Ethics statement

Institutional Animal Care and Use Committee (IACUC) review of all research involving animals was performed and procedures were approved by the Stanford University Administrative Panel on Laboratory Animal Care (Protocol ID: 11130). Stanford University complies with all federal and state regulations governing the humane care and use of laboratory animals, including the USDA Animal Welfare Act, and the Stanford University Assurance of Compliance with Public Health Service Policy on Humane Care and Use of Laboratory Animals. Acquisition and use of fetal material has been reviewed by the Stanford University Administrative Panel on Human Subjects in Medical Research and the scope of use does not meet the criteria for research involving human subjects. Anonymized fetal material is provided by the non-profit tissue supply organization Advanced Bioscience Resources, Inc. (ABR) in accordance with applicable federal and state regulations.

Cells

Human melanoma cells, LDL-GFP melanoma cells, and human embryonic lung fibroblasts (HELFs) were propagated in minimal essential media (MEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), nonessential amino acids (100 µM; Omega Scientific), and antibiotics (penicillin, 100 U/mL; streptomycin, 100 µg/mL; Invitrogen) [27]. CHO-K1 Cre cells, which stably express Cre recombinase, were propagated in F-12K Nutrient Mixture with Kaighn's modification (Invitrogen) supplemented with 10% FBS, penicillin, and puromycin (8 µg/mL; Invitrogen) [27].

Construction of gH(ORF37) expression vectors with mutations in the gHcyt

gH constructs were generated from the pME18s-gH[WT] vector (wildtype gH) or the pME18s-gH[TL] vector, which was a gift from Tadahiro Suenaga and Hisashi Arase (Osaka University, Osaka, Japan) [15], [27]. Primers containing the desired mutation were used to amplify two PCR products using AccuPrime Taq (Invitrogen). The amplicons were digested with either KpnI or AccI restriction enzymes (New England BioLabs), and blunt end ligated with digested pME18s-gH[TL] or the pME18s-gH[WT] vector. Ligated products were electroporated into TOP10F' Electrocomp E. Coli (Invitrogen). Clones were sequenced using the pME18s-KPN1 primer to confirm the mutation. (Table S1: primer list)

Mutagenesis of gH(ORF37) in pOka-DX bacterial artificial chromosome (BAC) and generation of recombinant viruses

The Y835A, Y835F, TL, Δ834-841, V5, and 834StopV5 mutations were generated in the self-excisable pOka-DX BAC as previously described [27]. Briefly, the mutations were generated in the shuttle vector, pCR669V1-gH-Kan [27]. The modified vector was digested with NaeI and PmeI enzymes (New England BioLabs) and the fragment of interest was inserted into pOka-DX-ΔORF37 BAC, which lacks ORF37 [27], by RED recombination. Fragment insertion was confirmed by PCR using primers {37}F65680-65700 and {37}R68697-68717. The pOka-TK-GFP-gB[Y881F] and pOka-TK-GFP-gH[Δ834-841] BAC constructs were generated from the pOka-TK-GFP-ΔORF37 BAC and -ΔORF31 BAC, respectively, using digested fragments from shuttle vectors following previously described methods [34]. The replacement of TK(ORF36) with TK-EGFP was performed by RED recombination using the previously generated pOka-DX-ΔORF37 and -ΔORF31 BAC and a PCR product amplified with primers 5′-TK-EGFP and 3′-TK-EGFP. The pOka-TK-GFP- gB[Y881F]/gH[Δ834-841] BAC was generated by deleting ORF31 in the pOka-gH[Δ834-841] BAC, replacing the gene with the gB[Y881F] cassette, followed by replacing the ORF36 with the TK-EGFP cassette. To confirm no spurious recombination, vectors were digested with HindIII enzyme (New England BioLabs), separated by gel electrophoresis, and compared to pOka-DX BAC. Recombinant viruses were generated by calcium chloride transfection of melanoma cells with mutant BAC DNA. The mutations in the viral genome were confirmed by PCR and sequencing. Recombinant viruses were passed in HELFs until the MiniF- sequences Cat and SopA were not detectable by PCR (Table S1), as previously described [35]. PCR of ORF31 further confirmed VZV genomic DNA.

Immunoprecipitation and western blotting

Briefly, CHO-K1 Cre cells were transfected with gH expressing vectors and the pcDNA3.1-gL vector, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions and lysates were harvested at 24 hours post transfection (hpt) [27]. Lysates from melanoma cells infected with recombinant VZV were harvested at 48 hours post infection (hpi). Immunoprecipitation and western blotting for gH used anti-gH monoclonal antibody SG3 (Meridian Life Science). Western blotting of viral proteins and cellular proteins was performed on infected-melanoma lysates as a control for infection and for sample loading. Membranes were probed for gE (mouse mAb 8612 anti-gE; Millipore), IE63 (rabbit anti-IE63; a kind gift of William Ruyechan, State University of New York, University at Buffalo, Buffalo, NY), and alpha-tubulin (clone B-5-1-2 mouse anti-α-tubulin; Sigma). All primary antibodies were detected using secondary horseradish peroxidase-conjugated antibodies to either anti-mouse or anti-rabbit and ECL Plus Detection Kit (GE Healthcare Bio-Sciences).

Quantitative Cre reporter assay to measure cell-cell fusion

Cell-cell fusion was measured using the quantitative Cre reporter assay described previously [27], [34]. Briefly, CHO-K1 Cre cells were transfected with equimolar amounts with pCAGGs-gB, a gift from Tadahiro Suenaga and Hisashi Arase (Osaka University, Osaka, Japan) [15], pcDNA3.1-gL, and pME18s-gH mutants and co-cultured with LDL-GFP melanoma cells. The frequency of GFP positive cells indicating fusion events was quantified on a modified Digital FACStar running Diva hardware and software (BD Bioscience). Analysis was performed using FlowJo and all results were normalized to gB/gH[TL]-gL. A negative control that contained only pME18s- and pcDNA3.1-empty vectors was used to establish background levels of GFP expression, which were then subtracted from the fusion frequency data obtained with the test constructs. Experiments were performed at minimum in duplicate.

Surface expression of gH

Analysis of gH surface expression was performed as previously described [27]. Briefly, CHO-K1 Cre cells were transfected with equimolar amounts of pcDNA3.1-gL and pME18s-gH. Cells were fixed at 24 hours post transfection, stained with anti-gH SG3 antibody, and detected with anti-mouse Alexa Fluor 488 antibody. Analysis was performed on a FACSCalibur controlled by CellQuest Pro (BD Bioscience) and gH expression was quantified with FlowJo (Tree Star). Experiments were performed at minimum in triplicate.

Confocal microscopy of transfected and infected cells

Confocal microscopy of melanoma cells transfected with pcDNA3.1-gL and either pME18s-gH[WT] or gH mutant constructs was performed as previously described [27]. For infection, melanoma cells were inoculated with 500 plaque forming units (PFU) of recombinant virus. For TK-GFP-BAC transfection, melanoma cells were transfected with pOka-TK-GFP-gB[Y881F], -gH[Δ834-841], and -gB[Y881F]/gH[Δ834-841] BACs using Lipofectamine 2000. Cells were fixed with 4% paraformaldehyde at post 24 hours for non-BAC transfected and infected cells and 72 hours for BAC transfected cells. Cells were probed for gH, early endosomes and the trans-Golgi-network using anti-gH mAb SG3 (mouse), anti-EEA1 mAb (rabbit; Novus Biological), and anti-TGN46 pAb (sheep; AbD Serotec), respectively. TK-GFP-BAC-transfected cells were probed with anti-IE62 (mouse; Chemicon International) and anti-ORF23 (polyclonal rabbit; [36]). Primary antibodies were detected with secondary antibodies, anti-mouse Alexa Fluor 555 (Invitrogen), anti-rabbit Alexa Fluor 488 (Invitrogen), and anti-sheep Alexa Fluor 647 (Invitrogen). Nuclei were stained with Hoechst 33342 (Invitrogen). Images were captured with a Leica SP2 AOBS Confocal Laser Scanning Microscope. Channel merging and image processing was performed with ImageJ and Photoshop.

Brightfield and fluorescence microscopy of syncytium

Melanoma cells were seeded in 6-well plates and infected with 500 PFU of pOka or recombinant gH mutant viruses. Images of syncytium were taken at 24, 36, and 48 hours post infection. Prior to each time point, infected cells were incubated with fresh media supplemented with Hoechst 33342 at 1∶1000 dilution for 10 minutes. Brightfield and fluorescence microscopy images were taken with an AX10 Microscope (Zeiss) equipped with an X-Cite Series 120 fluorescence excitation light source (Lumen Dynamics). Images were aligned and processed in Photoshop to enhance contrast for the cytoplasmic regions of the syncytium. To quantify the number of nuclei per syncytium at 36 hours post infection (hpi), 15 syncytia were randomly selected and the nuclei were visually counted in a single plane from images taken by a light microscope.

Replication kinetics, virus titration, and plaque size

As previously described [37], melanoma cells were infected with 1000 PFU of recombinant VZV and harvested at 24 hour intervals to examine replication kinetics. Viral titration was performed by 10-fold dilution on melanoma cells in triplicate. To access plaque size, stained plaques (n = 30) from titration plates at four days post infection were captured with an AX10 Microscope (Zeiss) with the plaques outlined and area calculated using ImageJ, as described [34].

Transmission electron microscopy of VZV-infected or VZV BAC-transfected melanoma cells

To examine infected cells, 12 mm glass coverslips were seeded with 2×10⁵ melanoma cells and infected with 1000 PFU of pOka-gH[Δ834-841] or pOka virus. To examine cells transfected with pOka-TK-GFP-gB[Y881F], -gH[Δ834-841], and -gB[Y881F]/gH[Δ834-841] BACs, 100 mm dishes seeded with 1.2×10⁶ melanoma cells were transfected with 30 µg of BAC DNA using Lipofectamine 2000. At 24 hours post transfection, cells were harvested and sorted for GFP positive cells using a BD InFlux Special Order sorter for enrichment. Sorted cells were then added to a cloning ring placed upon a 12 mm glass coverslip preseeded with 4×10⁵ melanoma cells. At 48 hours post reseeding for transfected cells and 72 hpi for infected cells, samples were fixed with 2% glutaraldehyde and 4% p-formaldehyde in 0.1M sodium cacodylate buffer pH 7.2 for 20 minutes. Cells were then treated with 1% osmium (OsO₄) for one hour and stained with 1% uranyl acetate overnight. Cells were dehydrated with multiple incubations of increasing concentrations of ethanol (50%, 70%, 95%, and 100%) followed by acetonitrile. Epon infiltration was performed by subsequent incubations with 1∶1 (Epon/Acetonitrile), 2∶1 (Epon/Acetonitrile), and 100% Epon. Samples were incubated at 65°C to allow for polymerization. Glass coverslips were dissolved by incubation in 49% hydrofluoric acid for 20 minutes. Areas of interest were trimmed. Ultrathin sections were prepared using an ultratome (Leica Microsystems) and placed upon formvar carbon film 100Mesh copper grids (Electron Microscopy Sciences). Grids were stained with 4% uranyl acetate and 0.2% lead citrate. Sections were visualized using a JEOL 1400 transmission electron microscope at 80 kV and digital photographs were captured with a Gatan Multiscan 701 digital camera.

Evaluation of gH mutant viruses in human skin xenografts

Skin xenografts were prepared in homozygous CB-17 scid/scid mice using human fetal skin tissue obtained according to federal and state regulations as previously described [12]. Briefly, implanted skin xenografts were inoculated with HELFs infected with wildtype pOka or mutant viruses. The inoculum titer for each virus was determined to confirm similar PFU/mL concentrations. Xenografts were harvested at 10 and 21 days post infection (dpi) and viral titers were determined. Confocal microscopy of sectioned infected skin xenografts was performed as previously described [34]. Briefly, 5 µm sections were deparaffinized and treated with citrate-based-Antigen Unmasking Solution (Vector) at high temperature following the manufacturer's instructions. Sections were then probed with primary antibodies, anti-gE Mab8612 (mouse; EMD Millipore), anti-ORF23 (polyclonal rabbit; [36]), and anti-TGN46 (sheep; AbD Serotec). Primary antibodies were detected with anti-mouse Alexa Fluor 555, anti-rabbit Alexa Fluor 488, and anti-sheep Alexa Fluor 647. Nuclei were stained with Hoechst 33342. Images were aligned and processed with Photoshop.

Transmembrane and cytoplasmic domain predictions

Amino acid sequences of gH homologues were obtained from protein searches on PubMed. Sequences were submitted to TOPCONS (http://topcons.cbr.su.se/) to identify transmembrane and cytoplasmic domains [38]. Hydrophobic residues in the predicted cytoplasmic domains were manually identified.

Statistical Analysis

All quantitative results were analyzed by either one-way or two-way ANOVA to determine statistical significance using Prism (Graphpad Software).

Amino acids 834-841 of the VZV gH cytoplasmic domain are critical for the regulation of cell fusion mediated by the gB/gH-gL complex

Expression of VZV gB, gL, and gH with amino acids 834-841 of its cytoplasmic domain absent (gH[TL]) because of a E834Stop mutation has been demonstrated to produce a high frequency of cell fusion events by the quantitative Cre reporter assay [27], [34]. This assay allows for the identification of domains that are required for fusion regulation based on enhanced fusogenicity (hyperfusogenicity) conferred by the mutations [34]. In contrast to gH[TL], the fusion frequencies for wildtype gH (gH[WT]) were only marginally greater than the vector alone indicating that the full length gH protein limits fusion under these conditions (Figure 1A and 2). Co-expression of gH[TL] with only gL did not induce fusion, further confirming that gB was a critical component of the cell fusion machinery. Expression levels and the molecular weights of gH[TL] were similar to gH[WT] in CHO cells, based on immunoprecipitation and western blots (Figure 1B), demonstrating that amino acids 834-841 in the gHcyt do not affect the synthesis, stability, or the post-translational processing of gH into its 118 or 130 kDa mature form in the transfected CHO cells used to evaluate cell fusion.

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Figure 1. Construction and in vitro expression of VZV gH mutants.

(A) VZV gH constructs with mutations in the predicted cytoplasmic domain (824-841) [29]. The solid black line indicates the length of the wildtype (WT) gHcyt (824-841) while the dotted line represents extension of the gHcyt length in the V5 and cMyc constructs. The predicted endocytosis YXXΦ motif is boxed in green. The V5 and cMyc sequences are boxed in solid red and white, respectively. The red characters indicate mutations made to the gHcyt. Asterisks represent stop codons. TM indicates the predicted transmembrane domain. (B) Western blot of gH from immunoprecipitation using anti-gH(SG3) antibody of lysates from CHO-K1 Cre cells transiently expressing vector only (Vector), wildtype gH only (WT only), gL only (gL only) or gH mutants with gL. Arrows indicate two molecular weights of gH, 118 and 130 kilodaltons (kDa). Molecular mass standard is indicated on the left.

https://doi.org/10.1371/journal.ppat.1004173.g001

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Figure 2. Truncation of the gHcyt causes enhanced in vitro cell-cell fusion.

Cell-cell fusion induced by gB and gH mutants were quantified by the Cre reporter fusion assay, with fusion events represented as a percentage relative to fusion events of gB/gH[TL]-gL. gB[WT], gB[Y881F], and gB[Y881/920F] were coexpressed with either gH[TL] or gH[WT] and gL. Vector only represents transfection with empty vectors. gB[WT] only represents expression of gB[WT] with empty vectors. gH[TL]-gL only and gH[WT]-gL only represent expression of gH[TL] and gH[WT] with gL, respectively, with an empty vector. The plus and minus symbols indicate the presence or absence of gL expressing vector, respectively. Data shown represents the mean of two independent experiments with the whiskers indicating the standard error of the mean (SEM). All values were evaluated by ANOVA analysis and found to be significantly different from gB/gH[TL]-gL (P<0.001).

https://doi.org/10.1371/journal.ppat.1004173.g002

To determine whether the gHcyt was critical for cell fusion regulation in combination with the gBcyt, the fusogenicity of the single substitution, gB[Y881F], and double substitution, gB[Y881/920F], were examined with gH[TL] and gH[WT] (Figure 2). When co-expressed with gH[TL] and gL, the gB[Y881F] and gB[Y881/920F] mutants induced levels of fusion at 60% and 137% greater than wildtype gB (gB[WT]), respectively, which was consistent with our previous report [34]. In contrast, both gBcyt mutants had fusion levels that were limited or below the level of detection when they were co-expressed with gH[WT]. Thus, the hyperfusion phenotype of gB[Y881F] and gB[Y881/920F] is dependent upon the absence of amino acids 834-841 of gH, suggesting that the cytoplasmic domains of gB and gH might function together in regulating cell fusion.

Regulation of gB/gH-gL mediated cell fusion by the gHcyt is dependent on the physical length of the domain

To further characterize regulation of cell fusion by the gHcyt, mutants were constructed that replaced the eight amino acids of 834-841 with either the 14 amino acid V5 epitope (gH[V5]) or the 10 amino acid cMyc epitope (gH[cMyc]) (Figure 1A). To confirm that there was no read through of the E834Stop nonsense mutation or disruption of the expression plasmid by the V5 sequence, a mutant that contained both the E834Stop and V5 epitope (gH[834StopV5]) was also constructed. All three gH mutants had protein expression profiles similar to gH[WT] in transfected CHO cells (Figure 1B). However, despite the absence of amino acids 834-841, the V5 and cMyc mutations resembled gH[WT] in having very limited capacity to induce cell fusion (Figure 3A). Similar to the increase in fusion levels observed by gH[TL] relative to gH[WT], gH[834StopV5] also had increased fusion in comparison to its corresponding control, the gH[V5]. Although a slight increase in fusion levels was observed between gH[834StopV5] and gH[TL], possibly because of the V5 sequence, the enhanced fusion levels caused by the gH[834StopV5] mutation demonstrated that read through past the 834Stop codon, like the gH[TL] mutation, did not occur. This strongly suggests that the gHcyt length is important for regulating cell fusion.

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Figure 3. Regulation of cell-cell fusion by gH is dependent on cytoplasmic domain length but motif independent.

(A) Cell-cell fusion between melanoma cells and CHO-K1 Cre cells expressing either gH[WT], gH[TL], gH[V5], gH[cMyc], gH[834stopV5], gH[Δ834-841], gH[Δ836-841], gH[Δ838-841], gH[Δ840-841], gH[834(IV)⁴], gH[834(QN)⁴], gH[Y835A], or gH[Y835F] with wildtype gB and gL. Fusion was quantified by the Cre reporter assay with fusion events represented as a percentage relative to fusion events of gB/gH[TL]-gL. Vector only represents transfection with empty vectors. gB only represents expression of gB with empty vectors. gH[TL]-gL only and gH[WT]-gL only represent expression of gH[TL] and gH[WT] with gL, respectively, with an empty vector. The plus and minus symbols indicate the presence or absence of gB or gL expressing vectors, respectively. (B) gH cell surface expression of melanoma cells transiently expressing gH mutants with gL quantified by nonpermeabilized staining for gH and flow cytometry. Surface expression is represented as a percentage relative to gH[WT]-gL. gL only represents expression of gL with empty vector. gH[TL] only and gH[WT] only represent expression of the two vectors with an empty vector. The plus and minus symbols indicate the presence or absence of gL expressing vectors, respectively. Data shown represents the mean of three independent experiments with the error bars indicating the SEM. Statistical differences between the mutants and gB/gH[TL]-gL for the cell-cell fusion assay and gH[WT]-gL for surface expression were evaluated by ANOVA (*P<0.05, **P<0.01, ***P<0.001). gH mutants with mean surface expression statistically (C) higher or (D) lower than gH[WL]-gL were examined on scatter plots comparing their mean surface expression relative to gH[WT]-gL and mean fusion levels relative to gB/gH[TL]-gL. The dotted line represents the trend line with Pearson r and R² values noted for each graph.

https://doi.org/10.1371/journal.ppat.1004173.g003

To further determine the importance of the physical length of the gHcyt rather than its sequence for regulating cell fusion, a series of truncation mutants was constructed: gH[Δ834-841], gH[Δ836-841], gH[Δ838-841], and gH[Δ840-841], containing eight, six, four, and two amino acid deletions from the carboxyl terminus of the gHcyt, respectively (Figure 1A). None of the truncation mutations changed the protein expression profile of gH in CHO cells (Figure 1B). The incremental deletions of the gHcyt caused stepwise increases in gB/gH-gL mediated cell fusion rather than an all-or-none increase in fusion levels that would be indicative of deletion of a specific functional motif (Figure 3A). As expected, the gH[Δ834-841] mutant induced levels of cell fusion similar to gH[TL], confirming again the importance of amino acids 834-841 for fusion regulation. This also demonstrated that the induction of cell fusion by gH[TL] was not caused by the type of mutation, whether nonsense or deletion, that was employed to eliminate expression of amino acids 834-841. The remaining Δ836-841, Δ838-841 and Δ840-841 deletions exhibited 56%, 73%, and 99% less cell fusion when compared with gH[TL], respectively (Figure 3A). Thus, this data provided further evidence that regulation of gB/gH-gL mediated cell-cell fusion by the gHcyt depends on the length of the cytoplasmic domain and not the specific sequence or intrinsic properties of the amino acids in the domain.

The presence of hydrophobic residues in the cytoplasmic domain of viral fusion-related proteins has been suggested to have a role in regulating cell fusion [39]. The predicted gHcyt of alphaherpesvirus homologues, including VZV, contain highly hydrophobic residues (leucine, isoleucine, and, phenylalanine) (Figure S1), suggesting that the presence of hydrophobic residues in the gHcyt might be a regulatory factor for cell fusion. To test this hypothesis, two gH mutants were constructed, gH[834(IV)⁴] and gH[834(QN)⁴], in which amino acids 834-841 were substituted with a series of alternating isoleucine and valine residues or glutamine and arginine residues, respectively (Figure 1A). The 834(IV)⁴ substitution was highly hydrophobic, while the 834(QN)⁴ was highly hydrophilic (Figure S2A). Similar to gH[WT], both mutations induced little or no detectable fusion (Figure 3A), which demonstrated that fusion regulation by the gHcyt was not dependent upon the presence of hydrophobic residues and further confirmed that the regulation was motif independent.

Given that expression of gH on the surface of the cell would be expected to influence cell fusion, FACS analysis with anti-gH antibody was performed on CHO cells expressing the gH[WT] or gH mutants along with gL (Figure S3) (Figure 3B). gH[TL] and gH[WT] protein were not detected on the cell surface in the absence of gL, which was consistent with previous findings [27]. Surface expression levels of the gH mutants with gL could be separated into three distinct groups, with mutants having significantly greater, equal, or reduced surface expression compared to gH[WT]. gH[TL], gH[834StopV5], gH[Δ834-841], and gH[Δ836-841] exhibited surface expression levels that were 12%, 20%, 24%, and 19% greater than gH[WT], respectively. gH[V5] and gH[834(QN)⁴] mutants had surface expression levels equivalent to gH[WT], while gH[cMyc], gH[Δ840-841], and gH[834(IV)⁴] had surface expression levels that were 15%, 30%, and 10% less than gH[WT] (Figure 3B). To determine if there was a correlation between the surface expression and cell fusion effects of these mutants, the mean levels of their surface expression were plotted against their cell fusion levels on a scatter plot along with gH[WT] and gH[TL]. For both the greater and lesser surface expression groups, no significant correlation was found between their surface expression and cell fusion levels. A Pearson R value of 0.53 and R² value of 0.28 was determined for the mutants with increased surface expression relative to gH[WT] with a P value of 0.29 (Figure 3C) and while a high R value of 0.713 and R² value of 0.54 was calculated for those mutants with reduced surface expression, the P value was 0.15, making the correlation not statistically significant (Figure 3D). Thus, while gB-gH/gL mediated cell fusion requires the presence of gH on the surface of the cell, greater levels of surface expression did not correlate with the induction of cell fusion and the induction or extent of fusion was not a result of increased gH surface expression.

Loss of cell fusion regulation is not a result of aberrant gH intracellular trafficking

To determine if the changes in cell fusion associated with the gHcyt mutations might be due to altered intracellular trafficking, the subcellular localization of gH mutants in melanoma cells co-expressing gL was examined by confocal microscopy. As expected from previous reports, gH[WT] had a pattern of juxta-nuclear localization as well as a punctate localization in the cytoplasm [27] (Figure 4). Co-staining with organelle markers confirmed the co-localization of gH with the trans-Golgi network (anti-TGN-46) at the juxta-nuclear position and with some early endosomes, detected with anti-EEA1 antibody, appearing as punctae in the cytoplasm. gH[WT]-like subcellular localization was also observed for the gH[V5], gH[cMyc], gH[834StopV5], gH[Δ834-841], gH[Δ836-841, gH[Δ838-841], and gH[Δ840-841] mutants (Figure 4) and for gH[834(IV)⁴] and gH[834(QN)⁴] (Figure S2B). Thus, amino acids 834-841 of the gHcyt did not have a functional role in gH intracellular trafficking or its internalization from the cell surface.

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Figure 4. Amino acids 834-841 of the gHcyt are dispensable for intracellular trafficking and endocytosis of gH in vitro.

Confocal microscopy of melanoma cells transiently expressing gH[WT] or gH mutants with gL at 24 hours post transfection. Cells were stained for gH (red), early endosome antigen (EEA1; green), trans-Golgi network (TGN46; green), and nuclei (Hoechst 33342; blue). White arrows indicate colocalization of EEA1 and gH, which highlight representative endocytic vesicles containing gH. The scale bars represent 20 µm.

https://doi.org/10.1371/journal.ppat.1004173.g004

The gHcyt YXXΦ motif does not regulate cell fusion

The role of the ⁸³⁵YNKI⁸³⁸ motif was examined in the virus-free transfection system by constructing two mutants with the tyrosine (Y835) residue substituted with either alanine (Y835A) or phenylalanine (Y835F). The alanine substitution fully disrupted the residue, while the phenylalanine substitution served to determine if the hydroxyl group had a functional role in endocytosis (Figure 1A). Neither substitution affected levels of gH protein expression or processing compared to gH[WT] (Figure 1B). Similar to gH[WT], both gH[Y835A] and gH[Y835F] were able to colocalize with EEA-1 markers, indicating that the protein was present in endocytic vesicles (Figure 4, white arrows). Thus, gH endocytosis was not prevented by disrupting the YXXΦ motif.

Similar to gH[WT], both the Y835A and Y835F substitutions induced little to no detectable fusion in the Cre reporter fusion assay (Figure 3A). Cell surface expression levels were different between the mutants, with gH[Y835A] having levels of surface expression greater than gH[WT] and gH[Y835F] having levels similar to the gH[WT] (Figure 3B). This further confirmed the lack of correlation between the induction of cell fusion observed in the Cre reporter fusion assay and increased gH surface expression. These results demonstrated the ⁸³⁵YNKI⁸³⁸ endocytosis motif in the gHcyt did not contribute to regulation of gB/gH-gL mediated cell fusion in the absence of other VZV proteins and was consistent with evidence that the regulation of gB/gH-gL mediated cell fusion depended on the physical length of the gHcyt and not the presence of specific residues or motifs.

Deletion of the gHcyt significantly impairs VZV pathogenesis in skin

To determine effects of the gHcyt mutations and dysregulation of syncytia formation on VZV pathogenesis in vivo, human skin xenografts were infected with pOka, pOka-gH[Δ834-841], pOka-gH[Y835A], pOka-gH[Y835F], pOka-gH[V5], and pOka-gH[TL] viruses at similar inoculum titers (Figure 12A). At 10 dpi, viral titers from skin xenografts infected with pOka-gH[Y835A] and pOka-gH[Y835F] mutants were not significantly different from pOka, indicating that the ⁸³⁵YNKI⁸³⁸ motif had no role in skin pathogenesis. The pOka-gH[V5] virus also replicated at similar levels to pOka. In contrast, infectious virus was not recovered from any of the six xenografts infected with pOka-gH[TL] or from five of the six pOka-gH[Δ834-841]-infected xenografts; the titer of the sixth implant was <1 log₁₀ (Figure 12B). Similar results were observed in xenografts recovered at 21 dpi after inoculation of pOka-gH[Y835A], pOka-gH[Y835F], and pOka-gH[V5] viruses, which had titers that were again comparable to pOka. pOka-gH[TL] virus was not isolated from five of the six xenografts with the sixth having a titer of <1 log₁₀. The pOka-gH[Δ834-841] infected implants showed some limited levels of viral replication with four of the six implants having a mean titer of 1.5 log₁₀, approximately 1.3 log₁₀ lower than the mean titers of pOka-infected xenografts. Thus, the gHcyt played a critical role in VZV skin pathogenesis and was dependent on amino acids 834-841 of gH.

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Figure 12. Truncation of the gHcyt restricts VZV pathogenesis in human skin xenografts.

(A) Mean titers of inoculum of pOka, pOka-gH[Y835A], -gH[Y835F], -gH[V5], -gH[Δ834-841], and -gH[TL] viruses used for infection of human skin xenografts. Inoculum was titrated in triplicate. The error bars indicate the SEM. (B) Box and whiskers plot of skin xenograft infection at 10 (white) and 21 (grey) days post infection (dpi) titrated in triplicate on melanoma cells. The boxes indicate the 10–90% percentile with the solid bar inside the box representing the median and the whiskers indicating the minimum and maximum of all titers. The numbers along the x-axis represent the frequency of virus-positive xenografts per number inoculated for each virus. Statistical differences were determined by two-way ANOVA by comparing the titers for the gH mutants to either pOka at 10 or 21 dpi. (**P<0.05, ***P<0.001).

https://doi.org/10.1371/journal.ppat.1004173.g012

To further examine VZV skin pathogenesis, confocal microscopy was performed on sections from xenografts at 21 dpi that yielded the highest viral titers. Infected cells were identified by ORF23 and gE expression, with the skin layers visualized by staining for TGN46 and nuclei (Figure 13). As previously reported [34], the lesion in the pOka-infected xenograft showed penetration and spread of the virus across the epidermal and basal layer into the dermal layer (Figure 13, top left). Infection was also visible in hair follicles and syncytia were identified scattered throughout the lesion (Figure 13, A). Similar to pOka, the single pOka-gH[Δ834-841]-infected xenograft that had a titer of 2 log₁₀ also showed penetration and spread through all three layers of the skin (Figure 13, B). In addition, differences in syncytia morphology and size were not apparent between pOka and pOka-gH[Δ834-841]. Lesions from xenografts infected with pOka-gH[Y835A], pOka-gH[Y835F], and pOka[gH-V5] viruses as well as ORF23 and gE staining were all morphologically similar to pOka. Lesions were not identified in the single xenograft from which pOka-gH[TL] was recovered, which correlated with the extremely low viral titer (Figure 12B).

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Figure 13. Pathogenesis of VZV with mutations in the gHcyt in human skin xenografts.

Confocal micrographs of 5 µm sections of human skin xenografts with the highest titers infected with either pOka or pOka-gH[Δ834-841], -gH[Y835A], -gH[Y835F], -gH[TL], and -gH[V5] at 21 dpi. The sections were stained for capsids (ORF23; green), gE (red), TGN46 (violet), and nuclei (Hoechst 33342; blue). The scale bar for skin implants infected with pOka (top left) and gH[Δ834-841] (top right) represent 200 µm. White squares are shown in higher magnification immediately below (A = pOka, B = Δ834-841). Higher magnification images consist of a four-color merge (top), two color-ORF23 and gE merge (bottom left), and two color-TGN46 and nuclei merge (bottom right). Lesions were not detected for pOka-gH[TL]-infected xenografts. The scale bars for higher magnification images represent 50 µm.

https://doi.org/10.1371/journal.ppat.1004173.g013

Skin pathogenesis and regulation of cell-cell fusion is mediated by the cytoplasmic domains of both gH and gB

This study elucidates the functional domains of gH and gB important for cell fusion and demonstrates that the regulation of syncytia formation is necessary to support effective skin pathogenesis. In the case of VZV, the current model of gB acting as the primary fusogen is supported by the requirement for gB as demonstrated by the inability of gH[TL] and gL in the absence of gB to induce the high levels of fusion observed with gB/gH[TL]-gL. Regulation of cell fusion by gH was determined to depend on the length of its cytoplasmic domain with truncations of the domain causing increased cell fusion, indicative of a loss in regulation. Deletions and substitutions of amino acids 834-841 of the gHcyt confirmed that the regulation was not dependent upon a unique motif or specific biochemical properties such as hydrophobicity. Given that direct protein interactions commonly depend upon the presence of a specific motif or domain, it is unlikely that the gHcyt functions by a direct interaction with either a host or viral protein [41]. However, while it might not interact directly, the gHcyt is proposed to operate as a gate keeper using its physical length to control access of functional domains within neighboring proteins.

The gB/gH-gL mediated cell-cell fusion is also regulated by the gBcyt ITIM which has the potential to be phosphorylated at Y881 [34]. VZV mutagenesis to substitute the tyrosine residue with a phenylalanine (gB[Y881F]), which prevents phosphorylation of the residue, resulted in exaggerated syncytia formation in melanoma cells and significantly reduced skin pathogenesis. In contrast to infected cells, the inability of the gB[Y881F] mutation to induce high levels of fusion when expressed with gH[WT] in the Cre reporter assay strongly supports the notion that the full length gHcyt has complex fusion regulatory properties that are apparent during VZV infection. While a direct interaction of gB and gH-gL has not been confirmed, cell fusion requires both to be present on the cell surface and it is also probable that these two transmembrane glycoproteins, and thus their cytoplasmic domains, are located within close proximity on cell membranes. As a gate keeper, the gHcyt could function as a regulator of phosphorylation of the ITIM of the gBcyt by controlling either kinase or phosphatase access to Y881 residue. Therefore, the absence of the gHcyt gate keeper function in the gH[TL] enables gB[Y881F] to readily enter a dysregulated state and enhance fusion. This dysregulated state was detrimental to the virus as melanoma cells transfected with pOka BAC containing the dual gB[Y881F]/gH[Δ834-841] mutation were unable to effectively establish a progressive infection, while retaining the capacity to induce exaggerated syncytia formation. These data confirm that losing syncytia regulation significantly limits viral replication and spread. The gBcyt might also contribute to regulation of the early steps of cell fusion, as demonstrated by the LL871/872AA substitution in the HSV-1 gBcyt initiating fusion significantly more rapidly than the wildtype, while having surface expression levels that were similar to the wildtype [42]. The VZV gBcyt also contains a similar dileucine motif (⁹⁰⁴LL⁹⁰⁵). Further investigation is needed to determine if the motif has a similar functional regulatory role in fusion initiation. Together, these data establish that VZV gB and gH participate in a complex regulatory system to limit syncytia formation.

Regulation of cell-cell membrane fusion differs from virion envelope and cell membrane fusion

Regulation of fusion between the virion envelope and cell membrane during entry has been postulated to be different from cell-cell fusion [43]. Our observations support this concept because while truncating the gHcyt increased cell fusion when transfected with gB and gL in vitro and caused exaggerated syncytia formation when introduced into the virus, the morphology of the viral particles and their presence on cell surfaces was similar to pOka. Since regulation of cell-cell fusion also depends upon phosphorylation of the gBcyt ITIM, identical regulation for both virion envelope and cell fusion would require the presence of kinases and phosphatases with the capacity to alter tyrosine phosphorylation within the virion as well as in infected cells. While mass spectrometry studies have not been performed on extracellular VZV virions, studies of HSV-1 virions have not shown incorporation of phosphatases or tyrosine kinases [44]. It has also been noted that the lipid and protein composition of the viral envelope membrane would be expected to be different from the cell membrane, which could also change the regulation of fusion [45]. While further studies are necessary, our findings support differences in how VZV gH and gB contribute to the regulation of cell fusion and virion entry.

Differences in the regulation of gB/gH-gL mediated cell fusion by the cytoplasmic domain of HSV and VZV gH

In contrast to VZV, syncytia formation is not a canonical characteristic of HSV-1 infection which suggests that the fusion machinery for HSV-1 controls cell fusion more tightly than VZV. While syncytial (syn) strains of HSV-1 have been identified with mutations in either gB (UL27), gK (UL53), UL20, or UL24 [46], [47], HSV syn viruses with mutations in the HSV gHcyt have not been reported. Regulation of syncytia formation by the HSV gHcyt has been demonstrated by the reduction of the syncytial phenotype in cells either infected with the HSV-1 gB[V855A] syn mutant virus when the gHcyt was truncated by nine amino acids or a gH null/gB[V855A] syn mutant virus was trans-complemented with a plasmid expressing the truncated gH [43], [48], [49]. This was also observed with in vitro fusion studies as expression of the syncytial gB[A885V] mutant with the truncated gH reduced its hyperfusion levels from 200% to 150% of wildtype fusion levels [50]. These findings suggest the V855A substitution in the HSV-1 gBcyt inhibited the regulatory control of the gBcyt over cell fusion that was then rescued by the truncation of the HSV gHcyt. This supports the model that cell fusion is jointly regulated by the gBcyt and gHcyt. In vitro cell fusion studies involving transient expression of HSV-1 glycoproteins have provided further evidence of the role of the gHcyt in fusion and its regulation. Cell fusion was inhibited when the HSV-1 gHcyt was either completely deleted and replaced with analogous domains from other glycoproteins or when both the gH transmembrane and cytoplasmic domains were substituted with a glycosylphosphatidylinositol (gpi)-addition and expressed with gB, gD, and gL [51],[52]. Similar to VZV gH, truncation of the final six amino acids of HSV-1 gHcyt increased levels of cell fusion by 50% relative to the wildtype gH when transfected with gB[WT], gD, and gL [50]. Whether inserting this gH mutation into the viral genome would produce a HSV syn mutant is not known. Also consistent with our observations about the VZV gHcyt, the length of the HSV-1 gHcyt might be important for its contribution to regulation of cell fusion [53]. An insertion of five amino acids at 824L, increasing the length of the gHcyt from 14 to 20 amino acids, inhibited cell fusion in the presence of gB[WT]. Nevertheless, in the context of infection, VZV and HSV regulation of cell fusion and syncytia formation differ since truncation of VZV gH caused exaggerated syncytia formation, while HSV gH truncation reduced syncytia formation by the gB syn mutant. Thus, the VZV gB/gH-gL core fusion machinery has characteristics that change its fusogenicity capacity compared to HSV.

While the VZV gHcyt has been predicted to be 18 amino acids in length, the HSV gHcyt is only 14 amino acids long and no motifs or sequences are conserved between the two homologues. Furthermore, induction of cell fusion by HSV in vitro requires not only gB/gH-gL, but also gD. These differences in sequence length and requirements for in vitro cell fusion make it challenging to compare the processes of HSV-1 and VZV cell fusion. However, it is apparent that the gHcyt domains of both viruses perform a significant function in cell fusion regulation which is linked to the regulation of their gB counterparts.

The gHcyt YNKI motif does not have a functional role in syncytia formation

The gH ⁸³⁵YNKI⁸³⁸ motif does not mediate gB/gH-gL cell-cell fusion in vitro, virus-induced syncytia formation, and spread in cultured cells or skin pathogenesis. Disruption of the motif also did not alter gH expression or trafficking in melanoma cells that were transiently expressing gH-gL or infected with recombinant mutant VZV. These results are in contrast to an earlier report which indicated that the ⁸³⁵YNKI⁸³⁸ motif was a functional endocytosis motif based on effects of an identical tyrosine to alanine substitution examined by confocal microscopy and using a biotin endocytosis assay. While confocal microscopy was used to examine endocytosis in both studies, the first observations were based upon infecting HeLa cells with recombinant vaccinia virus (VV) expressing the T7 polymerase and co-transfecting with pTM1 plasmids, a T7-driven vector, expressing gH and gL [29]. Given that VV can induce significant changes in the cytoskeletal network of host cells [54] and the cell proteome [55], the modified intracellular environment and the additional VV proteins might have contributed to the inhibition of endocytosis of gH[Y835A]. In addition, we have shown that gH-gL co-expression to be insufficient for cell-cell fusion [27], [34] in contrast to a previous report [56]. HeLa cells infected with VV expressing gH with the Y835A substitution and gL showed increased syncytia size, which might also be explained by VV effects [33]. Vaccinia virus expresses its own acid induced membrane fusion protein on the surface of the infected cell [57] and regulates actin polymerization in the host cell, which might prime the cell to be more fusogenic. Our approach to examine the ⁸³⁵YNKI⁸³⁸ motif more accurately models the function and activity of gH since the virus-free transfection system does not have additional molecules that could potentially affect gH trafficking. Furthermore, gH was also examined under the context of infection, which allows for usual expression of other VZV proteins that could facilitate gH trafficking in infected cells by mechanisms other than the ⁸³⁵YNKI⁸³⁸ motif.

Enhanced syncytia formation limits skin pathogenesis

The truncation of the VZV gHcyt increased syncytia formation and significantly reduced skin pathogenesis. A similar phenotype was observed with the gBcyt ITIM mutation indicating that exaggerated syncytia formation limits the ability of VZV to spread effectively in the skin. This demonstrates a direct link between skin pathogenesis and cell-cell fusion. The epidermis consists of three main layers, with the granular layer facing the surface, followed by the spinous layer and the inner basal layer. The epidermis is constantly in flux due to continued cell shedding that is quickly replaced by differentiated keratinocytes [58]. Exaggerated syncytia formation is proposed to limit the ability of the virus to effectively penetrate the skin, causing the lesion to remain near the surface (Figure 14A). If the rate of penetration is less than the rate of differentiation and migration of keratinocytes from the basal layer, then the epidermis would drive the infected cells out through the granular and spinous layers, preventing spread to deeper layers of the spinous and basal layer. When syncytia formation is regulated appropriately as in pOka-infected tissue, the virus is able to effectively spread and penetrate the skin creating the typical cutaneous lesions (Figure 14B). While this model requires further study, the present study demonstrates that tight regulation of syncytia formation through complementary functions of the cytoplasmic domains of gB and gH is critical for VZV pathogenesis in skin.

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Figure 14. Proposed model for limited skin pathogenesis of VZV caused by exaggerated syncytia formation.

Schematic of human skin with the three main layers denoted: G = Granular, S = Spinous, and B = Basal. (A) Exaggerated syncytia formation by pOka-gH[Δ834-841] and pOka-gH[TL] limits the virus from being able to spread effectively and penetrate the skin, causing the lesion to remain near the surface. (B) In contrast, normal syncytia formation by pOka allows the virus to easily spread and penetrate the basal layer allowing for expansion of the lesion.

https://doi.org/10.1371/journal.ppat.1004173.g014