Varicella-zoster virus glycoprotein E is a critical determinant of virulence in the SCID mouse-human model of neuropathogenesis

Varicella-zoster virus (VZV) is a neurotropic alphaherpesvirus. VZV infection of human dorsal root ganglion (DRG) xenografts in immunodeficient mice models the infection of sensory ganglia. We examined DRG infection with recombinant VZV (recombinant Oka [rOka]) and the following gE mutants: gEΔ27-90, gEΔCys, gE-AYRV, and gE-SSTT. gEΔ27-90, which lacks the gE domain that interacts with a putative receptor insulin-degrading enzyme (IDE), replicated as extensively as rOka, producing infectious virions and significant cytopathic effects within 14 days of inoculation. Since neural cells express IDE, the gE/IDE interaction was dispensable for VZV neurotropism. In contrast, gEΔCys, which lacks gE/gI heterodimer formation, was significantly impaired at early times postinfection; viral genome copy numbers increased slowly, and infectious virus production was not detected until day 28. Delayed replication was associated with impaired cell-cell spread in ganglia, similar to the phenotype of a gI deletion mutant (rOkaΔgI). However, at later time points, infection of satellite cells and other supportive nonneuronal cells resulted in extensive DRG tissue damage and cell loss such that cytopathic changes observed at day 70 were more severe than those for rOka-infected DRG. The replication of gE-AYRV, which is impaired for trans-Golgi network (TGN) localization, and the replication of gE-SSTT, which contains mutations in an acidic cluster, were equivalent to that of rOka, causing significant cytopathic effects and infectious virus production by day 14; genome copy numbers were equivalent to those of rOka. These experiments suggest that the gE interaction with cellular IDE, gE targeting to TGN sites of virion envelopment, and phosphorylation at SSTT are dispensable for VZV DRG infection, whereas the gE/gI interaction is critical for VZV neurovirulence.     

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

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-ΔCys 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.     

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

Like all alphaherpesviruses, varicella-zoster virus (VZV) infection proceeds by both cell-cell spread and virion production. Virions are enveloped within vacuoles located near the trans-Golgi network (TGN), while in cell-cell spread, surface glycoproteins fuse cells into syncytia. In this report, we delineate a potential role for serine/threonine phosphorylation of the cytoplasmic tail of the predominant VZV glycoprotein, gE, in these processes. The fact that VZV gE (formerly called gpI) is phosphorylated has been documented (E. A. Montalvo and C. Grose, Proc. Natl. Acad. Sci. USA 83:8967-8971, 1986), although respective roles of viral and cellular protein kinases have never been delineated. VZV ORF47 is a viral serine protein kinase that recognized a consensus sequence similar to that of casein kinase II (CKII). During open reading frame 47 (ORF47)-specific in vitro kinase assays, ORF47 phosphorylated four residues in the cytoplasmic tail of VZV gE (S593, S595, T596, and T598), thus modifying the known phosphofurin acidic cluster sorting protein 1 domain. CKII phosphorylated gE predominantly on the two threonine residues. In wild-type-virus-infected cells, where ORF47-mediated phosphorylation predominated, gE endocytosed and relocalized to the TGN. In cells infected with a VZV ORF47-null mutant, internalized VZV gE recycled to the plasma membrane and did not localize to the TGN. The mutant virus also formed larger syncytia than the wild-type virus, linking CKII-mediated gE phosphorylation with increased cell-cell spread. Thus, ORF47 and CKII behaved as "team players" in the phosphorylation of VZV gE. Taken together, the results showed that phosphorylation of VZV gE by ORF47 or CKII determined whether VZV infection proceeded toward a pathway likely involved with either virion production or cell-cell spread.     

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

Varicella-zoster virus (VZV) glycoprotein E (gE) is a multifunctional protein important for cell-cell spread, envelopment, and possibly entry. In contrast to other alphaherpesviruses, gE is essential for VZV replication. Interestingly, the N-terminal region of gE, comprised of amino acids 1 to 188, was shown not to be conserved in the other alphaherpesviruses by bioinformatics analysis. Mutational analysis was performed to investigate the functions associated with this unique gE N-terminal region. Linker insertions, serine-to-alanine mutations, and deletions were introduced in the gE N-terminal region in the VZV genome, and the effects of these mutations on virus replication and cell-cell spread, gE trafficking and localization, virion formation, and replication in vivo in the skin were analyzed. In summary, mutagenesis of the gE N-terminal region identified a new functional region in the VZV gE ectodomain essential for cell-cell spread and the pathogenesis of VZV skin tropism and demonstrated that different subdomains of the unique N-terminal region had specific roles in viral replication, cell-cell spread, and secondary envelopment.     

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.

Previous studies suggested that varicella-zoster virus (VZV) envelope glycoproteins (gps) are selectively transported to the trans-Golgi network (TGN) and that the cytosolic domain of gpI (gE) targets it to the TGN. To identify targeting signals in the gpI cytosolic domain, intracellular protein trafficking was studied in transfected cells expressing chimeric proteins in which a full-length or mutated gpI cytosolic domain was fused to the gpI transmembrane domain and interleukin-2 receptor (tac) ectodomain. Expressed protein was visualized with antibodies to tac. A targeting sequence (AYRV) and a second, acidic amino acid-rich region of the gpI cytosolic domain (putative signal patch) were each sufficient to cause expressed protein to colocalize with TGN markers. This targeting was lost when the tyrosine of the AYRV sequence was replaced with glycine or lysine, when arginine was replaced with glutamic acid, or when valine was substituted with lysine. In contrast, tyrosine could be replaced by phenylalanine and valine could be substituted with leucine. Mutation of alanine to aspartic acid or deletion of alanine abolished TGN targeting. Exposure of transfected cells to antibodies to the tac ectodomain revealed that the TCN targeting of expressed tac-gpI chimeric proteins occurred as a result of selective retrieval from the plasmalemma. These data suggest that the AYRV sequence and a second signaling patch in the cytosolic domain of gpI are responsible for its targeting to the TGN. The observations also support the hypothesis that the TGN plays a critical role in the envelopment of VZV.     

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

Varicella-zoster virus (VZV) is enveloped in the trans-Golgi network (TGN). Here we report that glycoprotein I (gI) is required within the TGN for VZV envelopment. Enveloping membranous TGN cisternae were microscopically identified in cells infected with intact VZV. These sacs curved around, and ultimately enclosed, nucleocapsids. Tegument coated the concave face of these sacs, which formed the viral envelope, but the convex surface was tegument-free. TGN cisternae of cells infected with VZV mutants lacking gI (gI(Delta)) or its C (gI(DeltaC))- or N-terminal (gI(DeltaN))-terminal domains were uniformly tegument coated and adhered to one another, forming bizarre membranous stacks. Viral envelopment was compromised, and no virions were delivered to post-Golgi structures. The TGN was not gI-immunoreactive in cells infected with the gI(Delta) or gI(DeltaN) mutants, but it was in cells infected with gI(DeltaC) (because the ectodomains of gI and gE interact). The presence in the TGN of gI lacking a C-terminal domain, therefore, was not sufficient to maintain enveloping cisternae. In cells infected with intact VZV or with gI(Delta), gI(DeltaN), or gI(DeltaC) mutants, ORF10p immunoreactivity was concentrated on the cytosolic face of TGN membranes, suggesting that it interacts with the cytosolic domains of glycoproteins. Because of the gE-gI interaction, cotransfected cells that expressed gE or gI were able to target truncated forms of the other to the TGN. Our data suggest that the C-terminal domain of gI is required to segregate viral and cellular proteins in enveloping TGN cisternae.     

Mutagenesis of varicella-zoster virus glycoprotein I (gI) identifies a cysteine residue critical for gE/gI heterodimer formation, gI structure, and virulence in skin cells

Varicella-zoster virus (VZV) is the alphaherpesvirus that causes chicken pox (varicella) and shingles (zoster). The two VZV glycoproteins gE and gI form a heterodimer that mediates efficient cell-to-cell spread. Deletion of gI yields a small-plaque-phenotype virus, ΔgI virus, which is avirulent in human skin using the xenograft model of VZV pathogenesis. In the present study, 10 mutant viruses were generated to determine which residues were required for the typical function of gI. Three phosphorylation sites in the cytoplasmic domain of gI were not required for VZV virulence in vivo. Two deletion mutants mapped a gE binding region in gI to residues 105 to 125. A glycosylation site, N116, in this region did not affect virulence. Substitution of four cysteine residues highly conserved in the Alphaherpesvirinae established that C95 is required for gE/gI heterodimer formation. The C95A and Δ105-125 (with residues 105 to 125 deleted) viruses had small-plaque phenotypes with reduced replication kinetics in vitro similar to those of the ΔgI virus. The Δ105-125 virus was avirulent for human skin in vivo. In contrast, the C95A mutant replicated in vivo but with significantly reduced kinetics compared to those of the wild-type virus. In addition to abolished gE/gI heterodimer formation, gI from the C95A or the Δ105-125 mutant was not recognized by monoclonal antibodies that detect the canonical conformation of gI, demonstrating structural disruption of gI in these viruses. This alteration prevented gI incorporation into virus particles. Thus, residues C95 and 105 to 125 are critical for gI structure required for gE/gI heterodimer formation, virion incorporation, and ultimately, effective viral spread in human skin.     

Mutational analysis of the role of glycoprotein I in varicella-zoster virus replication and its effects on glycoprotein E conformation and trafficking.

The contributions of the glycoproteins gI (ORF67) and gE (ORF68) to varicella-zoster virus (VZV) replication were investigated in deletion mutants made by using cosmids with VZV DNA derived from the Oka strain. Deletion of both gI and gE prevented virus replication. Complete deletion of gI or deletions of 60% of the N terminus or 40% of the C terminus of gI resulted in a small plaque phenotype as well as reduced yields of infectious virus. Melanoma cells infected with gI deletion mutants formed abnormal polykaryocytes with a disrupted organization of nuclei. In the absence of intact gI, gE became localized in patches on the cell membrane, as demonstrated by confocal microscopy. A truncated N-terminal form of gI was transported to the cell surface, but its expression did not restore plaque morphology or infectivity. The fusogenic function of gH did not compensate for gI deletion or the associated disruption of the gE-gI complex. These experiments demonstrated that gI was dispensable for VZV replication in vitro, whereas gE appeared to be required. Although VZV gI was dispensable, its deletion or mutation resulted in a significant decrease in infectious virus yields, disrupted syncytium formation, and altered the conformation and distribution of gE in infected cells. Normal cell-to-cell spread and replication kinetics were restored when gI was expressed from a nonnative locus in the VZV genome. The expression of intact gI, the ORF67 gene product, is required for efficient membrane fusion during VZV replication.     

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

Herpes simplex virus (HSV) glycoprotein heterodimer gE/gI is necessary for virus spread in epithelial and neuronal tissues. Deletion of the relatively large gE cytoplasmic (CT) domain abrogates the ability of gE/gI to mediate HSV spread. The gE CT domain is required for the sorting of gE/gI to the trans-Golgi network (TGN) in early stages of virus infection, and there are several recognizable TGN sorting motifs grouped near the center of this domain. Late in HSV infection, gE/gI, other viral glycoproteins, and enveloped virions redistribute from the TGN to epithelial cell junctions, and the gE CT domain is also required for this process. Without the gE CT domain, newly enveloped virions are directed to apical surfaces instead of to cell junctions. We hypothesized that the gE CT domain promotes virus envelopment into TGN subdomains from which nascent enveloped virions are sorted to cell junctions, a process that enhances cell-to-cell spread. To characterize elements of the gE CT domain involved in intracellular trafficking and cell-to-cell spread, we constructed a panel of truncation mutants. Specifically, these mutants were used to address whether sorting to the TGN and redistribution to cell junctions are necessary, and sufficient, for gE/gI to promote cell-to-cell spread. gE-519, lacking 32 C-terminal residues, localized normally to the TGN early in infection and then trafficked to cell junctions at late times and mediated virus spread. By contrast, mutants gE-495 (lacking 56 C-terminal residues) and gE-470 (lacking 81 residues) accumulated in the TGN but did not traffic to cell junctions and did not mediate cell-to-cell spread. A fourth mutant, gE-448 (lacking most of the CT domain), did not localize to cell junctions and did not mediate virus spread. Therefore, the capacity of gE/gI to promote cell-cell spread requires early localization to the TGN, but this is not sufficient for virus spread. Additionally, gE CT sequences between residues 495 and 519, which contain no obvious cell sorting motifs, are required to promote gE/gI traffic to cell junctions and cell-to-cell spread.     

Endocytosis and recycling of varicella-zoster virus Fc receptor glycoprotein gE: internalization mediated by a YXXL motif in the cytoplasmic tail.

Varicella-zoster virus (VZV) encodes a cell surface Fc receptor, glycoprotein gE. VZV gE has previously been shown to display several features common to nonviral cell surface receptors. Most recently, VZV gE was reported to be tyrosine phosphorylated on a dimeric form (J. K. Olson, G. A. Bishop, and C. Grose, J. Virol. 71:110-119, 1997). Thereafter, attention focused on the ability of VZV gE to undergo receptor-mediated endocytosis. The current transient transfection studies demonstrated by confocal microscopy and internalization assays that VZV gE was endocytosed when expressed in HeLa cells. Endocytosis of gE was shown to be dependent on clathrin-coated vesicle formation within the cells. Subsequent colocalization studies showed that endocytosis of VZV gE closely mimicked endocytosis of the transferrin receptor. The gE cytoplasmic tail and more specifically tyrosine residue 582 were determined by mutagenesis studies to be important for efficient internalization of the protein; this tyrosine residue is part of a conserved YXXL motif. The amount of gE internalized at any given time reached a steady state of 32%. In addition, like the transferrin receptor, internalized gE recycled to the cell surface. The finding of gE endocytosis provided insight into earlier documentation of gE serine/threonine and tyrosine phosphorylation, since these phosphorylation events may serve as sorting signals for internalized receptors. Taken together with the previous discovery that both human and simian immunodeficiency virus envelope proteins can undergo endocytosis, the gE findings suggest that endocytosis of envelope components may be a posttranslational regulatory mechanism among divergent families of enveloped viruses.     

Varicella-Zoster Virus Glycoprotein I Is Essential for Spread in Dorsal Root Ganglia and Facilitates Axonal Localization of Structural Virion Components in Neuronal Cultures

Neurons of the sensory ganglia are the major site of varicella-zoster virus (VZV) latency and may undergo productive infection during reactivation. Although the VZV glycoprotein E/glycoprotein I (gE/gI) complex is known to be critical for neurovirulence, few studies have assessed the roles of these proteins during infection of dorsal root ganglia (DRG) due to the high human specificity of the virus. Here, we show that the VZV glycoprotein I gene is an important neurotropic gene responsible for mediating the spread of virus in neuronal cultures and explanted DRG. Inoculation of differentiated SH-SY5Y neuronal cell cultures with a VZV gI gene deletion strain (VZV rOkaΔgI) showed a large reduction in the percentage of cells infected and significantly smaller plaque sizes in a comparison with cultures infected with the parental strain (VZV rOka). In contrast, VZV rOkaΔgI was not significantly attenuated in fibroblast cultures, demonstrating a cell type-specific role for VZV gI. Analysis of rOkaΔgI protein localization by immunofluorescent staining revealed aberrant localization of viral glycoprotein and capsid proteins, with little or no staining present in the axons of differentiated SH-SY5Y cells infected with rOkaΔgI, yet axonal vesicle trafficking was not impaired. Further studies utilizing explanted human DRG indicated that VZV gI is required for the spread of virus within DRG. These data demonstrate a role for VZV gI in the cell-to-cell spread of virus during productive replication in neuronal cells and a role in facilitating the access of virion components to axons.     

Varicella-zoster virus glycoprotein I is essential for growth of virus in Vero cells.

Varicella-zoster virus (VZV) encodes at least six glycoproteins. Glycoprotein I (gI), the product of open reading frame 67, is a 58- to 62-kDa glycoprotein found in VZV-infected cells. We constructed two VZV gI deletion mutants. Immunoprecipitation of VZV gE from infected cells indicated that cells infected with VZV deleted for gI expressed a gE that was larger (100 kDa) than that expressed in cells infected with the parental virus (98 kDa). Cell-associated or cell-free VZV deleted for gI grew to lower titers in melanoma cells than did parental VZV. While VZV deleted for gI replicated in other human cells, the mutant virus replicated to very low titers in primary guinea pig and monkey cells and did not replicate in Vero cells. When compared with the parental virus, rescued viruses, in which the gI deletion was restored with a wild-type allele, showed a similarly sized gE and comparable growth patterns in melanoma and Vero cells. VZV deleted for gI entered Vero cells; however, viral DNA synthesis was impaired in these cells. The VZV gI mutant was slightly impaired for adsorption to human cells. Thus, VZV gI is required for replication of the virus in Vero cells, for efficient replication of the virus in nonhuman cells, and for normal processing of gE.     

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

Alphaherpesviruses express a heterodimeric glycoprotein, gE/gI, that facilitates cell-to-cell spread between epithelial cells and neurons. Herpes simplex virus (HSV) gE/gI accumulates at junctions formed between polarized epithelial cells at late times of infection. However, at earlier times after HSV infection, or when gE/gI is expressed using virus vectors, the glycoprotein localizes to the trans-Golgi network (TGN). The cytoplasmic (CT) domains of gE and gI contain numerous TGN and endosomal sorting motifs and are essential for epithelial cell-to-cell spread. Here, we swapped the CT domains of HSV gE and gI onto another HSV glycoprotein, gD. When the gD-gI(CT) chimeric protein was expressed using a replication-defective adenovirus (Ad) vector, the protein was found on both the apical and basolateral surfaces of epithelial cells, as was gD. By contrast, the gD-gE(CT) chimeric protein, gE/gI, and gE, when expressed by using Ad vectors, localized exclusively to the TGN. However, gD-gE(CT), gE/gI, and TGN46, a cellular TGN protein, became redistributed largely to lateral surfaces and cell junctions during intermediate to late stages of HSV infection. Strikingly, gE and TGN46 remained sequestered in the TGN when cells were infected with a gI(-)HSV mutant. The redistribution of gE/gI to lateral cell surfaces did not involve widespread HSV inhibition of endocytosis because the transferrin receptor and gE were both internalized from the cell surface. Thus, gE/gI accumulates in the TGN in early phases of HSV infection then moves to lateral surfaces, to cell junctions, at late stages of infection, coincident with the redistribution of a TGN marker. These results are related to recent observations that gE/gI participates in the envelopment of nucleocapsids into cytoplasmic vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004-4016, 2000) and that gE/gI can sort nascent virions from cytoplasmic vesicles specifically to the lateral surfaces of epithelial cells (D. C. Johnson, M. Webb, T. W. Wisner, and C. Brunetti, J. Virol. 75:821-833, 2000). Therefore, gE/gI localizes to the TGN, through interactions between the CT domain of gE and cellular sorting machinery, and then participates in envelopment of cytosolic nucleocapsids there. Nascent virions are then sorted from the TGN to cell junctions.     

Insulin Degrading Enzyme Induces a Conformational Change in Varicella-Zoster Virus gE,and Enhances Virus Infectivity and Stability

Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for virus infectivity and binds to a cellular receptor, insulin-degrading enzyme (IDE), through its unique amino terminal extracellular domain. Previous work has shown IDE plays an important role in VZV infection and virus cell-to-cell spread, which is the sole route for VZV spread in vitro. Here we report that a recombinant soluble IDE (rIDE) enhances VZV infectivity at an early step of infection associated with an increase in virus internalization, and increases cell-to-cell spread. VZV mutants lacking the IDE binding domain of gE were impaired for syncytia formation and membrane fusion. Pre-treatment of cell-free VZV with rIDE markedly enhanced the stability of the virus over a range of conditions. rIDE interacted with gE to elicit a conformational change in gE and rendered it more susceptible to proteolysis. Co-incubation of rIDE with gE modified the size of gE. We propose that the conformational change in gE elicited by IDE enhances infectivity and stability of the virus and leads to increased fusogenicity during VZV infection. The ability of rIDE to enhance infectivity of cell-free VZV over a wide range of incubation times and temperatures suggests that rIDE may be useful for increasing the stability of varicella or zoster vaccines.     

The immediate-early 63 protein of Varicella-Zoster virus: analysis of functional domains required for replication in vitro and for T-cell and skin tropism in the SCIDhu model in vivo

The immediate-early 63-kDa (IE63) protein of varicella-zoster virus (VZV) is a phosphoprotein encoded by open reading frame (ORF) ORF63/ORF70. To identify functional domains, 22 ORF63 mutations were evaluated for effects on IE63 binding to the major VZV transactivator, IE62, and on IE63 phosphorylation and nuclear localization in transient transfections, and after insertion into the viral genome with VZV cosmids. The IE62 binding site was mapped to IE63 amino acids 55 to 67, with R59/L60 being critical residues. Alanine substitutions within the IE63 center region showed that S165, S173, and S185 were phosphorylated by cellular kinases. Four mutations that changed two putative nuclear localization signal (NLS) sequences altered IE63 distribution to a cytoplasmic/nuclear pattern. Only three of 22 mutations in ORF63 were compatible with recovery of infectious VZV from our cosmids, but infectivity was restored by inserting intact ORF63 into each mutated cosmid. The viable IE63 mutants had a single alanine substitution, altering T171, S181, or S185. These mutants, rOKA/ORF63rev[T171], rOKA/ORF63rev[S181], and rOKA/ORF63rev[S185], produced less infectious virus and had a decreased plaque phenotype in vitro. ORF47 kinase protein and glycoprotein E (gE) synthesis was reduced, indicating that IE63 contributed to optimal expression of early and late gene products. The three IE63 mutants replicated in skin xenografts in the SCIDhu mouse model, but virulence was markedly attenuated. In contrast, infectivity in T-cell xenografts was not altered. Comparative analysis suggested that IE63 resembled the herpes simplex virus type 1 U(S)1.5 protein, which is expressed colinearly with ICP22 (U(S)1). In summary, most mutations of ORF63 made with our VZV cosmid system were lethal for infectivity. The few IE63 changes that were tolerated resulted in VZV mutants with an impaired capacity to replicate in vitro. However, the IE63 mutants were attenuated in skin but not T cells in vivo, indicating that the contribution of the IE63 tegument/regulatory protein to VZV pathogenesis depends upon the differentiated human cell type which is targeted for infection within the intact tissue microenvironment.     

Intracellular Traffic of Herpes Simplex Virus Glycoprotein gE: Characterization of the Sorting Signals Required for Its trans-Golgi Network Localization

Herpes simplex virus (HSV) and varicella-zoster virus (VZV) are two pathogenic human alphaherpesviruses whose intracellular assembly is thought to follow different pathways. VZV presumably acquires its envelope in the trans-Golgi network (TGN), and it has recently been shown that its major envelope glycoprotein, VZV-gE, accumulates in this compartment when expressed alone. In contrast, the envelopment of HSV has been proposed to occur at the inner nuclear membrane, although to which compartment the gE homolog (HSV-gE) is transported is unknown. For this reason, we have studied the intracellular traffic of HSV-gE and have found that this glycoprotein accumulates at steady state in the TGN, both when expressed from cloned cDNA and in HSV-infected cells. In addition, HSV-gE cycles between the TGN and the cell surface and requires a conserved tyrosine-containing motif within its cytoplasmic tail for proper trafficking. These results show that VZV-gE and HSV-gE have similar intracellular trafficking pathways, probably reflecting the presence of similar sorting signals in the cytoplasmic domains of both molecules, and suggest that the respective viruses, VZV and HSV, could use the same subcellular organelle, the TGN, for their envelopment.     

Trafficking of varicella-zoster virus glycoprotein gI: T(338)-dependent retention in the trans-Golgi network, secretion, and mannose 6-phosphate-inhibitable uptake of the ectodomain

The trans-Golgi network (TGN) is putatively the site where varicella-zoster virus is enveloped. gE is targeted to the TGN by selective retrieval from the plasmalemma in response to signaling sequences in its endodomain. gI lacks these sequences but forms a complex with gE. We now find that gI is targeted to the TGN and plasma membrane when expressed in Cos-7 cells; nevertheless, surface labeling revealed that gI is not retrieved from the plasma membrane. TGN targeting of gI depended on the T(338) of its endodomain and was lost when T(338) was deleted or mutated to A, S, or D. The endodomain of gI was sufficient, if it contained T(338), to target a fusion protein containing the ectodomain of the human interleukin-2 receptor to the TGN. A truncated protein consisting only of the gI ectodomain was secreted and taken up by nontransfected cells. This uptake of the secreted gI ectodomain was blocked by mannose 6-phosphate. Following cotransfection, both gI and gE were retrieved to the TGN from the plasma membrane in 26.7% of cells, neither gI nor gE was internalized in 18.3%, and gE was retrieved to the TGN while gI remained at the plasma membrane in 55%. We suggest that the T(338) of its endodomain is necessary to retain gI in the TGN; moreover, because gI and gE interact, the signaling sequences of each glycoprotein reinforce one another in ensuring that both glycoproteins are concentrated in the TGN yet remain on the cell surface.     

The amino terminus of varicella-zoster virus (VZV) glycoprotein E is required for binding to insulin-degrading enzyme, a VZV receptor

Varicella-zoster virus (VZV) glycoprotein E (gE) is required for VZV infection. Although gE is well conserved among alphaherpesviruses, the amino terminus of VZV gE is unique. Previously, we showed that gE interacts with insulin-degrading enzyme (IDE) and facilitates VZV infection and cell-to-cell spread of the virus. Here we define the region of VZV gE required to bind IDE. Deletion of amino acids 32 to 71 of gE, located immediately after the predicted signal peptide, resulted in loss of the ability of gE to bind IDE. A synthetic peptide corresponding to amino acids 24 to 50 of gE blocked its interaction with IDE in a concentration-dependent manner. However, a chimeric gE in which amino acids 1 to 71 of VZV gE were fused to amino acids 30 to 545 of herpes simplex virus type 2 gE did not show an increased level of binding to IDE compared with that of full-length HSV gE. Thus, amino acids 24 to 71 of gE are required for IDE binding, and the secondary structure of gE is critical for the interaction. VZV gE also forms a heterodimer with glycoprotein gI. Deletion of amino acids 163 to 208 of gE severely reduced its ability to form a complex with gI. The amino portion of IDE, as well an IDE mutant in the catalytic domain of the protein, bound to gE. Therefore, distinct motifs of VZV gE are important for binding to IDE or to gI.     

Insulin degrading enzyme is a cellular receptor mediating varicella-zoster virus infection and cell-to-cell spread

Varicella-zoster virus (VZV) causes chickenpox and shingles. While varicella is likely spread as cell-free virus to susceptible hosts, the virus is transmitted by cell-to-cell spread in the body and in vitro. Since VZV glycoprotein E (gE) is essential for virus infection, we postulated that gE binds to a cellular receptor. We found that insulin-degrading enzyme (IDE) interacts with gE through its extracellular domain. Downregulation of IDE by siRNA, or blocking of IDE with antibody, with soluble IDE protein extracted from liver, or with bacitracin inhibited VZV infection. Cell-to-cell spread of virus was also impaired by blocking IDE. Transfection of cell lines impaired for VZV infection with a plasmid expressing human IDE resulted in increased entry and enhanced infection with cell-free and cell-associated virus. These studies indicate that IDE is a cellular receptor for both cell-free and cell-associated VZV.