full complement of four glycoproteins and therefore not every transfected cell was able to fuse (55).

The above data from numerous virology laboratories studying fusion establish that VV infection has never been associated with polykaryon formation. In fact, a concern by some investigators has been that VV infection may inhibit syncytium formation. A remaining question relates to the relevance of our VZV data toward reinterpretation of the negative HSV fusion results in a recombinant VV system. Since we observed greater fusion when two glycoprotein genes were inserted into one VV genome, as opposed to simultaneous infection with two recombinant VVs, it is possible that an experimental protocol with four HSV-1 recombinant viruses (each expressing a different glycoprotein) limits the number of cells infected with and expressing simultaneously all four HSV glycoproteins. In addition, the investigators studying HSV-1 fusion used a considerably larger total inoculum of VV, namely 10 to 20 PFU/cell (two to four HSV recombinant VVs per experiment), than was used in the VZV experiments with one double recombinant virus (0.6 PFU/cell). When we added additional nonrecombinant VV strain Praha equivalent to 5 to 15 PFU/cell to the well containing the VZV double recombinant virus, the degree of fusion in the monolayer was diminished.

The fusogenic potential of VZV gB had not been directly assessed in earlier publications. The gB glycoprotein exhibits many features described for fusion proteins: it is a homodimeric type 1 N-glycosylated membrane protein which is proteolytically cleaved into disulfide-linked subunits in many herpesviruses (40). Sequence alignments of carboxy-terminal hydrophobic regions of HSV, human cytomegalovirus, pseudorabies virus, and VZV gBs reveal similarities with known fusion peptides in influenza A and B viruses, Sendai virus, and human immunodeficiency virus type 1 (4, 44). Several syncytial mutations have been mapped to the cytoplasmic domain of HSV-1 gB (1, 18). Surprisingly, introduction of a disrupted gE gene into the HSV-1 ANG strain containing syncytial mutation A855V in the gB gene resulted in a nonsyncytial phenotype, implying a role for gE in HSV-1 gB-mediated fusion (3). For pseudorabies virus, glycoproteins gB, gH, and gL are sufficient to mediate fusion, a property which was enhanced when a carboxy-terminally truncated version of gB was used instead of wild-type gB (24). In human cytomegalovirus, cells constitutively producing gB formed syncytia containing 5 to 25 cells (54).

Our prior fusion data with the VZV gH-gL complex led to the hypothesis that gB may require a second protein in order to cause extensive cell-to-cell fusion. The selection of gE as a candidate for the second protein was based on a prior experiment in which gE was shown to substitute for gL and facilitate the trafficking of gH to the cell surface (13, 14). Comparative analyses demonstrated that VZV gE maturation and gE surface expression were significantly enhanced under conditions of gE-gB coexpression. This dramatic effect of the gE-gB interaction further expanded the multifunctional role of gE in the VZV life cycle and demonstrated a redundancy to VZV-induced fusion apparently not seen in the HSV-1 system.