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Journal of Virology, January 2009, p. 228-240, Vol. 83, No. 1
0022-538X/09/$08.00+0     doi:10.1128/JVI.00913-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Deletion of the First Cysteine-Rich Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain Abolishes the gE and gI Interaction and Differentially Affects Cell-Cell Spread and Viral Entry{triangledown}

Barbara Berarducci,* Jaya Rajamani, Mike Reichelt, Marvin Sommer, Leigh Zerboni, and Ann M. Arvin

Departments of Pediatrics and Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305

Received 1 May 2008/ Accepted 8 October 2008


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ABSTRACT
 
Varicella-zoster virus (VZV) glycoprotein E (gE) is the most abundant glycoprotein in infected cells and, in contrast to those of other alphaherpesviruses, is essential for viral replication. The gE ectodomain contains a unique N-terminal region required for viral replication, cell-cell spread, and secondary envelopment; this region also binds to the insulin-degrading enzyme (IDE), a proposed VZV receptor. To identify new functional domains of the gE ectodomain, the effect of mutagenesis of the first cysteine-rich region of the gE ectodomain (amino acids 208 to 236) was assessed using VZV cosmids. Deletion of this region was compatible with VZV replication in vitro, but cell-cell spread of the rOka-{Delta}Cys mutant was reduced significantly. Deletion of the cysteine-rich region abolished the binding of the mutant gE to gI but not to IDE. Preventing gE binding to gI altered the pattern of gE expression at the plasma membrane of infected cells and the posttranslational maturation of gI and its incorporation into viral particles. In contrast, deletion of the first cysteine-rich region did not affect viral entry into human tonsil T cells in vitro or into melanoma cells infected with cell-free VZV. These experiments demonstrate that gE/gI heterodimer formation is essential for efficient cell-cell spread and incorporation of gI into viral particles but that it is dispensable for infectious varicella-zoster virion formation and entry into target cells. Blocking gE binding to gI resulted in severe impairment of VZV infection of human skin xenografts in SCIDhu mice in vivo, documenting the importance of cell fusion mediated by this complex for VZV virulence in skin.


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INTRODUCTION
 
Varicella-zoster virus (VZV) is a human alphaherpesvirus and the causative agent of varicella (chicken pox). VZV infects the sensory ganglia, where it establishes lifelong latency, and causes herpes zoster (shingles) upon reactivation (8). VZV exhibits tropism for T cells (28, 29), which appear to transport the virus from the site of inoculation to the skin during the primary infection through a cell-associated viremia; cell fusion during skin infection results in the formation of characteristic large polykaryocytes and vesicular lesions (8, 27).

The VZV genome (~125 kb) encodes nine putative glycoproteins, which are known or presumed to contribute to the different steps of VZV replication: attachment and entry into the target cell, envelopment of the viral particles, cell-cell spread, and egress (8). Glycoprotein E (gE), the product of open reading frame 68 (ORF68), is a 623-amino-acid (aa) type I membrane protein that is essential for viral replication (34, 40) and involved in cell-cell fusion and secondary envelopment (3, 9, 35, 36, 50, 53).

gE, which is conserved among the alphaherpesviruses, is the most abundant glycoprotein expressed in VZV-infected cells (19). The cytosolic C terminus of gE (aa 562 to 623) contains sequences important for gE trafficking between the plasma membrane and the trans-Golgi network (TGN) of infected cells (1, 25, 49, 62, 65, 66). Alteration of the proper gE trafficking during VZV infection by deletion of the cytoplasmic C-terminal domain or mutation of the endocytosis motif, YAGL, located in this region had lethal effects (43); this motif mediates recycling of gE from the plasma membrane to the TGN, the site of secondary envelopment (17, 38, 49, 65). The cytosolic domain is important in the regulation of gE trafficking and secondary envelopment in other alphaherpesviruses, as well (5, 15, 16, 37, 59).

As we have reported, VZV gE differs from its homologues in the alphaherpesviruses because the extracellular domain of VZV gE (aa 1 to 544) contains a large nonconserved N-terminal region (aa 1 to 188). This unique domain is essential for VZV replication, and its mutagenesis alters cell-cell spread and secondary envelopment (3). A single amino acid change in the N-terminal region (D150N) of the spontaneously occurring VZV mutant VZV-MSP has been shown to accelerate cell-cell spread in vitro and in vivo (53), further indicating the involvement of the unique gE N-terminal region in VZV-induced cell fusion. Interestingly, the unique gE N-terminal domain has been recently shown to bind to the cellular protein insulin-degrading enzyme (IDE) (31); this interaction has been reported to have functions in VZV entry and cell-cell spread (30).

As in the other alphaherpesviruses, VZV gE forms noncovalent heterodimers with gI (ORF67). While not essential for VZV replication in vitro, gI is involved in posttranslational modification and trafficking of gE, cell-cell spread, and secondary envelopment of virions (34, 40, 48, 57, 61). Deletion or mutation of gI affected gE conformation and cellular localization and disrupted the extensive syncytium formation that is the hallmark of VZV replication (7, 34, 40). Importantly, whereas gI is dispensable for VZV replication in vitro, studies with the SCIDhu mouse system (44, 63) showed that gI is essential for VZV infection of human skin and T cells (21, 42). Surprisingly, while gI is important for the pathogenesis of VZV infection of human dorsal root ganglion (DRG) xenografts, it is not essential for viral replication and cell-cell spread in human neurons and satellite cells in vivo (64).

In herpes simplex virus (HSV), the extracellular region of gE mediates binding to gI (2, 52). Formation of the HSV gE/gI heterodimer contributes to the sorting of nascent virions to the lateral surface in epithelial cells and cell-cell spread (10-13, 24, 51, 59). The HSV gE/gI complex, as well as gD, is also involved in secondary envelopment of cytoplasmic virions in the TGN (14). HSV gE/gI on the lateral surfaces of epithelial cells colocalized with the adherent junction component β-catenin, but not with ZO-1, a protein of the tight junction (13). In contrast, VZV gE with or without gI accelerated tight-junction formation between polarized epithelial cells and colocalized with the tight-junction protein ZO-1 (41). The VZV and HSV gE/gI complexes function as receptors for the Fc portion of human immunoglobulin G (IgG) (22, 23, 32, 33). The HSV and pseudorabies virus gE/gI heterodimer is also a determinant of neuroinvasion and neurovirulence in vivo in animal models (12, 39, 58).

Our interest is in dissecting the functions associated with the large extracellular region of VZV gE during viral replication in vitro and in VZV pathogenesis in human xenografts in the SCIDhu mouse model in vivo (3). Here, we used mutagenesis of the VZV genome to investigate the possible functions associated with the first cysteine-rich region of the gE ectodomain, aa 208 to 236, during VZV replication and pathogenesis. This region contains 4 of the 10 gE extracellular cysteines. We show that this motif is required for gE binding to gI, but not to IDE. While it was not essential for VZV entry into primary T cells or melanoma cells, this region was necessary for efficient cell-cell spread in vitro and for VZV virulence in human skin xenografts in vivo. Mutagenesis of this cysteine-rich region showed that domains of the VZV gE ectodomain required in cell-to-cell spread and viral entry are distinct; residues involved in gE/gI heterodimer formation are important in the former but not in the latter process.


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MATERIALS AND METHODS
 
Construction of the {Delta}Cys208-236 gE mutant cosmid. The complete genome of the VZV parental Oka strain (P-Oka) is contained in four overlapping SuperCos1 cosmid vectors (Stratagene, La Jolla, CA) designated pFsp73 (nucleotides [nt] 1 to 33128), pSpe14 (nt 21795 to 61868), pPme2 (nt 53755 to 96035), and pSpe23 (nt 94055 to 125124) (46). ORF68 (nt 115911 to 117782 in P-Oka; nt 115808 to 117679 in the Dumas strain) is located in the unique short region of the VZV genome in the pSpe23 cosmid. The pBSK{Delta}KpnI-EcoRI-SacI plasmid containing the ORF68 sequence in the SacI fragment from the pSpe23 cosmid (nt 112015 to 118091 in P-Oka) was previously described (3). In this plasmid, the unique BglII and EcoRI sites were used to introduce the mutation into ORF68. The mutant {Delta}Cys208-236 contains a deletion from aa 208 to 236 and a 12-nt linker containing a NotI site (underlined) (5'-TTGCGGCCGCAA-3') (3) inserted at the site of deletion, between aa 207 and 237. The deletion/linker insertion mutant was constructed in two PCR steps as previously described (3), using the following primers: Cys{Delta}-fw external primer (5'-GATAGCGGGGAACGGTTAATG-3'), Cys{Delta}-rev internal primer (5'-CGCTTGCGGCCGCAAGGTTAATGACGGCAAAAAGCTC-3'), Cys{Delta}-rev external primer (5'-GTACAACCGGAATTCATATGAGA-3'), and Cys{Delta}-fw internal primer (5'-CGCTTGGCGGCCGCAAGCGGAAAATACTAAAGAGGATCA-3'). The PCR product was cloned into the pCR4-TOPO vector (Invitrogen, Carlsbad, CA) and sequenced to verify the mutation.

The PCR product was cut with BglII and EcoRI and cloned into the pBSK{Delta}KpnI-EcoRI-SacI plasmid, replacing the wild-type (wt) fragment. The pBSK{Delta}KpnI-EcoRI-SacI plasmid, containing the gE mutation, was restricted with AvrII and SgrAI and inserted into the pSpe23{Delta}AvrII cosmid, in which the AvrII site in position 112956 in the P-Oka genome (Dumas strain, nt 112854) is a unique site, replacing the wt AvrII-SgrAI fragment (nt 112956 to 117355 in P-Oka). Two independently derived mutant pSpe23-{Delta}Cys208-236 cosmids were sequenced to verify the mutation (Elim Biopharmaceuticals, Inc., Hayward, CA) and used to produce recombinant VZV.

Cosmid transfection, DNA isolation, and infectious-focus assay. Cosmid DNA preparation and transfection were done as previously described (34, 54). DNA was isolated from transfected melanoma cells using DNAzol (Gibco BRL, Grand Island, NY), and PCR and sequencing were performed to confirm the expected mutations. Sequencing was performed by Elim Biopharmaceuticals, Inc., Hayward, CA. Infectious focus assays were performed with melanoma cells and human embryonic lung fibroblasts (HELF) as previously described (3). Cells were fixed in 4% paraformaldehyde and stained with polyclonal anti-VZV human immune serum (44) and secondary anti-human biotin (Vector Laboratories, Inc., Burlingame, CA). The cells were stained with the Fast Red substrate (Sigma). Two sets of experiments were performed, and the titers of samples collected at each time point were determined in triplicate. Statistical differences in the titers of the mutant and the control viruses at each time point were determined by Student's t test.

Construction of expression plasmids and transfection. The wt gE and the {Delta}Cys208-236 mutant gE sequences were cloned in the expression plasmid pCDNA3.1+ (Invitrogen, Carlsbad, CA). Wt gE and mutant {Delta}Cys208-236 sequences were amplified using the gEp-fw primer containing the Kozak sequence after the HindIII site (5'-CGCAAGCTTGCCACCATGGGGACAGTTAATAAACCTG-3') and the gEp-rev primer (5'-CGCCTCGAGTCACCGGGTCTTATCTATATAC-3') containing an XhoI site (the restriction enzyme sites are underlined in the sequences above). The PCR fragments were cloned into the pCR4-TOPO plasmid (Invitrogen, Carlsbad, CA) and sequenced. The pCR4-TOPO plasmids containing the different PCR fragments were digested with HindIII and XhoI, and the fragments were inserted into pCDNA3.1+ in the HindIII and XhoI sites.

To analyze gE dimer formation, the hemagglutinin (HA) tag (YPYDVPDYA peptide) or the FLAG tag (DYKDDDDK peptide) was inserted by PCR at the C terminus of the wt gE and mutant gE before the stop codon using the EcoRI-fw primer (5'-CTCTCTCATATGAATTCCGGTTG-3'; the EcoRI site, nt 1227 to 1232 of ORF68 in P-OKA, is underlined) and the HA-rev primer (5'-CGCCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGGGTACCGGGT CTTATCTATATAC-3'; the XhoI site is underlined, and the HA sequence is indicated in boldface) or the FLAG-rev primer (5'-CGCCTCGAGTCACTTATCGTCGTCATCCTTGTAATCCCGGGTCTTATCTATATAC-3'; the XhoI site is underlined, and the FLAG sequence is indicated in boldface). The PCR fragments were cut with EcoRI and XhoI and cloned into pCDNA3.1+gE or pCDNA3.1+{Delta}Cys208-236, replacing the EcoRI-XhoI wt fragment. Hek-293 cells (ATCC CRL-1573) were plated in 100-mm dishes at a density of 8 x 106 cells; 24 h later, the cells were transfected with 26 µg of plasmid with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Forty-eight hours posttransfection, the cells were harvested in radioimmunoprecipitation (RIPA) buffer and analyzed by immunoprecipitation.

Immunoprecipitation and Western blotting. Melanoma cells were infected with the recombinant rOka-{Delta}Cys mutant, along with the rOka control, and protein lysates were collected in RIPA buffer for analyzing the gE/gI interaction or in 25 mM Tris-HCl, pH 7.4, 5 mM EDTA, 15 mM NaCl, and 0.1% NP-40 for studying the gE/IDE interaction (30); lysis buffer was supplemented with protease inhibitor cocktail (Complete minitablet; Roche Inc., Indianapolis, IN). Protein lysates from uninfected melanoma cells were used as a negative control. The cell lysates were precleared by incubating them with 30 µl of protein A-Sepharose beads (GE Healthcare Life Sciences, New Jersey, and Pierce Biotechnology, Inc., Illinois) for 1 h at 4°C. In the analysis of the gE/IDE and gI/IDE interaction, IDE was immunoprecipitated using a mouse anti-IDE monoclonal antibody (MAb) (Covance) at a dilution of 1:250, gE with 1 µg/ml anti-gE MAb (Chemicon, Temecula, CA), and gI with an anti-gI MAb (6B5) (60). For analysis of the gI/gE interaction, immunoprecipitation of gI was performed using the anti-gI MAb 6B5; for immunoprecipitation of gE, MAb 3B3 (53) was cross-linked to the beads using the ExactaCruz immunoprecipitation kit (Santa Cruz Biotechnology, Inc., California) to avoid interference with the detection of gI by the antibody heavy chain in immunoblotting. For the gE-gE interaction analysis, Hek-293 cells were transfected with pCDNA3.1+gE-HA, pCDNA3.1+gE-FLAG, pCDNA3.1+{Delta}Cys-HA, or pCDNA3.1+{Delta}Cys-FLAG. Lysates were collected in RIPA buffer containing the protease inhibitor cocktail (Complete minitablet; Roche Inc., Indianapolis, IN), and immunoprecipitation was performed with rabbit anti-HA antibody (Sigma) at a dilution of 1:200 or with 5 µg/ml of MAb anti-FLAG M2 antibody (Sigma). Mouse IgG (Vector Laboratories, Inc., Burlingame, CA) or the beads used for preclearing the lysates were used as a negative control. Immunoprecipitations were performed by incubating the antibody with 30 µl of beads for 2 h at 4°C; the beads were washed in lysis buffer and then incubated with the precleared lysates overnight at 4°C. Alternatively, the antibody was incubated with the precleared lysates for 2 h at 4°C, and the complexes were immunoprecipitated by adding 30 µl of beads and incubating them overnight at 4°C. The samples were separated on a 7.5% or 10% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). For immunoblotting, the anti-gE MAb 3B3 was used at a dilution of 1:15,000 to 1:20,000, rabbit polyclonal anti-IDE antibody (Covance) was used at a dilution of 1:1,000, rabbit polyclonal anti-gI (kindly provided by S. Silverstein, Columbia University, New York) was used at a dilution of 1:1,500, anti-FLAG M2 MAb (Sigma) was used at a dilution of 1:750, and rabbit anti-HA antibody (Sigma) was used at a dilution of 1:1,000. Primary antibodies were detected with horseradish peroxidase-conjugated sheep anti-mouse and sheep anti-rabbit secondary antibodies (Amersham) at a dilution of 1:10,000 to 1:20,000 and visualized by ECL Plus (Amersham) and X-ray film (Kodak).

For analysis of the effect of the {Delta}Cys mutation on gE expression, melanoma cells were infected with rOka-{Delta}Cys and with the rOka control, and cell lysates were collected in RIPA buffer at 24 h and 72 h postinfection. Samples were analyzed by Western blotting using the anti-gE MAb 3B3, rabbit polyclonal anti-IE63 antibody (a kind gift from W. Ruyechan, University of Buffalo, Buffalo, NY) at a dilution of 1:2,500, and anti-{alpha}-tubulin MAb (Sigma) at a 1:10,000 dilution. The analysis was performed with protein lysates obtained from two separate preparations of infected cells.

Confocal microscopy. Melanoma cells infected with rOka and rOka-{Delta}Cys viruses were fixed in 4% paraformaldehyde and processed for immunofluorescence as previously described (3). Uninfected melanoma cells were used as a control. gE was detected with the anti-gE MAb purchased from Chemicon and the rabbit polyclonal anti-gI antibody (a gift of S. Silverstein, Columbia University, New York). Confocal analysis was performed at the Cell Sciences Imaging Facility (Stanford, CA) with a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss, Inc.).

Analysis of glycoprotein maturation. Analysis of gE and gI glycosylation in infected melanoma cells was performed with endoglycosidase H (EndoH), PNGase F, and neuraminidase. Twenty to 40 µg of total protein lysates from rOka- or rOka-{Delta}Cys-infected melanoma cells was digested with EndoH or PNGase F (New England BioLabs) for 1 h at 37°C, according to the manufacturer's instructions. For the neuraminidase (Sigma) and PNGase F cotreatment, protein lysates were first incubated with neuraminidase for 1 h at 37°C and then with PNGase F for an additional 1 h. Lysates from uninfected cells were used as negative controls in all experiments. Samples (1 µg for gE and 20 µg for gI) were analyzed by Western blotting using the MAb 3B3 to detect gE and the rabbit polyclonal anti-gI.

Infection of human skin xenografts in SCIDhu mice. Skin xenografts were made in homozygous CB-17scid/scid mice, using human fetal tissue supplied by Advanced Bioscience Resources (Alameda, CA) according to federal and state regulations (44, 45). Animal use was in accordance with the Animal Welfare Act and was approved by the Stanford University Administrative Panel on Laboratory Animal Care. rOka and rOka-{Delta}Cys mutant viruses were passed three times in primary HELF before inoculation of the xenografts. The infectious-virus titer was determined for each inoculum at the time of inoculation. Skin xenografts were harvested at 10 and 21 or 22 days postinoculation and analyzed by infectious focus assay and immunohistochemistry with polyclonal anti-VZV human immune serum (44). Virus recovered from the tissues was tested by PCR and sequencing to confirm the expected mutations.

Primary human tonsil cell preparation and fluorescence-activated cell sorting analysis. Primary tonsil T cells were prepared from human tonsils obtained from the Department of Pathology, Stanford University Medical Center, according to a protocol approved by the Stanford University Committee on Human Subjects in Research. Suspensions of T cells were prepared as previously described (28). Primary HELF infected with rOka or the rOka-{Delta}Cys mutant were overlaid with 1 x 107 tonsil cells; uninfected fibroblasts overlaid with tonsil cells were used as a negative control. The tonsil cells were processed for fluorescence-activated cell sorting staining 48 h postinfection as previously described (4), and the titers of the infected fibroblasts were determined on melanoma cells. Samples were analyzed on a FACSCalibur apparatus (Becton Dickinson, Inc.).

Preparation of cell-free virus. Subconfluent HELF monolayers were infected with rOka-{Delta}Cys or the rOka control virus in T150 flasks. The cells were washed three times in cold phosphate-buffered saline (PBS) and dislodged in 10 ml of PBS using glass beads 4 mm in diameter (Fisher Scientific). The cell pellet was resuspended in PSGC medium (5% sucrose, 0.1% sodium glutamate, 10% fetal bovine serum in PBS) and sonicated for 30 s three times. Cell debris was eliminated by centrifugation at 3,000 rpm for 10 min at 4°C. The titer of the cell-free virus was determined by infectious focus assay on melanoma cells. For the cell-free virus entry experiments, the cell-free preparation obtained from infected HELF was used to infect melanoma cells. Three separate experiments were performed, and from three to six replicates were tested in each experiment.

Electron microscopy. For the study of virion morphogenesis, HELF were seeded on glass microscope coverslips in 24 wells at a density of 3 x 105 cells/well and infected with 1 x 103 PFU of rOka or rOka-{Delta}Cys virus. Samples were collected at 48 h or 96 h postinfection and processed as previously described (3). For immunogold staining, HELF were seeded in 100-mm dishes at a density of 5 x 106 cells/dish and infected with 1.6 x 104 PFU of rOka or rOka-{Delta}Cys virus. Samples were collected at 2 or 6 days postinfection; the cells were fixed in 2% paraformaldehyde/0.5% glutaraldehyde in PBS for 45 min at 4°C and then dislodged with a scraper and spun. The cell pellet was washed twice in PBS for 10 min, and samples were stained in 0.5% uranyl acetate overnight at 4°C. Samples were dehydrated through a series of increasing ethanol concentrations, embedded in LR White resin, and polymerized at 55°C for 2 days. Specimens were sectioned with an Ultra microtome (Leica), and the sections were collected on copper grids. For immunogold staining, ultrathin sections (60 nm) were incubated in digoxigenin-blocking solution (Roche Diagnostics) for 30 min and then for 1 h with anti-gE MAb (Chemicon) at a dilution of 1:1 or with rabbit polyclonal anti-gI (a gift of S. Silverstein, Columbia University, New York) at a dilution of 1:5. Samples were washed in PBS and incubated with protein A conjugated to 10-nm colloidal gold (PAG10 nm) from CMC (Utrecht, The Netherlands) at a dilution of 1:60. For gE staining, samples were incubated with a rabbit anti-mouse antibody (Cappel Laboratories) that served as a bridging antibody for secondary detection with protein A gold (protein A binds much more strongly to rabbit than to mouse antibodies). The use of the bridging antibody also amplified the signal. Samples were stained with 3.5% aqueous uranyl acetate for 10 min and with 0.2% lead citrate for 3 min and examined on a Joel 1230 transmission electron microscope.


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RESULTS
 
Deletion of the first cysteine-rich region of the gE ectodomain and effect on VZV replication in vitro. To identify potential functions associated with the first cysteine-rich region of the gE ectodomain, we first tested the effect of deletion of this region on VZV replication. In the gE-{Delta}Cys mutant, the region from cysteine 208 to cysteine 236 was deleted and replaced by a dodecameric linker coding for the 4-aa peptide Leu-Arg-Pro-Gln (3) (Fig. 1A). The pSpe23-{Delta}Cys208-236 cosmid containing the gE mutation was transfected with the intact pFsp73, pPme2, and pSpe14 cosmids in melanoma cells; pSpe23 containing the wt sequence of gE was transfected along with the other cosmids as a control. Recombinant virus, named rOka-{Delta}Cys, was recovered from the transfection of two independently derived clones of pSpe23-{Delta}Cys208-236, indicating that the first cysteine-rich region of the gE ectodomain was not essential for viral replication in vitro. When the growth kinetics of the rOka-{Delta}Cys mutant was analyzed in melanoma cells, we found that plaque numbers were similar to those for the rOka control during the first 4 days; however, rOka-{Delta}Cys titers were slightly higher than those of the rOka control at days 5 and 6 (a 1.6- to 1.7-fold difference) (Fig. 1B) because the cells were depleted in the control due to more rapid replication. In addition, the {Delta}Cys mutation was associated with a reduction of the plaque size (rOka, 1.17 ± 0.23 mm; {Delta}Cys, 0.81 ± 0.22 mm; P < 0.0001), suggesting impairment of the ability of the mutant virus to spread from cell to cell (Fig. 1C and D). When the growth kinetics was analyzed in HELF, the rOka-{Delta}Cys mutant had lower titers than the control (Fig. 1E); this decrease in rOka-{Delta}Cys mutant replication was associated with a small-plaque phenotype (Fig. 1F and G), as observed in the melanoma cells (Fig. 1C and D).


Figure 1
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  		FIG. 1. Characterization of the rOka-{Delta}Cys mutant. (A) Schematic representation of VZV gE protein and the {Delta}Cys mutation. The position of the deletion from cysteine 208 to cysteine 236 ({Delta}) is indicated. Amino acids are numbered from the N terminus to the C terminus of the gE protein. The hatched box indicates the unique N-terminal region (aa 1 to 188) (3), and the black box represents the transmembrane domain. The two cysteine-rich regions and the cysteines (C) in the gE ectodomain are indicated. (B) Replication of the gE-{Delta}Cys mutant in vitro. Melanoma cells were inoculated on day 0 with 1 x 103 PFU of rOka or rOka-{Delta}Cys. Aliquots were harvested from day 1 to day 6. Each point represents the mean of three wells. The error bars represent the standard deviations. The significance of the difference in titers between rOka and the rOka-{Delta}Cys mutant at each time point was determined by Student's t test; one asterisk indicates a P value of <0.01, and two asterisks indicate a P value of <0.001. (C and D) Analysis of plaque morphology in melanoma cells. Cells infected with rOka (C) or the rOka-{Delta}Cys mutant (D) were fixed and stained with polyclonal anti-VZV human immune serum. (E) Replication of the gE-{Delta}Cys mutant in HELF. Cells were inoculated on day 0 with 1 x 103 PFU of rOka or rOka-{Delta}Cys. Aliquots were harvested from day 1 to day 5. Each point represents the mean of three wells. The error bars represent the standard deviations. One asterisk indicates a P value of <0.01; two asterisks indicate a difference with a P value of <0.001. (F and G) Plaque morphology of the gE-{Delta}Cys mutant in HELF. Representative plaques formed by rOka (F) or rOka-{Delta}Cys (G) are compared. (H) Analysis of gE expression. Melanoma cells were inoculated with the rOka-{Delta}Cys mutant or rOka control, harvested at 24 h and 72 h postinfection, and analyzed by Western blotting with anti-gE 3B3 MAb (top) or rabbit polyclonal anti-IE63 antibody (center). The anti-{alpha}-tubulin MAb was used for normalization (bottom). Lanes 1, uninfected cell lysate; lanes 2, rOka-infected cell lysate; lanes 3, rOka-{Delta}Cys-infected cell lysate. The marker for molecular weight is on the left.

To assess if the deletion of the first cysteine-rich region affected gE expression, gE levels were analyzed in melanoma cells infected with rOka-{Delta}Cys at 24 and 72 h postinfection by immunoblotting. A delay in the accumulation of gE-{Delta}Cys was observed compared to the levels of wt gE at both time points (Fig. 1H, top). The decreased expression of mutant gE was related to a reduced level of replication, as indicated by a decreased level of the immediate-early protein IE63 (Fig. 1H, middle). The reduced level of expression of viral proteins in the rOka-{Delta}Cys-infected cells was consistent with the impairment in cell-cell spread of the mutant virus, indicated by the reduced plaque size.

Analysis of gE and gI cellular localization, interaction, and maturation in rOka-{Delta}Cys-infected cells. To further investigate the effect of the {Delta}Cys mutation on cell-cell spread, we analyzed the cellular localization of gE-{Delta}Cys and its heterodimer partner, gI, in melanoma cells. At 24 h postinfection, gE localization in the rOka control and in the {Delta}Cys mutant was predominantly in the perinuclear region, consistent with the TGN, where it colocalized with gI (Fig. 2A to F). At 72 h postinfection, mutant gE was localized at the plasma membrane but in a very patchy distribution compared to the rOka control (Fig. 2G and J); in addition, colocalization of gE-{Delta}Cys and gI at this site was minimal, if any (Fig. 2H and I, and K and L). Coimmunoprecipitation of gE and gI was performed to determine if the deletion of the cysteine-rich region aa 208 to 236 affected gE/gI heterodimer formation in the infected cells. Immunoprecipitation of gI or gE confirmed the interaction of the two proteins in rOka-infected cells (Fig. 3A, lane 2). In contrast, no binding was observed in the rOka-{Delta}Cys-infected cell lysates (Fig. 3A, line 3), indicating that deletion of the first cysteine-rich domain of the gE ectodomain impaired binding to gI.


Figure 2
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  		FIG. 2. Localization of gE and gI in rOka-{Delta}Cys-infected cells. Melanoma cells were infected with rOka (A to C and G to I) or the rOka-{Delta}Cys mutant (D to F and J to L) and processed for immunofluorescence at 24 and 72 h postinfection (p.i.). The cells were labeled with anti-gE MAb (Chemicon) (red; left column) and with rabbit anti-gI antibody (green; middle column). (Right column) Colocalization of gE and gI signals. The insets (C, F, I, and L) are enlargements of the images in the panels. Open arrowheads indicate colocalization of gE and gI in the perinuclear region in the rOka control and gE mutant (C and F) and colocalization at the plasma membrane in the rOka control (I) but not in the gE mutant (L). Nuclei were stained with 4',6'-diamidino-2-phenylindole (DAPI) (blue). Magnification, x400.


Figure 3
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  		FIG. 3. Analysis of gE and gI interaction and glycosylation in melanoma cells. (A) Coimmunoprecipitation of gE and gI in infected cells. Melanoma cells were inoculated with the rOka-{Delta}Cys mutant or rOka control, and lysates were collected in RIPA buffer. The anti-gI MAb 6B5 or the anti-gE MAb 3B3 was used for immunoprecipitation, while rabbit anti-gI and the anti-gE MAb 3B3 were used for Western blotting analysis. Lanes 1, uninfected cells; lanes 2, rOka-infected cells; lanes 3, rOka-{Delta}Cys-infected cells. (B and C) Analysis of gE and gI glycosylation. (B) Cell lysates from rOka-infected (lanes 1 and 2 and lanes 5 and 6) or rOka-{Delta}Cys-infected (lanes 3 and 4 and lanes 7 and 8) cells were digested with EndoH (lanes 2 and 4) or PNGase F (lanes 6 and 8). (C) Cell lysates from rOka- or rOka-{Delta}Cys-infected cells were treated with neuraminidase alone or in combination with PNGase F. Lane 1, untreated lysates; lane 2, neuraminidase-treated lysates; lane 3, neuraminidase- and PNGase F-treated lysates. Samples were analyzed by Western blotting to detect gE and gI. Markers of molecular weight are on the left. The asterisks indicate the altered form of mature gI. The black arrowhead indicates the faster-migrating gI band obtained after treatment with PNGase F alone (B) or in combination with neuraminidase (C).

In addition, we observed that the mature form of gI in the rOka-{Delta}Cys-infected cells had a molecular weight that was higher than that of gI in wt-virus-infected cells (Fig. 2A, right, lane 3). It was previously shown that posttranslational modification of gE and gI independently expressed in transfected cells differed from that of the two proteins when coexpressed in transfected or infected cells (61). We hypothesized that the increased molecular weight of gI observed in rOka-{Delta}Cys-infected cells was due to altered gI maturation in the absence of gE binding to gI. To test this hypothesis, we analyzed the glycosylation of gE and gI in rOka-{Delta}Cys-infected cells. Cell lysates from rOka and rOka-{Delta}Cys-infected cells were digested with EndoH, which cleaves N-linked oligosaccharides of the high-mannose and hybrid types, or PNGase F, which releases the high-mannose, complex, and hybrid oligosaccharides; in addition, neuraminidase treatment to remove the sialic residues and subsequent digestion with PNGase F were performed. In agreement with the previous analysis (18, 61), when lysates from rOka-infected cells were digested with EndoH, migration of the mature form of wt gE and gI changed little or not at all (Fig. 3B, lanes 1 to 4), while the complete removal of the N-linked oligosaccharides by PNGase F resulted in the appearance of products of decreased molecular weight (Fig. 3B, lanes 5 to 8). Treatment with neuraminidase was associated with the appearance of faster-migrating products (Fig. 3C, lane 2), indicating that sialic residues were present on the oligosaccharide chains, and sequential digestion of neuraminidase and PNGase F resulted in a further decrease in the molecular weights of these products (Fig. 3C, lane 3). Analysis of the lysates from the rOka-{Delta}Cys-infected cells showed that the altered mature form of gI had the same pattern of resistance or sensitivity to the different enzymes as the wt gI. Digestion with PNGase F alone or in combination with neuraminidase produced a faster-migrating gI band that had a molecular weight similar that of the one in the rOka lysates, suggesting that the increased molecular weight observed was the result of different glycosylation, likely N-linked type (Fig. 3B, lanes 6 and 8, and 3C, lane 3). Mutant gE-{Delta}Cys showed no difference from the wt gE in its resistance or sensitivity to the endoglycosidases, suggesting that neither the mutation nor the lack of interaction with gI altered maturation of the mutant gE.

Analysis of virion morphogenesis in rOka-{Delta}Cys-infected cells and incorporation of gE and gI into viral particles. Given the fact that VZV gE and gI are involved in secondary envelopment of viral particles (17, 57, 65), we asked if the {Delta}Cys mutation affected virion morphogenesis. HELF were infected with rOka or rOka-{Delta}Cys and analyzed by transmission electron microscopy (TEM). Since samples of rOka-{Delta}Cys examined at 48 h postinfection showed a clear delay in virion accumulation (data not shown), samples of the rOka control at 48 h postinfection were compared to the rOka-{Delta}Cys mutant at 96 h postinfection. In these comparisons, nucleocapsids were observed in the nuclei of cells infected with either rOka (Fig. 4A) or rOka-{Delta}Cys (Fig. 4B). Cytoplasmic vacuoles containing viral particles were present in rOka-{Delta}Cys- and rOka-infected cells (Fig. 4C and D). However, while viral particles emerging from the cells infected with rOka were abundant and easily detectable (Fig. 4E), extracellular viral particles were less frequent at the surfaces of cells infected with rOka-{Delta}Cys (Fig. 4F). The delay in virion formation observed in rOka-{Delta}Cys-infected cells was consistent with the decreased replication of the mutant virus in HELF (1E) and the slower accumulation of viral proteins (Fig. 1H).


Figure 4
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  		FIG. 4. Virion morphogenesis of the rOka-{Delta}Cys mutant. HELF were infected with rOka (A, C, and E) for 48 h or with rOka-{Delta}Cys (B, D, and F) for 96 h and processed for TEM. Nucleocapsids in the nuclei of infected cells (black arrowheads) were observed with rOka (A) and rOka-{Delta}Cys (B). Viral particles in cytoplasmic vacuoles were present in the rOka-infected (C) and rOka-{Delta}Cys-infected (D) cells; extracellular virions (open arrowheads) were abundant and easily detectable at 48 h postinfection (p.i.) in the rOka-infected cells (E), while they were scarce in the rOka-{Delta}Cys-infected cells and were detected at later time points (96 h postinfection) (F). cyt, cytoplasm; nuc, nucleus. Magnifications are indicated. The insets in panels A and B are enlargements of the boxed areas.

To determine whether eliminating gE binding to gI impaired the incorporation of mutant gE-{Delta}Cys and gI into viral particles, HELF were infected with rOka-{Delta}Cys and the rOka control and analyzed by TEM and immunogold labeling to detect gE or gI. Because of the evidence of slower accumulation of viral particles in the previous experiments, rOka-{Delta}Cys-infected cells were analyzed at 6 days postinfection, when virion numbers were comparable to those in rOka-infected cells at 48 h postinfection. The pattern of incorporation of the {Delta}Cys mutant form of gE into viral particles was similar to that observed in the rOka control (Fig. 5A to F). In contrast, the incorporation of gI into viral particles was significantly reduced in cells infected with the rOka-{Delta}Cys mutant compared to rOka (Fig. 5G to L); gI labeling was weak, and only a minor fraction of virions were decorated with the gold particles (Fig. 5J to L). These data indicated that gE interaction with gI is important for the incorporation of gI, but not gE, into the viral envelope.


Figure 5
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  		FIG. 5. Incorporation of gE and gI in the envelopes of rOka-{Delta}Cys mutant virions. Immunogold labeling and TEM of rOka-infected (A to C and G to I) and rOka-{Delta}Cys-infected (D to F and J to L) HELF. Labeling was performed using the anti-gE MAb from Chemicon to detect gE (A to F) and the rabbit polyclonal anti-gI to detect gI (G to L). The open arrowheads in panels G and I and the black arrowheads in panels J and L indicate gold-decorated particles. Scale bars, 0.2 µm (A, C, D, F, G, I, J, and L) and 0.5 µm (B, E, H, and K). The boxed areas in the middle column are enlarged in the right and left columns as indicated.

Pathogenesis of infection with rOka-{Delta}Cys in human skin xenografts in vivo. The ability of the rOka-{Delta}Cys mutant to replicate in skin xenografts in vivo was evaluated in two independent experiments. Only 1 of 10 skin xenografts infected with rOka-{Delta}Cys yielded infectious virus at day 10, and virus was recovered from only one of 11 rOka-{Delta}Cys-infected xenografts at days 21 and 22 (Fig. 6A). The titers of rOka-{Delta}Cys from both of these xenografts were also lower than titers in those infected with rOka. While the inoculum titer of the rOka-{Delta}Cys mutant was lower than that of rOka (P = 0.02), this 1-log-unit difference does not explain the nearly complete failure of rOka-{Delta}Cys to replicate in vivo. As expected, the two rOka-{Delta}Cys viruses recovered from skin xenografts retained their small-plaque morphology compared rOka (Fig. 6B). Persistence of the mutation in these viruses after skin replication was confirmed by sequencing. This severe impairment of virulence in skin associated with the deletion of the first cysteine-rich region in the gE ectodomain and the lack of gE/gI interaction can be attributed to the resulting decreased capacity for cell-cell spread.


Figure 6
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  		FIG. 6. Effect of {Delta}Cys mutation on VZV replication in the human skin in vivo. (A) Skin xenografts were inoculated with HELF infected with rOka or rOka-{Delta}Cys mutant virus. The infected xenografts were collected 10 and 21 or 22 days postinoculation. The results from two independent experiments were combined. The number of samples from which infectious virus was recovered per total number of xenografts inoculated is indicated on the horizontal axis. Each bar represents the mean titer, and the error bar indicates the standard error. The asterisk indicates statistical significance (P < 0.05). (B) Immunohistochemistry of melanoma cells infected with the skin samples in the infectious-focus assay. Cells were fixed and stained with polyclonal anti-VZV human immune serum. A typical plaque formed by the rOka control virus is compared to the rOka-{Delta}Cys mutant plaque.

Effect of the {Delta}Cys deletion on VZV entry. To assess whether the first cysteine-rich region of gE, and therefore the capacity to form gE/gI heterodimers, was required for VZV entry, experiments were done using primary human tonsil T cells as target cells and by inoculating melanoma cells with cell-free preparations of rOka-{Delta}Cys and rOka. Infection of primary tonsil T cells provides an assay for VZV entry because these cells do not undergo VZV-induced cell fusion (27). T cells were infected by coculturing them with rOka- or rOka-{Delta}Cys-infected HELF and analyzed by flow cytometry using CD3 as the T-cell marker and polyclonal anti-VZV human immune serum to detect VZV-infected cells. The percentage of CD3+/VZV+ T cells was somewhat higher when T cells were infected with the rOka-{Delta}Cys mutant than when they were infected with rOka (rOka, 15.8%; rOka-{Delta}Cys, 24.6%; P = 0.04) (Fig. 7A). However, this difference can be accounted for by the higher titer of rOka-{Delta}Cys in the HELF monolayer used to infect the T cells compared to that in rOka-infected HELF (rOka, 1.37 x 105 PFU/ml; rOka-{Delta}Cys, 6.7 x 105 PFU/ml; P = 0.02). We concluded that deletion of the first cysteine-rich region of gE, which blocks gI binding, did not block VZV entry into human T cells in vitro.


Figure 7
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  		FIG. 7. Analysis of rOka-{Delta}Cys mutant entry into target cells. (A) Infection of human T cells in vitro. Primary human tonsil T cells were infected by coculturing them with HELF-infected monolayers for 48 h, stained with anti-CD3 antibody and anti-VZV immune serum, and analyzed by flow cytometry. The bars represent the percentages of CD3+/VZV+ cells for each virus compared to the uninfected control. The error bars represent the standard deviations. (B) Cell-free VZV infection of melanoma cells. Cell-free rOka-{Delta}Cys or rOka control was used to infect melanoma cells. The medium was replaced 2 h postinfection (A) or was not replaced (B). Three days postinfection, cells were processed for immunohistochemistry using anti-VZV immune serum. The error bars represent the standard deviations. No statistically significant difference was observed between the results from the sets of experiments in panels A and B.

To further test the ability of the rOka-{Delta}Cys mutant to enter target cells, melanoma cells were infected with a cell-free preparation of rOka-{Delta}Cys or rOka and plaques were counted 3 days postinfection. No difference in the number of plaques was observed between the rOka- and the rOka-{Delta}Cys-infected melanoma cells (Fig. 7B); plaques in the rOka-{Delta}Cys-infected cells remained consistently smaller than in the rOka-infected samples (data not shown).

Effect of the {Delta}Cys mutation on binding to IDE during VZV replication. IDE has been identified as contributing to VZV entry and was shown to interact with the unique gE N-terminal region in vitro using expression plasmids (30, 31). We tested whether the binding between gE and IDE was preserved in cells infected with rOka-{Delta}Cys. Immunoprecipitation with gE (Fig. 8A, top) or IDE (Fig. 8A, bottom) antibodies confirmed the gE-IDE interaction in rOka-infected cells and showed that the {Delta}Cys mutation did not abolish this interaction (Fig. 8A, lane 3). Immunoprecipitation of gI from lysates of cells infected with the rOka-{Delta}Cys mutant showed almost no interaction between gI and IDE (Fig. 8B, lane 3), suggesting that the contribution of gI to the gE-IDE complex is not important in the context of viral infection. These results indicated that the complex observed with the rOka lysate (Fig. 8B, lane 2) was secondary to the binding of IDE to gE, which is pulled down by immunoprecipitation of gI when gE/gI binding is intact. These findings are in agreement with data using gE and gI expression constructs (30, 31).


Figure 8
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  		FIG. 8. Effect of {Delta}Cys mutation on gE/IDE interaction. (A) Coimmunoprecipitation of gE and IDE in infected cells. Melanoma cells were inoculated with the rOka-{Delta}Cys mutant or rOka control virus, and the lysates were processed for immunoprecipitation (IP). Anti-gE MAb (Chemicon) (top) or anti-IDE MAb (Covance) (bottom) was used for immunoprecipitation, while rabbit anti-IDE (Covance) and anti-gE MAb 3B3 were used for Western blotting analysis. Mouse IgG was used as the immunoprecipitation control. (B) Coimmunoprecipitation of gI and IDE. Cell lysates from rOka-{Delta}Cys mutant- or rOka control-infected melanoma cells were immunoprecipitated with anti-gI MAb 6B5 and analyzed by Western blotting with rabbit anti-IDE (Covance). The asterisk indicates the weak pull-down of IDE observed in the rOka-{Delta}Cys mutant. Lanes 1, uninfected cells; lanes 2, rOka-infected cells; lanes 3, rOka-{Delta}Cys-infected cells. The markers of molecular weight are on the left.

Analysis of gE-gE interaction in vitro. It has been shown that VZV gE exists in monomeric and dimeric forms in transfected cells (47), and recent scanning electron microscopy and immunogold labeling experiments demonstrated that gE dimers are present on the VZV envelope (6). These observations suggest a possible function for dimeric gE during VZV replication. We tested gE-gE binding in vitro and the effect of the {Delta}Cys mutation on the interaction. The HA or FLAG epitope tag was inserted in frame at the C termini of intact gE and the {Delta}Cys mutant form of gE, and the constructs were transfected into HEK-293 cells. Immunoprecipitation of the HA-tagged (Fig. 9A) or the FLAG-tagged (Fig. 9B) proteins showed gE-gE interaction with both the wt gE (Fig. 9A and B, lane 3) and the gE {Delta}Cys constructs (Fig. 9A and B, lane 4), indicating that gE-gE binding does not depend on the first cysteine-rich region of the gE N-terminal region.


Figure 9
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  		FIG. 9. Analysis of gE-gE interaction. Hek-293 cells were transfected with wt or mutant gE tagged with the HA or FLAG peptide; 48 h posttransfection, the cell lysates were harvested and processed for immunoprecipitation. Immunoprecipitation was performed with the rabbit anti-HA (top) or anti-FLAG MAb (bottom) and analyzed by Western blotting with anti-FLAG (top) or anti-HA (bottom) antibodies. Lanes 1, gE-HA; lanes 2, gE-FLAG; lanes 3, gE-HA plus gE-FLAG; lanes 4, gE-{Delta}Cys-HA plus gE-{Delta}Cys-FLAG. Molecular weight markers are indicated.


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DISCUSSION
 
VZV gE differs from its homologues in the alphaherpesviruses because it is required for replication and has a large unique N-terminal region (3, 4, 34, 40). However, VZV gE shares the formation of noncovalently linked gE/gI heterodimers with its homologues in these related viruses (22, 26, 61, 67). The unique short segment of the VZV genome encodes only gE and gI, whereas the other alphaherpesviruses have gD, as well as gE and gI, at this locus. Since these other viruses require gD for viral entry, one hypothesis is that VZV gE has gD-like functions that are involved in initiating infection. Recent evidence suggests that gE binding to the cellular protein IDE is involved in VZV entry (30, 31). In our experiments using targeted mutagenesis of the VZV genome, we showed that deletion of the first cysteine-rich region of the gE ectodomain was compatible with VZV replication but that this motif was essential for gE binding to gI. Analyses of gE/gI binding using VZV gE plasmid constructs showed that removing the second cysteine-rich region (aa 342 to 446) in the highly conserved C-terminal region of the ectodomain reduced but did not abolish the interaction, suggesting that the deletion of this cysteine-rich region might alter gE conformation and interfere with gE/gI interactions but that these residues were not essential (61). Similarly, deletion of aa 163 to 208 from gE, which removes only one of the four cysteines of the first cysteine-rich region, impaired but did not abrogate gE/gI complex formation (31). In our experiments, the complete deletion of the first cysteine-rich region of gE (aa 208 to 236) completely blocked gE/gI complex formation during viral replication in infected cells. Whether this mutation deleted a specific gI binding domain from gE or created a more severe gE conformational change than the cysteine mutations evaluated in plasmid constructs, thereby preventing gE/gI heterodimer formation, is not known. Of interest, the {Delta}Cys deletion did not alter gE expression and maturation, and the mutant protein was able to traffic to the plasma membrane. Moreover, gE with the {Delta}Cys mutation was still able to bind to IDE in VZV-infected cells. These observations suggest that the mutation did not cause a global change in gE conformation, but three-dimensional structural data are required to confirm this prediction. The crystal structure of HSV gE has been reported (56); however, the important sequence differences between these homologues, particularly in the N-terminal region, preclude predictions about VZV gE based on the HSV gE structure.

Preventing the formation of VZV gE/gI heterodimers by deletion of the first cysteine-rich region of gE made it possible to analyze gE functions that were associated with or independent of the gE/gI complex in VZV-infected cells. VZV growth was not affected in melanoma cells, as measured by numbers of plaques, but was reduced in primary human fibroblasts; however, plaque size was reduced in both cell types infected with the {Delta}Cys mutant, indicating alteration of cell-cell spread. TEM analysis showed a delay in the accumulation of mutant virions in the infected fibroblasts, consistent with the decreased replication and cell-cell spread. Importantly, the replication of this gE mutant that was defective for gI binding was almost completely blocked in skin xenografts in vivo. Taken together, these observations indicate that the usual pattern of VZV-induced cell fusion requires the gE/gI complex and that the capacity to form typical VZV polykaryocytes as a mechanism of VZV replication is not only an in vitro characteristic that depends on gE/gI complex formation, but appears to be critical for its virulence as a skin pathogen. The delay in accumulation of mutant viral particles might also contribute to the severe impairment of replication in the skin observed in the absence of the gE/gI complex.

Experiments using the {Delta}Cys mutant virus revealed that preventing gE interaction with gI altered the posttranslational modification of gI in VZV-infected cells, even though the levels of the mutant form of gE were not diminished. Differences in the processing of gI and gE when expressed alone or as a complex in transfected cells have been shown previously (61). The pattern of sensitivity to endoglycosidases that we observed suggested that gI glycosylation was abnormal when gI was not associated with gE during rOka-{Delta}Cys replication, even though both proteins were present in infected cells. In contrast, no obvious changes in the molecular weight of mutant gE and its sensitivity to endoglycosidases were observed after preventing its binding to gI. Thus, preventing gE/gI heterodimer formation has consequences for the posttranslational modification of gI but has little or no impact on gE maturation.

VZV gE is typically expressed abundantly over the plasma membranes of infected cells. While gE maturation was not affected, blocking the gE/gI interaction changed gE localization to a more punctate pattern on cells infected with the {Delta}Cys mutant. These observations were consistent with the abnormal pattern of gE expression on the plasma membranes of cells infected with our VZV gI null mutant in vitro and in neural cells in DRG xenografts infected with rOka-{Delta}gI in vivo (34, 64). Deleting gI also reduces VZV-induced cell fusion. Since gI was present in cells infected with rOka-{Delta}Cys, the current experiments demonstrate that proper gE trafficking to the plasma membrane during VZV replication depends specifically on gE/gI binding. It has been reported that while gI had no effect on gE targeting to the TGN (57), the gI-mediated enhancement of gE endocytosis was more efficient when gE was complexed with gI (48). Thus, either the absence of gI in the gI null mutant (34) or targeted mutagenesis that prevented gE/gI complex formation in the {Delta}Cys mutant affected gE expression on the plasma membrane, inhibited cell fusion in vitro, and blocked skin virulence in vivo (34, 42). These experiments demonstrate that the presence of the other VZV glycoproteins or gE and gI in noninteractive forms does not compensate for disruption of gE/gI heterodimer formation in the pathogenesis of VZV skin infection.

Interestingly, the analysis of virion morphogenesis in cells infected with the {Delta}Cys mutant did not indicate any obvious alteration of varicella-zoster virion envelopment. In contrast, as shown with the gI null mutant and mutants lacking the gI N-terminal or C-terminal domain, viral envelopment was severely compromised and was associated with fusion of the TGN cisternae, forming membranous stacks both in vitro (57) and in differentiated human neural cells in DRG xenografts in vivo (64). Of interest, the fact that this consequence was not observed in rOka-{Delta}Cys-infected cells is indirect evidence that gI has a specific function at this step of varicella-zoster virion morphogenesis that is independent of its binding to gE. Our data further indicate that loss of the gE/gI complex does not affect secondary envelopment as long as both proteins are present; however, complex formation is required for efficient incorporation of gI in the viral particles, as demonstrated by immunogold labeling and TEM analysis.

The HSV gE/gI complex promotes cell-cell spread, but it is not involved in the attachment and fusion of the viral envelope required for entry (11, 12). Recently, VZV gE has been shown to bind to IDE and IDE has been reported to function as a VZV cellular receptor (30, 31). Experiments using gE plasmid expression constructs demonstrated that the gE binding site for interactions with IDE was in the unique extracellular N-terminal region (31), which is not present in the alphaherpesvirus gE homologues (3). In the current experiments, we found that deletion of the first cysteine region and consequent failure to form the gE/gI complex did not block the ability of the {Delta}Cys mutant virus to enter primary human tonsil T cells in vitro or to infect melanoma cells when the virus was prepared as cell-free VZV. In addition, mutant gE expressed in cells infected with rOka-{Delta}Cys retained its capacity to bind to IDE. Our data showing that gE/gI heterodimers are not required for entry into target cells or for binding to IDE during viral infection suggest that any role that is played by gE in VZV entry is a function separate from its interaction with gI. This conclusion is also consistent with the finding that viral entry was not affected even though incorporation of gI into rOka-{Delta}Cys viral particles was decreased. VZV gE and gI have been shown to be present in the VZV envelope in similar amounts (6), although whether the two glycoproteins form a complex in viral particles is not known. Our data favor a model in which the gE/gI heterodimer is not required for VZV entry. However, we have not excluded the possibility that the presence of gI in complex with gE may affect the efficiency of VZV entry.

In addition to its interactions with gI, VZV gE has the capacity to form homodimers (47). Recent high-magnification ultrastructural studies indicate that gE/gE homodimers are present in the envelopes of VZV particles on the surfaces of infected cells (6). The dimeric form of gE was identified in cells transfected with a gE expression vector; these 130-kDa underglycosylated gE homodimers were present on the plasma membrane and exhibited tyrosine phosphorylation, as is characteristic of cellular receptors (47). Our experiments showed that the deletion of the first cysteine-rich region did not disrupt gE/gE interaction and viral entry. HSV gD, which binds the cellular HSV receptors, HVEM (herpesvirus entry mediator), nectin-1, nectin-2, and 3-O-sulfated heparan sulfate (55), is dimeric in HSV particles (20). The detection of gE dimers in the VZV envelope (6) and our experiments with the {Delta}Cys mutant showing that dimerization is independent of gE/gI interaction and that entry is preserved despite this mutation are consistent with the hypothesis that gE homodimers in the viral envelope may interact with cellular receptors during VZV entry.

In summary, we have shown that the first cysteine-rich region of the gE ectodomain is required for gE/gI complex formation. The loss of the functional VZV gE/gI heterodimer differentially affected cell-cell spread and viral entry, establishing that there is a differential requirement for gE/gI interaction in these processes and indicating that functions associated with the first cysteine-rich region, which is in the conserved portion of the gE extracellular domain, and those of the unique N-terminal domain of gE are independent.


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ACKNOWLEDGMENTS
 
This work was supported by grants from the National Institute of Allergy and Infectious Diseases (AI20459) and the National Cancer Institute (CA49605).

We thank Nafisa Ghori at the Department of Microbiology and Immunology, Stanford University, for assistance with TEM.


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FOOTNOTES
 
* Corresponding author. Present address: Institut Pasteur, Départment de Virologie, 25 rue du Dr Roux, 75015 Paris, France. Phone: 33 (0) 1 45 68 87 43. Fax: 33 (0) 1 45 68 89 93. E-mail: barbara.berarducci{at}pasteur.fr Back

{triangledown} Published ahead of print on 22 October 2008. Back


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REFERENCES
 
    1
  1. Alconada, A., U. Bauer, and B. Hoflack. 1996. A tyrosine-based motif and a casein kinase II phosphorylation site regulate the intracellular trafficking of the varicella-zoster virus glycoprotein I, a protein localized in the trans-Golgi network. EMBO J. 15:6096-6110.[Medline]
    2. 2
  2. Basu, S., G. Dubin, M. Basu, V. Nguyen, and H. M. Friedman. 1995. Characterization of regions of herpes simplex virus type 1 glycoprotein E involved in binding the Fc domain of monomeric IgG and in forming a complex with glycoprotein I. J. Immunol. 154:260-267.[Abstract]
    3. 3
  3. Berarducci, B., M. Ikoma, S. Stamatis, M. Sommer, C. Grose, and A. M. Arvin. 2006. Essential functions of the unique N-terminal region of the varicella-zoster virus glycoprotein E ectodomain in viral replication and in the pathogenesis of skin infection. J. Virol. 80:9481-9496.[Abstract/Free Full Text]
    4. 4
  4. Berarducci, B., M. Sommer, L. Zerboni, J. Rajamani, and A. M. Arvin. 2007. Cellular and viral factors regulate the varicella-zoster virus gE promoter during viral replication. J. Virol. 81:10258-10267[Abstract/Free Full Text]
    5. 5
  5. Brack, A. R., B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter. 2000. Role of the cytoplasmic tail of pseudorabies virus glycoprotein E in virion formation. J. Virol. 74:4004-4016.[Abstract/Free Full Text]
    6. 6
  6. Carpenter, J. E., J. A. Hutchinson, W. Jackson, and C. Grose. 2008. Egress of light particles among filopodia on the surface of varicella-zoster virus-infected cells. J. Virol. 82:2821-2835.[Abstract/Free Full Text]
    7. 7
  7. Cohen, J. I., and H. Nguyen. 1997. Varicella-zoster virus glycoprotein I is essential for growth of virus in Vero cells. J. Virol. 71:6913-6920.[Abstract]
    8. 8
  8. Cohen, J. I., S. E. Straus, and A. M. Arvin. 2007. Varicella-zoster virus replication, pathogenesis, and management, p. 2773-2818. In D. N. K. and P. M. Howley (ed.), Fields virology, 5th ed., vol. 2. Lippincott Wlliams & Wilkins, Philadelphia, PA.
    9. 9
  9. Cole, N. L., and C. Grose. 2003. Membrane fusion mediated by herpesvirus glycoproteins: the paradigm of varicella-zoster virus. Rev. Med. Virol. 13:207-222.[CrossRef][Medline]
    10. 10
  10. Collins, W. J., and D. C. Johnson. 2003. Herpes simplex virus gE/gI expressed in epithelial cells interferes with cell-to-cell spread. J. Virol. 77:2686-2695.[Abstract/Free Full Text]
    11. 11
  11. Dingwell, K. S., C. R. Brunetti, R. L. Hendricks, Q. Tang, M. Tang, A. J. Rainbow, and D. C. Johnson. 1994. Herpes simplex virus glycoproteins E and I facilitate cell-to-cell spread in vivo and across junctions of cultured cells. J. Virol. 68:834-845.[Abstract/Free Full Text]
    12. 12
  12. Dingwell, K. S., L. C. Doering, and D. C. Johnson. 1995. Glycoproteins E and I facilitate neuron-to-neuron spread of herpes simplex virus. J. Virol. 69:7087-7098.[Abstract]
    13. 13
  13. Dingwell, K. S., and D. C. Johnson. 1998. The herpes simplex virus gE-gI complex facilitates cell-to-cell spread and binds to components of cell junctions. J. Virol. 72:8933-8942.[Abstract/Free Full Text]
    14. 14
  14. Farnsworth, A., K. Goldsmith, and D. C. Johnson. 2003. Herpes simplex virus glycoproteins gD and gE/gI serve essential but redundant functions during acquisition of the virion envelope in the cytoplasm. J. Virol. 77:8481-8494.[Abstract/Free Full Text]
    15. 15
  15. Farnsworth, A., and D. C. Johnson. 2006. Herpes simplex virus gE/gI must accumulate in the trans-Golgi network at early times and then redistribute to cell junctions to promote cell-cell spread. J. Virol. 80:3167-3179.[Abstract/Free Full Text]
    16. 16
  16. Farnsworth, A., T. W. Wisner, and D. C. Johnson. 2007. Cytoplasmic residues of herpes simplex virus glycoprotein gE required for secondary envelopment and binding of tegument proteins VP22 and UL11 to gE and gD. J. Virol. 81:319-331.[Abstract/Free Full Text]
    17. 17
  17. Gershon, A. A., D. L. Sherman, Z. Zhu, C. A. Gabel, R. T. Ambron, and M. D. Gershon. 1994. Intracellular transport of newly synthesized varicella-zoster virus: final envelopment in the trans-Golgi network. J. Virol. 68:6372-6390.[Abstract/Free Full Text]
    18. 18
  18. Grose, C. 1990. Glycoproteins encoded by varicella-zoster virus: biosynthesis, phosphorylation, and intracellular trafficking. Annu. Rev. Microbiol. 44:59-80.[CrossRef][Medline]
    19. 19
  19. Grose, C. 2002. The predominant varicella-zoster virus gE and gI glycoprotein complex, p. 195-223. In A. Holzenburg and E. Bogner (ed.), Structure-function relationships of human pathogenic viruses. Kluwer Academic Publishers, New York, NY.
    20. 20
  20. Handler, C. G., R. J. Eisenberg, and G. H. Cohen. 1996. Oligomeric structure of glycoproteins in herpes simplex virus type 1. J. Virol. 70:6067-6070.[Abstract]
    21. 21
  21. Ito, H., M. H. Sommer, L. Zerboni, H. He, D. Boucaud, J. Hay, W. Ruyechan, and A. M. Arvin. 2003. Promoter sequences of varicella-zoster virus glycoprotein I targeted by cellular transactivating factors Sp1 and USF determine virulence in skin and T cells in SCIDhu mice in vivo. J. Virol. 77:489-498.[CrossRef][Medline]
    22. 22
  22. Johnson, D. C., and V. Feenstra. 1987. Identification of a novel herpes simplex virus type 1-induced glycoprotein which complexes with gE and binds immunoglobulin. J. Virol. 61:2208-2216.[Abstract/Free Full Text]
    23. 23
  23. Johnson, D. C., M. C. Frame, M. W. Ligas, A. M. Cross, and N. D. Stow. 1988. Herpes simplex virus immunoglobulin G Fc receptor activity depends on a complex of two viral glycoproteins, gE and gI. J. Virol. 62:1347-1354.[Abstract/Free Full Text]
    24. 24
  24. Johnson, D. C., M. Webb, T. W. Wisner, and C. Brunetti. 2001. Herpes simplex virus gE/gI sorts nascent virions to epithelial cell junctions, promoting virus spread. J. Virol. 75:821-833.[Abstract/Free Full Text]
    25. 25
  25. Kenyon, T. K., J. I. Cohen, and C. Grose. 2002. Phosphorylation by the varicella-zoster virus ORF47 protein serine kinase determines whether endocytosed viral gE traffics to the trans-Golgi network or recycles to the cell membrane. J. Virol. 76:10980-10993.[Abstract/Free Full Text]
    26. 26
  26. Kimura, H., S. E. Straus, and R. K. Williams. 1997. Varicella-zoster virus glycoproteins E and I expressed in insect cells form a heterodimer that requires the N-terminal domain of glycoprotein I. Virology 233:382-391.[CrossRef][Medline]
    27. 27
  27. Ku, C. C., J. Besser, A. Abendroth, C. Grose, and A. M. Arvin. 2005. Varicella-zoster virus pathogenesis and immunobiology: new concepts emerging from investigations with the SCIDhu mouse model. J. Virol. 79:2651-2658.[Free Full Text]
    28. 28
  28. Ku, C. C., J. A. Padilla, C. Grose, E. C. Butcher, and A. M. Arvin. 2002. Tropism of varicella-zoster virus for human tonsillar CD4+ T lymphocytes that express activation, memory, and skin homing markers. J. Virol. 76:11425-11433.[Abstract/Free Full Text]
    29. 29
  29. Ku, C. C., L. Zerboni, H. Ito, B. S. Graham, M. Wallace, and A. M. Arvin. 2004. Varicella-zoster virus transfer to skin by T Cells and modulation of viral replication by epidermal cell interferon-alpha. J. Exp. Med. 200:917-925.[Abstract/Free Full Text]
    30. 30
  30. Li, Q., M. A. Ali, and J. I. Cohen. 2006. Insulin degrading enzyme is a cellular receptor mediating varicella-zoster virus infection and cell-to-cell spread. Cell 127:305-316.[CrossRef][Medline]
    31. 31
  31. Li, Q., T. Krogmann, M. A. Ali, W. J. Tang, and J. I. Cohen. 2007. The amino terminus of varicella-zoster virus (VZV) glycoprotein E is required for binding to insulin-degrading enzyme, a VZV receptor. J. Virol. 81:8525-8532.[Abstract/Free Full Text]
    32. 32
  32. Litwin, V., W. Jackson, and C. Grose. 1992. Receptor properties of two varicella-zoster virus glycoproteins, gpI and gpIV, homologous to herpes simplex virus gE and gI. J. Virol. 66:3643-3651.[Abstract/Free Full Text]
    33. 33
  33. Litwin, V., M. Sandor, and C. Grose. 1990. Cell surface expression of the varicella-zoster virus glycoproteins and Fc receptor. Virology 178:263-272.[CrossRef][Medline]
    34. 34
  34. Mallory, S., M. Sommer, and A. M. Arvin. 1997. Mutational analysis of the role of glycoprotein I in varicella-zoster virus replication and its effects on glycoprotein E conformation and trafficking. J. Virol. 71:8279-8288.[Abstract]
    35. 35
  35. Maresova, L., T. J. Pasieka, and C. Grose. 2001. Varicella-zoster virus gB and gE coexpression, but not gB or gE alone, leads to abundant fusion and syncytium formation equivalent to those from gH and gL coexpression. J. Virol. 75:9483-9492.[Abstract/Free Full Text]
    36. 36
  36. Maresova, L., T. J. Pasieka, E. Homan, E. Gerday, and C. Grose. 2005. Incorporation of three endocytosed varicella-zoster virus glycoproteins, gE, gH, and gB, into the virion envelope. J. Virol. 79:997-1007.[Abstract/Free Full Text]
    37. 37
  37. McMillan, T. N., and D. C. Johnson. 2001. Cytoplasmic domain of herpes simplex virus gE causes accumulation in the trans-Golgi network, a site of virus envelopment and sorting of virions to cell junctions. J. Virol. 75:1928-1940.[Abstract/Free Full Text]
    38. 38
  38. Mettenleiter, T. C. 2004. Budding events in herpesvirus morphogenesis. Virus Res. 106:167-180.[CrossRef][Medline]
    39. 39
  39. Mettenleiter, T. C. 2003. Pathogenesis of neurotropic herpesviruses: role of viral glycoproteins in neuroinvasion and transneuronal spread. Virus Res. 92:197-206.[CrossRef][Medline]
    40. 40
  40. Mo, C., J. Lee, M. Sommer, C. Grose, and A. M. Arvin. 2002. The requirement of varicella zoster virus glycoprotein E (gE) for viral replication and effects of glycoprotein I on gE in melanoma cells. Virology 304:176-186.[CrossRef][Medline]
    41. 41
  41. Mo, C., E. E. Schneeberger, and A. M. Arvin. 2000. Glycoprotein E of varicella-zoster virus enhances cell-cell contact in polarized epithelial cells. J. Virol. 74:11377-11387.[Abstract/Free Full Text]
    42. 42
  42. Moffat, J., H. Ito, M. Sommer, S. Taylor, and A. M. Arvin. 2002. Glycoprotein I of varicella-zoster virus is required for viral replication in skin and T cells. J. Virol. 76:8468-8471.[Abstract/Free Full Text]
    43. 43
  43. Moffat, J., C. Mo, J. J. Cheng, M. Sommer, L. Zerboni, S. Stamatis, and A. M. Arvin. 2004. Functions of the C-terminal domain of varicella-zoster virus glycoprotein E in viral replication in vitro and skin and T-cell tropism in vivo. J. Virol. 78:12406-12415.[Abstract/Free Full Text]
    44. 44
  44. Moffat, J. F., M. D. Stein, H. Kaneshima, and A. M. Arvin. 1995. Tropism of varicella-zoster virus for human CD4+ and CD8+ T lymphocytes and epidermal cells in SCID-hu mice. J. Virol. 69:5236-5242.[Abstract]
    45. 45
  45. Moffat, J. F., L. Zerboni, P. R. Kinchington, C. Grose, H. Kaneshima, and A. M. Arvin. 1998. Attenuation of the vaccine Oka strain of varicella-zoster virus and role of glycoprotein C in alphaherpesvirus virulence demonstrated in the SCID-hu mouse. J. Virol. 72:965-974.[Abstract/Free Full Text]
    46. 46
  46. Niizuma, T., L. Zerboni, M. H. Sommer, H. Ito, S. Hinchliffe, and A. M. Arvin. 2003. Construction of varicella-zoster virus recombinants from parent Oka cosmids and demonstration that ORF65 protein is dispensable for infection of human skin and T cells in the SCID-hu mouse model. J. Virol. 77:6062-6065.[Abstract/Free Full Text]
    47. 47
  47. Olson, J. K., G. A. Bishop, and C. Grose. 1997. Varicella-zoster virus Fc receptor gE glycoprotein: serine/threonine and tyrosine phosphorylation of monomeric and dimeric forms. J. Virol. 71:110-119.[Abstract]
    48. 48
  48. Olson, J. K., and C. Grose. 1998. Complex formation facilitates endocytosis of the varicella-zoster virus gE:gI Fc receptor. J. Virol. 72:1542-1551.[Abstract/Free Full Text]
    49. 49
  49. Olson, J. K., and C. Grose. 1997. Endocytosis and recycling of varicella-zoster virus Fc receptor glycoprotein gE: internalization mediated by a YXXL motif in the cytoplasmic tail. J. Virol. 71:4042-4054.[Abstract]
    50. 50
  50. Pasieka, T. J., L. Maresova, K. Shiraki, and C. Grose. 2004. Regulation of varicella-zoster virus-induced cell-to-cell fusion by the endocytosis-competent glycoproteins gH and gE. J. Virol. 78:2884-2896.[Abstract/Free Full Text]
    51. 51
  51. Polcicova, K., K. Goldsmith, B. L. Rainish, T. W. Wisner, and D. C. Johnson. 2005. The extracellular domain of herpes simplex virus gE is indispensable for efficient cell-to-cell spread: evidence for gE/gI receptors. J. Virol. 79:11990-12001.[Abstract/Free Full Text]
    52. 52
  52. Rizvi, S. M., and M. Raghavan. 2001. An N-terminal domain of herpes simplex virus type Ig E is capable of forming stable complexes with gI. J. Virol. 75:11897-11901.[Abstract/Free Full Text]
    53. 53
  53. Santos, R. A., C. C. Hatfield, N. L. Cole, J. A. Padilla, J. F. Moffat, A. M. Arvin, W. T. Ruyechan, J. Hay, and C. Grose. 2000. Varicella-zoster virus gE escape mutant VZV-MSP exhibits an accelerated cell-to-cell spread phenotype in both infected cell cultures and SCID-hu mice. Virology 275:306-317.[CrossRef][Medline]
    54. 54
  54. Sommer, M. H., E. Zagha, O. K. Serrano, C. C. Ku, L. Zerboni, A. Baiker, R. Santos, M. Spengler, J. Lynch, C. Grose, W. Ruyechan, J. Hay, and A. M. Arvin. 2001. Mutational analysis of the repeated open reading frames, ORFs 63 and 70 and ORFs 64 and 69, of varicella-zoster virus. J. Virol. 75:8224-8239.[Abstract/Free Full Text]
    55. 55
  55. Spear, P. G. 2004. Herpes simplex virus: receptors and ligands for cell entry. Cell Microbiol. 6:401-410.[CrossRef][Medline]
    56. 56
  56. Sprague, E. R., C. Wang, D. Baker, and P. J. Bjorkman. 2006. Crystal structure of the HSV-1 Fc receptor bound to Fc reveals a mechanism for antibody bipolar bridging. PLoS Biol. 4:e148.[CrossRef][Medline]
    57. 57
  57. Wang, Z. H., M. D. Gershon, O. Lungu, Z. Zhu, S. Mallory, A. M. Arvin, and A. A. Gershon. 2001. Essential role played by the C-terminal domain of glycoprotein I in envelopment of varicella-zoster virus in the trans-Golgi network: interactions of glycoproteins with tegument. J. Virol. 75:323-340.[Abstract/Free Full Text]
    58. 58
  58. Whealy, M. E., J. P. Card, A. K. Robbins, J. R. Dubin, H. J. Rziha, and L. W. Enquist. 1993. Specific pseudorabies virus infection of the rat visual system requires both gI and gp63 glycoproteins. J. Virol. 67:3786-3797.[Abstract/Free Full Text]
    59. 59
  59. Wisner, T., C. Brunetti, K. Dingwell, and D. C. Johnson. 2000. The extracellular domain of herpes simplex virus gE is sufficient for accumulation at cell junctions but not for cell-to-cell spread. J. Virol. 74:2278-2287.[Abstract/Free Full Text]
    60. 60
  60. Yao, Z., and C. Grose. 1994. Unusual phosphorylation sequence in the gpIV (gI) component of the varicella-zoster virus gpI-gpIV glycoprotein complex (VZV gE-gI complex). J. Virol. 68:4204-4211.[Abstract/Free Full Text]
    61. 61
  61. Yao, Z., W. Jackson, B. Forghani, and C. Grose. 1993. Varicella-zoster virus glycoprotein gpI/gpIV receptor: expression, complex formation, and antigenicity within the vaccinia virus-T7 RNA polymerase transfection system. J. Virol. 67:305-314.[Abstract/Free Full Text]
    62. 62
  62. Yao, Z., W. Jackson, and C. Grose. 1993. Identification of the phosphorylation sequence in the cytoplasmic tail of the varicella-zoster virus Fc receptor glycoprotein gpI. J. Virol. 67:4464-4473.[Abstract/Free Full Text]
    63. 63
  63. Zerboni, L., C. C. Ku, C. D. Jones, J. L. Zehnder, and A. M. Arvin. 2005. Varicella-zoster virus infection of human dorsal root ganglia in vivo. Proc. Natl. Acad. Sci. USA 102:6490-6495.[Abstract/Free Full Text]
    64. 64
  64. Zerboni, L., M. Reichelt, C. D. Jones, J. L. Zehnder, H. Ito, and A. M. Arvin. 2007. Aberrant infection and persistence of varicella-zoster virus in human dorsal root ganglia in vivo in the absence of glycoprotein I. Proc. Natl. Acad. Sci. USA 104:14086-14091.[Abstract/Free Full Text]
    65. 65
  65. Zhu, Z., M. D. Gershon, Y. Hao, R. T. Ambron, C. A. Gabel, and A. A. Gershon. 1995. Envelopment of varicella-zoster virus: targeting of viral glycoproteins to the trans-Golgi network. J. Virol. 69:7951-7959.[Abstract]
    66. 66
  66. Zhu, Z., Y. Hao, M. D. Gershon, R. T. Ambron, and A. A. Gershon. 1996. Targeting of glycoprotein I (gE) of varicella-zoster virus to the trans-Golgi network by an AYRV sequence and an acidic amino acid-rich patch in the cytosolic domain of the molecule. J. Virol. 70:6563-6575.[Abstract/Free Full Text]
    67. 67
  67. Zuckermann, F. A., T. C. Mettenleiter, C. Schreurs, N. Sugg, and T. Ben-Porat. 1988. Complex between glycoproteins gI and gp63 of pseudorabies virus: its effect on virus replication. J. Virol. 62:4622-4626.[Abstract/Free Full Text]

Journal of Virology, January 2009, p. 228-240, Vol. 83, No. 1
0022-538X/09/$08.00+0     doi:10.1128/JVI.00913-08
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