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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2009 Dec 29;107(1):282–287. doi: 10.1073/pnas.0912373107

Functions of the unique N-terminal region of glycoprotein E in the pathogenesis of varicella-zoster virus infection

Barbara Berarducci 1,1, Jaya Rajamani 1, Leigh Zerboni 1, Xibing Che 1, Marvin Sommer 1, Ann M Arvin 1,2
PMCID: PMC2806775  PMID: 19966293

Abstract

Varicella-zoster virus (VZV) is an alphaherpesvirus that infects skin, lymphocytes, and sensory ganglia. VZV glycoprotein E (gE) has a unique N-terminal region (aa1-188), which is required for replication and includes domains involved in secondary envelopment, efficient cell-cell spread, and skin infection in vivo. The nonconserved N-terminal region also mediates binding to the insulin-degrading enzyme (IDE), which is proposed to be a VZV receptor. Using viral mutagenesis to make the recombinant rOka-ΔP27-G90, we showed that amino acids in this region are required for gE/IDE binding in infected cells; this deletion reduced cell-cell spread in vitro and skin infection in vivo. However, a gE point mutation, linker insertions, and partial deletions in the aa27-90 region, and deletion of a large portion of the unique N-terminal region, aa52-187, had similar or more severe effects on VZV replication in vitro and in vivo without disrupting the gE/IDE interaction. VZV replication in T cells in vivo was not impaired by deletion of gE aa27-90, suggesting that these gE residues are not essential for VZV T cell tropism. However, the rOka-ΔY51-P187 mutant failed to replicate in T cell xenografts as well as skin in vivo. VZV tropism for T cells and skin, which is necessary for its life cycle in the human host, requires this nonconserved region of the N-terminal region of VZV gE.

Keywords: herpesvirus, insulin degrading enzyme, entry, tropism, xenograft

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella as the primary infection, and zoster upon reactivation from latency in the sensory ganglia (1). In addition to its tropism for skin and neural cells, VZV infects T cells, which provides a mechanism for viral transport to the skin (2, 3). VZV skin infection progresses by cell-cell spread and polykaryocyte formation, whereas VZV-infected T cells do not undergo fusion. Functions of VZV gene products can be determined by mutagenesis of the genome (4, 5). Evaluating VZV mutants in human tissue xenografts in SCID mice provides an approach to define functional domains of VZV proteins that are important for infection of skin, T cells, and sensory ganglia (6, 7).

Among the nine putative VZV glycoproteins, glycoprotein E (gE), which is a 623-amino acid type I membrane protein, is the most abundant in infected cells and functions in cell-cell spread and secondary envelopment (8–12). All alphaherpesviruses have gE homologs but VZV gE differs in being essential (12–15). Further, we showed that VZV gE has a large nonconserved N-terminal region (aa1-188) that is required for replication (12). Mutations in this domain altered cell-cell spread, secondary envelopment, and skin infection (12). The gE cytosolic domain has an essential endocytosis and a gE trafficking motif (16–19) that contributes to VZV virulence (15).

Like its homologs, VZV gE forms noncovalent heterodimers with glycoprotein I (gI) (20), which is important for gE trafficking and maturation, cell-cell spread, and secondary envelopment (13, 14, 20–23). VZV gI is not required in vitro (13, 24) but is essential for infection of skin and T cells in vivo and deleting gI disrupts neurotropism (25, 26). We identified the first cysteine-rich region of gE within the conserved region of the gE ectodomain as being necessary for gE/gI complex formation in VZV-infected cells (21). Disrupting this domain altered gE expression on plasma membrane, decreased cell-cell spread, and blocked skin infection (21). Deleting gI had similar consequences (13, 25), indicating that the gE/gI complex is necessary for these functions. In contrast, gE in a form that did not bind gI was sufficient for secondary envelopment of virions (21), whereas virion assembly is aberrant without gI (22). Infection of T cells allows assessment of residues involved in VZV entry because cell fusion is not induced (27). The gE ΔCys mutant infected T cells (21), indicating that gE/gI binding was not required.

The gE/gI complex is important for cell-cell spread and envelopment of herpes simplex viruses (HSV) 1 and 2, which are related to VZV (28–30). In HSV, as in other alphaherpesviruses, gD binding to a cellular receptor is an essential step for entry (31, 32), whereas VZV lacks gD. It has been suggested that gE and gD derive from gene duplication (33) and that VZV gE may have gD-like functions (8). VZV gE has been proposed to act as a receptor binding protein by its interaction with insulin degrading enzyme (IDE) (34). In transient expression experiments, gE interaction with IDE involved aa24-71 in the unique N-terminal region; deleting aa32-71 blocked the interaction, and gE/IDE binding was independent of gI (35). gE/IDE binding is independent of gE/gI heterodimer formation during VZV replication because it was preserved in the gE ΔCys mutant (21).

Based on these observations, we used our gE linker insertion and deletion mutants (12) to generate new gE mutants with partial and complete deletions of the IDE-binding domain and to evaluate the consequences of interference with the gE/IDE interaction as well as effects of other changes in the unique gE N-terminal region on VZV pathogenesis in the SCID mouse model.

Results

Effects of Mutagenesis of gE Residues from aa27-90 on VZV Replication In Vitro.

Mutant pSpe23 cosmids pSpe23-ΔP27-G90 and pSpe23-ΔY51-G90, deleting aa28-90 and aa52-90, were constructed (Fig. 1A), and viruses rOka-ΔP27-G90 and rOka-ΔY51-G90 were recovered by using two independently derived mutant cosmids, indicating that these two regions of gE were not essential. rOka-P27, rOka-Y51, rOka-S31A, and rOka-ΔP27-Y51 (Fig. 1A) were made as described in ref. 12. The growth kinetics of rOka-ΔP27-G90 showed a minor decrease on days 2, 3, 5, and 6 compared to rOka (Fig. 1B); the ΔY51-G90 deletion had no effect (Fig. 1C). When compared to rOka plaque size (1.12 ± 0.42 mm2), plaque sizes of rOka-ΔP27-G90 (0.54 ± 0.26 mm2, P < 0.0001) and rOka-ΔY51-G90 (0.77 ± 0.29 mm2, P < 0.001) were decreased (Fig. 1 D–F). Growth kinetics of rOka-ΔP27-Y51 and rOka-Y51 did not differ from rOka, whereas rOka-ΔP27-Y51 had a small plaque phenotype and rOka-P27 showed delayed growth on days 1–3 and slightly smaller plaques; rOka-S31A titers were lower on days 3–6, with no change in plaque size (12).

Fig. 1.

Fig. 1.

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Effect of the gE ΔP27-G90 and ΔY51-G90 mutations on VZV replication in vitro. (A) Schematic of gE N-terminal region mutagenesis. The aa position of the linker insertions (∇, line 1), serine to alanine substitution (tick bar, line 2), and deletions with linker insertion (∆, line 3) are indicated. The black box indicates the transmembrane domain. (B and C) Replication of the gE mutants in vitro. Melanoma cells were inoculated on day 0 with 1 × 103 pfu of rOka or rOka-ΔP27-G90 (B), and rOka or rOka-ΔY51-G90 (C), and aliquots harvested from day 1 to day 6 were titered on melanoma cells. Each point represents the mean titer of three wells. The error bars represent the standard deviation (SD). (D–F) Analysis of plaque morphology. Melanoma cells infected with rOka (D), rOka-ΔY51-G90 (E), or rOka-ΔP27-G90 (F) were stained with polyclonal anti-VZV human immune serum. (Magnification: ×50.)

Effect of Mutations Involving gE aa27-90 on Binding to IDE and gI.

Coimmunoprecipitation of gE and IDE from cells infected with rOka-P27, rOka-S31A, rOka-Y51, and rOka-ΔP27-Y51 showed that the gE/IDE interaction was preserved (Fig. 2A). The P27 and S31A mutations are adjacent to and the Y51 is within the region aa32-71, reported to be involved in IDE binding (35); the deletion of aa28-51 eliminated most of this region. None of these mutations impaired the gE/gI interaction (Fig. 2B) (12). Coimmunoprecipitations with rOka-ΔP27-G90 and rOka-ΔY51-G90 showed that the ΔP27-G90 mutation disrupted the gE/IDE interaction, whereas the partial deletion ΔY51-G90 did not (Fig. 2C); these two gE mutations did not affect the gE/gI interaction (Fig. 2D). Coimmunoprecipitation of gE and IDE from lysates of Hek293 cells transfected with plasmids expressing gE ΔP27-Y51, ΔY51-G90, and ΔP27-G90 confirmed these results and showed that the effect of these mutations on gE/IDE binding was independent of the presence of gI (Fig. 2E).

Fig. 2.

Fig. 2.

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Effect of mutagenesis of the gE N-terminal region on gE/IDE and gE/gI interactions. Analysis of gE/IDE (A) and gE/gI (B) interactions in melanoma cells infected with the gE N-terminal mutants. Analysis of gE/IDE (C) and gE/gI (D) interactions in rOka-ΔP27-G90 and rOka-ΔY51-G90-infected melanoma cells. L, lysate; IP, immunoprecipitation; C, IP control. (E) Coimmunoprecipitation of gE and IDE in Hek293 cells transfected with the expression plasmids carrying the wt gE, gE ΔP27-Y51, gE ΔY51-G90, or gE ΔP27-G90. gE was immunoprecipitated with MAb anti-gE (Chemicon) (A and E) or MAb 3B3 (B and C), whereas gI was immunoprecipitated with MAb anti-gI 6B5 (D). gI was detected with rabbit anti-gI v67 (B), IDE with rabbit anti-IDE antibody (A, C, and E), and gE with MAb 3B3 (D). Ui, uninfected cells lysates; vector, lysates from cells transfected with the empty vector.

Effect of Mutations of the gE aa27-90 region on gE Intracellular Localization and Expression, and T Cell Entry.

Analysis 24 h and 72 h after infection showed that gE-ΔY51-G90 localization was similar to wt gE in rOka-infected cells (Fig. 3 A, C, D, and F). gE ΔP27-G90 localization was similar to rOka at 24 h (Fig. 3 A and B), whereas it was more evident at the perinuclear region (ER and TGN) than at the cell surface at 72 h (Fig. 3 D, E, G, and H); gI localization was similar. By immunoblot gE expression was similar in rOka-ΔY51-G90 and rOka-infected cells at 24 h and 72 h (Fig. 3I, lanes 2 and 4), whereas the ΔP27-G90 mutation was associated with decreased gE at 24 h and 72 h (Fig. 3I, lane 3). IE63 expression was similar in cells infected with rOka-ΔY51-G90 and rOka (Fig. 3I, lanes 2 and 4), whereas some decrease in IE63 was observed in rOka-ΔP27-G90-infected cells (Fig. 3I, lane 3). The P27, Y51, S31A, and ΔP27-Y51 mutations did not alter gE trafficking but gE expression was decreased in rOka-P27 and rOka-ΔP27-Y51-infected cells (12).

Fig. 3.

Fig. 3.

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Effect of mutagenesis of the gE aa27-90 region on gE cellular localization and expression and VZV entry into human T cells in vitro. Melanoma cells were infected with rOka (A, D, and G), rOka-ΔP27-G90 (B, E, and H), or rOka-ΔY51-G90 (C and F), and fixed at 24 h (A–C) and 72 h (D–H) after infection. Cells were labeled with MAb anti-gE (Chemicon) (red), and nuclei were stained with Hoechst 33342 (blue). Insets are enlargements of the images in the panels. (Magnfication: A–F, ×400; G and H, ×630.) (I) Lysates from melanoma cells infected with rOka or gE mutant viruses were collected at 24 and 72 h after infection (p.i.) and analyzed by Western blot using MAb anti-gE 3B3 (Top), or rabbit anti-IE63 (Middle). MAb anti-αtubulin was used for normalization (Bottom). Lane 1, uninfected cells; lane 2, rOka; lane 3, rOka-ΔP27-G90; lane 4, rOka-ΔY51-G90. (J) Primary human tonsil T cells were infected by coculturing with HELF-infected monolayers for 26 h, stained with anti-CD3 antibody and anti-VZV immune serum, and analyzed by flow cytometry. Error bars indicate SD. The titer of the infected monolayers is indicated in the table.

To further evaluate the consequences of mutations in the gE IDE-binding region, we analyzed the capacity of the rOka-ΔP27-Y51, rOka-ΔP27-G90, and rOka-ΔY51-G90 mutants to enter T cells in vitro. There were no differences in percentages of VZV+CD3+ T cells when mutants with partial and complete deletions of the gE region aa27-90 were compared to rOka (Fig. 3J). Results were comparable in two additional analyses. Thus, T cell entry of rOka-ΔP27-G90, in which gE/IDE binding was disrupted, did not differ from that of gE mutants that retained IDE binding.

Role of the gE aa27-90 Region in Skin Infection.

When assessed in SCIDhu skin xenografts, the growth of rOka-ΔP27-Y51 was significantly less than rOka at both 10 and 22 days after infection (Fig. 4A); in addition, at day 22, virus was recovered from only 3 of 6 xenografts compared to 6 of 6 infected with rOka. At day 10, rOka-ΔP27-G90 replication was significantly lower, whereas rOka-ΔY51-G90 was equivalent to rOka (Fig. 4B). Replication of rOka-ΔP27-G90 was also significantly decreased at 22 days; rOka-ΔY51-G90 showed a slight increase (Fig. 4B). Thus, although the ΔP27-G90 mutation, which disrupted IDE binding, diminished VZV virulence, the partial deletion, ΔP27-Y51, also reduced VZV pathogenesis in skin even though gE/IDE binding was not disrupted.

Fig. 4.

Fig. 4.

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Effect of the partial and complete deletions of the gE aa27-90 region on VZV replication in human skin in vivo. Skin xenografts were inoculated with HELF infected with rOka or rOka-ΔP27-Y51 (A) and rOka, rOka-ΔP27-G90, or rOka-ΔY51-G90 (B). 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 for those yielding virus, and the error bar indicates the standard error (SE). *, P < 0.05; **, P < 0.01. The titers of the inoculum are indicated as pfu/mL.

Effect of Mutations of the gE aa51-187 Region on VZV T Cell Tropism.

The gE ΔY51-P187 mutation, which removed most of the unique N-terminal region, reduced replication, had a small plaque phenotype, and altered secondary envelopment (12). To test its effect on gE/IDE interaction, wt gE and gE ΔY51-P187 were tagged with the HA epitope. Both gE ΔY51-P187 and wt gE were coimmunoprecipitated with IDE in transfected Hek293 cells (Fig. 5A), indicating that the interaction was preserved; however, IDE appeared to be immunoprecipitated less efficiently with the ΔY51-P187 construct. Similar results were obtained with the reverse immunoprecipitation. When effects on T cell entry were assessed, 20.8% of the CD3+ cells cocultured with rOka-infected HELF were VZV+ compared to 13.7% in the rOka-ΔY51-P187 preparation (Fig. 5B). Although this difference in the percentage of VZV+ T cells was marginally significant (P = 0.049), these experiments indicated that the rOka-ΔY51-P187 mutant retained the capacity to enter T cells in vitro. However, when the replication of rOka-ΔY51-P187, rOka-Y51, and rOka-S31A mutants was compared with rOka in T cell xenografts in SCID mice, rOka-ΔY51-P187 was not recovered at 14 or 21 days after infection (Fig. 5C). No significant differences were observed between rOka-Y51 and rOka at day 14 (Fig. 5C); rOka-Y51 was not recovered by day 21, consistent with rapid growth and depletion of T cells from the xenografts. The growth of rOka-S31A resembled rOka at both 14 and 21 days (Fig. 5C). Finally, when replication of rOka-ΔP27-G90 was evaluated in T cell xenografts, viral titers were equivalent at day 12 (P = 0.42) (Fig. 5D). By day 21, T cells were severely depleted and virus yield had dropped significantly for both viruses; at this late time point, virus was recovered from 1 of 3 rOka-infected and 1 of 4 rOka-ΔP27-G90-infected xenografts at equivalent titers (Fig. 5D). Thus, although gE residues altered by the ΔY51-P187 mutation were essential, gE/IDE interaction was not required for VZV infection and replication in T cells in vivo.

Fig. 5.

Fig. 5.

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Effect of mutagenesis of gE aa51-187 region on VZV entry and replication in human T cells. (A) Coimmunoprecipitation of gE-ΔY51-P187 and IDE in transfected Hek293 cells. gE was immunoprecipitated with rabbit anti-HA; MAb anti-IDE was used to detect IDE. L, lysate; IP, immunoprecipitation; C, IP control; vector, lysates from cells transfected with the empty vector. (B) Human T cells from uninfected thymus/liver xenografts were infected by coculturing with rOka- or rOka-ΔY51-P187-infected HELF for 30 h. The T cells were stained with anti-CD3 antibody and anti-VZV immune serum and analyzed by flow cytometry. The error bars indicate SD. The titer of the infected monolayers is indicated in the table. *, P = 0.049. Number of replicates, n = 2. (C) Thymus/liver xenografts were inoculated with HELF infected with rOka, rOka-Y51, rOka-S31A, or rOka-ΔY51-P187. The number of samples from which infectious virus was recovered per total xenografts inoculated is on the horizontal axis. Each bar represents the mean titer for those yielding virus ± SE (D) Thymus/liver xenografts inoculated with rOka- or rOka-ΔP27-G90-infected HELF. Each bar represents the mean titer ± SD for samples yielding virus and containing > 5% CD3+ cells. The titers of the inoculum are shown as pfu/mL.

Discussion

VZV gE is an essential protein and contains a nonconserved 188-amino acid N-terminal region (12). Using mutagenesis of the VZV genome, we showed that gE binding to IDE is mediated by residues in the unique gE N-terminal region during VZV replication; however, VZV virulence for skin and T cells in human tissue xenografts in SCID mice was altered by targeted mutations in the unique gE N-terminal region whether or not IDE binding was affected (Table 1). During VZV replication, gE interactions with IDE were disrupted by the ΔP27-G90 mutation, which removes aa28-90, which includes the IDE binding domain, aa32-71, as defined in protein expression experiments (35). While this manuscript was in preparation, deleting gE aa32-71 from the VZV genome by using a different set of cosmids was reported to disrupt IDE binding (36).

Table 1.

Phenotypes of VZV recombinants with gE mutations in vitro and in vivo

Virus Replication in vitro Plaque size gE/IDE binding T cell entry in vitro Infection of xenografts in vivo
Skin T cells
S31A Slight decrease NL + ND Decrease NL
∇P27 Delayed Slight decrease + ND Slight decrease ND
∇Y51 NL NL + ND NL NL
∆P27-Y51 NL Decrease + + Decrease ND
∆Y51-G90 NL Decrease + + NL ND
∆P27-G90 Slight decrease Decrease — + Decrease NL
∆Y51-P187 Decrease Decrease +/− + None None
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ND: not done; NL: equivalent to parent virus.

Comparing the mapping of gE residues that mediate IDE binding by our viral mutagenesis with the analysis done by Li and colleagues using gE peptides (35) and the recent data by Ali et al. (36) confirms the importance of residues in the region from aa32-71. However, IDE binding was not abolished in cells infected with rOka-ΔP27-Y51 or rOka-ΔY51-G90, which differed from the observation that the aa24-50 peptide blocked the IDE/gE interaction and the aa51-71 peptide had a partial effect (35). Our experiments evaluated whether gE/IDE binding was preserved in the gE N-terminal mutants and did not assess the efficiency of this interaction; thus, an effect on the affinity of the gE/IDE interaction detected with the partial deletions ΔP27-Y51 and ΔY51-G90 was not excluded. The VZV gE region aa1-71 was not sufficient for IDE binding when fused to aa30-545 of HSV-2 gE, suggesting that the secondary structure of this portion of the gE N-terminal region is also important for binding to IDE (35). The fact that deletion of both portions of the aa27-90 region is required to abolish IDE binding during VZV replication further suggests a role for gE conformation in its IDE binding function.

The ΔY51-G90 and ΔP27-G90 mutations had little or no effect on growth kinetics in vitro, but cell-cell spread was reduced; effects of the ΔP27-Y51 mutation were similar (12) (Table 1). Removing aa52-187 with the ΔY51-P187 mutation markedly reduced both growth kinetics and plaque size. Thus, the characteristic VZV syncytia formation was diminished by mutations in the unique gE N-terminal region whether or not IDE binding was preserved. Some mutations that disrupted gE residues in the aa27-90 region, including ΔP27-G90, ΔP27-Y51, and P27, also resulted in less gE expression in infected cells; this effect was independent of IDE binding. It is possible that mutagenesis of aa27-90 altered gE stability. Reduced gE synthesis and accumulation at the plasma membrane at late times in cells infected with rOka-ΔP27-G90, which lacks IDE binding capacity, can explain diminished cell-cell spread. Similarly, Ali et al. showed that deletion of gE aa32-71 in ROka68D32-71 reduced gE expression and affected cell-cell spread (36). As in HSV, VZV gE enhanced the tight junction formation that contributes to cell-cell spread (37, 38). The reduced rOka-ΔP27-G90 syncytia formation might reflect less production or a role for gE/IDE interactions in cell-cell spread. IDE might also modulate the interaction of gE with other molecules, like the MPRci, which has been shown to be important for cell-free VZV infection in cultured cells (39).

The mutations altering gE in the aa27-90 region had variable effects on VZV pathogenesis in skin (Table 1). Although reduced syncytia formation in vitro often accompanies diminished skin virulence (12, 13, 21, 25), rOkaΔY51-G90 was not impaired. Blocking the gE/IDE interaction in rOka-ΔP27-G90 was associated with some reduction in skin infection but the partial deletion, ΔP27-Y51 that retains gE/IDE binding had similar effects. Although the lack of the gE/IDE complex was not uniquely correlated with impaired replication in skin, a contribution of the gE/IDE interaction to this phenotype is not excluded. The decrease in gE expression and accumulation on plasma membrane observed in vitro may contribute to the somewhat reduced virulence of rOka-ΔP27-G90 because skin polykaryocyte formation is aberrant when gE is mislocalized (40); the reduced gE expression in rOka-ΔP27-Y51 may have similar effects. Notably, both the ΔP27-G90 and ΔP27-Y51 deletions had much less severe consequences for skin virulence than the alanine substitution at S31 (12). Disrupting gE binding to gI also had a much greater impact on VZV infection in skin than blocking gE/IDE interactions (21). The large gE deletion, ΔY51-P187, was lethal, whereas ΔY51-G90 had no effect. These observations suggest that the portion of gE from aa90-187 is critical for skin pathogenesis, with functions that may include maintaining gE conformation or interactions with viral or cellular proteins.

In contrast to HSV, VZV exhibits lymphotropism that allows viral transport to skin (2, 27). None of the mutations of the unique gE N-terminal region, including ΔP27-G90 that prevented IDE binding and the large ΔY51-P187 deletion that may diminish efficiency of gE/IDE binding, blocked VZV entry into human T cells in vitro (Table 1). Some gE binding to IDE expressed by T cells has been shown by pull-down with soluble gE but whether the interaction was occurring at the plasma membrane or during infection was not determined (34). Deletion of the gE IDE binding domain in ROka68D32-71 impaired cell-free virus infection of melanoma cells (36); however, it was not determined if the entry defect resulted from blocking the gE/IDE interaction, reducing gE synthesis or interference with other gE functions that require this domain. Although our in vitro assay measures entry but not the efficiency of entry, which could be modulated by gE interactions with cell surface proteins, including IDE if it is present, the fact that replication of rOka-ΔP27-G90 in the T cell xenografts was not diminished is strong evidence that IDE does not function as a VZV T cell receptor. VZV encodes three glycoproteins, gB, gH and gL, that are critical for fusion of the virion envelope with the cell membrane in other herpesviruses, e.g., EBV infection of B lymphocytes (41). This highly conserved glycoprotein complex often functions with accessory proteins that seem to enhance or confer target cell specificity, e.g., gp42 has this function for EBV B cell entry but not epithelial cell entry (41). One hypothesis is that gE binding to IDE is an accessory function in fibroblasts, whereas VZV infection of T cells occurs through a pathway that does not involve gE/IDE interaction.

Nevertheless, our experiments indicate that the unique gE N-terminal region is an important determinant of VZV tropism for T cells because the ΔY51-P187 mutation was lethal for VZV replication in T cell xenografts in vivo. The failure of rOka-ΔY51-P187 to replicate in T cells in vivo does not appear to result from a block of T cell entry although entry efficiency might be diminished. The ΔY51-P187 mutation was associated with limited virion formation and intracytoplasmic aggregates, suggesting aberrant secondary envelopment (12); because VZV T cells tropism relies on virion assembly and release, the lethal phenotype observed in T cells xenografts is likely related to alterations of these processes.

In conclusion, the gE unique N-terminal region has important functions for VZV pathogenesis in skin and T cells in vivo and residues from aa51-187 appear to be indispensable. gE binding to IDE contributes to skin virulence but is not essential whereas gE/gI heterodimer formation is required (21). Importantly, our findings suggest that gE/IDE interaction is not necessary for VZV infection of T cells in vivo. Further investigation of the glycoproteins and cellular receptors involved in VZV entry into various differentiated cell types that are important for pathogenesis is needed.

Materials and Methods

Construction of ORF68 Mutant Cosmids.

The parental VZV Oka strain (P-Oka) genome is contained in four cosmids (pFsp73, pSpe14, pPme2, and pSpe23) (4). ORF68 (nt 115911–117782), which encodes gE, is in the unique short region (pSpe23) (nt 94055–125124). The pBSKΔKpnI-SacI plasmid containing ORF68 in the SacI fragment from pSpe23 was used for mutagenesis (12). The linker insertion mutants P27, Y51, and G90 (12) were used to produce deletions ΔP27-G90 (aa 28–90) and ΔY51-G90 (aa 52–90), containing the linker sequence inserted at the site of deletion, between aa27-91 and aa51-91, respectively. These mutants were generated by restrictions using the NotI site in the linker sequence and inserted in pSpe23 (12).

Cosmid Transfections, Infectious Focus Assay, and Plaque Size.

Cosmid transfections were done as described in ref. 13. DNA was isolated from transfected melanoma cells (DNAzol, Invitrogen); PCR and sequencing were performed to confirm the expected mutations. For infectious focus assay (12), cells were fixed in 4% paraformaldehyde, stained with polyclonal anti-VZV human immune serum (7), biotinylated anti-human IgG (Vector Laboratories), and alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories), and detected with the Fast Red substrate (Sigma). Images of 30 plaques per virus were acquired, and average plaque area was calculated with ImageJ (http://rsb.info.nih.gov/ij/).

Expression Plasmid Construction and Transfection of Hek293 Cells.

The pCDNA3.1+ plasmids containing wt gE or wt gE-HA (HA tag: YPYDVPDYA) have been described (12, 21); plasmids with gE mutations ΔP27-Y51, ΔY51-G90, ΔP27-G90, and ΔY51-P187-HA were generated with this procedure. Transfections of Hek293 cells were done as described in ref. 21.

Antibodies.

Antibodies were mouse monoclonal (MAb) anti-gE (Chemicon), MAb anti-gE 3B3 and MAb anti-gI 6B5 (kindly provided by C. Grose, University of Iowa, Iowa City, IA), rabbit anti-gI v67 (a gift from S. Silverstein, Columbia University, New York), MAb and rabbit anti-IDE antibodies (Covance), rabbit anti-HA antibody (Sigma), rabbit anti-IE63 antibody (a gift from W. Ruyechan, University of Buffalo, Buffalo, NY), and MAb anti-αtubulin antibody (Sigma).

Immunoprecipitation and Western Blot.

Protein lysates from melanoma cells infected with the gE N-terminal mutants, or Hek293 cells transfected with plasmids expressing gE mutants, were collected in lysis buffer supplemented with protease inhibitor mixture (Roche). Radioimmunoprecipitation (RIPA) buffer was used for analyzing the gE/gI interaction, whereas 25 mM Tris·HCL (pH 7.4), 5 mM EDTA, 15 mM NaCl, and 0.1% Nonidet P-40 was used for studying the gE/IDE interaction (34). Immunoprecipitations were performed as previously described in ref. 21. Mouse IgG (Vector Laboratories) or the beads used for preclearing the lysates were used as negative control for the immunoprecipitation. To avoid interference with the detection of gI or gE ΔY51-P187 by the antibody heavy chain in immunoblot, the ExactaCruz Immunoprecipitation kit (Santa Cruz Biotechnology) was used to cross-link the antibody to the beads. For Western blotting analysis, protein lysates obtained from two separate preparations of infected cells were processed as described in refs. 12 and 21.

Confocal Microscopy.

Melanoma cells infected with rOka or the gE mutants viruses were fixed in 4% paraformaldehyde and processed for immunofluorescence as described in ref. 12. Confocal analysis was performed with a Leica TCS SP2 confocal laser scanning microscope.

Infection of CD3+ T Cells In Vitro.

HELF infected with rOka-ΔP27-Y51, rOka-ΔY51-G90, rOka-ΔP27-G90, or rOka were overlaid with 1 × 107 tonsil T cells (21). HELF infected with rOka-ΔY51-P187 or rOka were overlaid with 3.5 × 106 T cells obtained from uninfected human thymus/liver xenografts. As negative control, uninfected fibroblasts were also overlaid with T cells. T cells were stained and analyzed by flow cytometry as described in ref. 21. Samples were analyzed on a FACSCalibur apparatus.

Infection of Human Xenografts in SCIDhu Mice.

Skin xenografts were made in homozygous CB-17scid/scid mice (7) by using human fetal tissue obtained according to federal and state regulations. Animal use was in accordance with the Animal Welfare Act and approved by the Stanford University Administrative Panel on Laboratory Animal Care. rOka and rOka-gE mutant viruses were passed three times in primary human lung fibroblasts (MRC-5 or HELF) before inoculation. Inoculum titers were determined at inoculation. Skin xenografts were harvested at 10 and 22 days and T cell xenografts at 12 or 14, and 21 days after inoculation, and viral titers were determined by infectious focus assay (7). T cell xenografts infected with rOka and rOka-ΔP27-G90 were also analyzed by flow cytometry using the T cell marker CD3 and the anti-VZV immune serum. Viruses recovered from the xenografts were tested by PCR and sequencing.

Statistical Analysis.

Statistical differences were determined by the Student t test and considered significant at P < 0.05.

Acknowledgments

SCID-hu (thy/liv) mice were kindly provided by Cheryl Stoddard (Gladstone Institute, University of California, San Francisco). We thank Mike Reichelt for valuable assistance with confocal microscopy and Linda Lew for technical assistance. This work was supported by AI20459 and CA49605.

Footnotes

The authors declare no conflict of interest.

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