Varicella-Zoster Virus Fc Receptor Component gI Is Phosphorylated on Its Endodomain by a Cyclin-Dependent Kinase

Varicella-zoster virus (VZV) glycoprotein gI is a type 1 transmembrane glycoprotein which is one component of the heterodimeric gE:gI Fc receptor complex. Like VZV gE, VZV gI was phosphorylated in both VZV-infected cells and gI-transfected cells. Preliminary studies demonstrated that a serine 343-proline 344 sequence located within the gI cytoplasmic tail was the most likely phosphorylation site. To determine which protein kinase catalyzed the gI phosphorylation event, we constructed a fusion protein, consisting of glutathione-S-transferase (GST) and the gI cytoplasmic tail, called GST-gI-wt. When this fusion protein was used as a substrate for gI phosphorylation in vitro, the results demonstrated that GST-gI-wt fusion protein was phosphorylated by a representative cyclin-dependent kinase (CDK) called P-TEFb, a homologue of CDK1 (cdc2). When serine 343 within the serine-proline phosphorylation site was replaced with an alanine residue, the level of phosphorylation of the gI fusion protein was greatly reduced. Subsequent experiments with individually immunoprecipitated mammalian CDKs revealed that the VZV gI fusion protein was phosphorylated best by CDK1, to a lesser degree by CDK2, and not at all by CDK6. Transient-transfection assays carried out in the presence of the specific CDK inhibitor roscovitine strongly supported the prior results by demonstrating a marked decrease in gI phosphorylation while gI protein expression was unaffected. Finally, the possibility that VZV gI contained a CDK phosphorylation site in its endodomain was of further interest because its partner, gE, contains a casein kinase II phosphorylation site in its endodomain; prior studies have established that CDK1 can phosphorylate casein kinase II.     

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.     

Varicella-zoster virus glycoprotein gpI/gpIV receptor: expression, complex formation, and antigenicity within the vaccinia virus-T7 RNA polymerase transfection system.

The unique short region of the varicella-zoster virus (VZV) genome contains two open reading frames which encode glycoproteins designated gpI and gpIV (herpes simplex virus homologs gE and gI, respectively). Like its herpesviral counterpart gE, the VZV gpI gene product functions as a cell surface receptor (V. Litwin, W. Jackson, and C. Grose, J. Virol. 66:3643-3651, 1992). To evaluate the biosynthesis of the two VZV glycoproteins and further explore their relationship to one another, the two glycoprotein genes were individually cloned into a pTM1 vector under control of the T7 promoter. Transfection of the cloned gpI or gpIV construct into HeLa cells previously infected with vaccinia recombinant virus expressing bacteriophage T7 polymerase resulted in a much higher level expression of each VZV glycoprotein than previously achieved. Synthesis of both gpI and gpIV included intermediary partially glycosylated forms and mature N- and O-linked final product. Transfections in the presence of 32Pi demonstrated that the mature forms of both gpI and gpIV were phosphorylated, while similar experiments with [35S]sulfate showed that only the mature gpI was sulfated. When gpI and gpIV were coexpressed in the same cell, the two glycoproteins were complexed to each other, as both proteins could be immunoprecipitated by antibodies against either gpI or gpIV. Coprecipitation did not occur as a result of a shared epitope, because gpI expressed alone was not precipitated by antibody to gpIV, and gpIV expressed alone was not precipitated by antibody to gpI. Pulse-chase analysis demonstrated that the gpI-gpIV association occurred early in processing; furthermore, this complex formation interfered with posttranslational modifications and thereby reduced the M(r)s of the mature forms of both gpI and gpIV. Similarly, the molecular masses of the cotransfected gene products corresponded with those of the infected cell glycoproteins, a result which suggested that authentic gpI and gpIV were ordinarily found within a complex. Thus, the adjacent open reading frames 67 and 68 code for two glycoproteins which in turn form a distinctive sulfated and phosphorylated cell surface complex with receptor properties.     

Identification of the phosphorylation sequence in the cytoplasmic tail of the varicella-zoster virus Fc receptor glycoprotein gpI.

Varicella-zoster virus (VZV) glycoprotein gpI, the homolog of herpes simplex virus gE, functions as a receptor for the Fc portion of immunoglobulin G. Like other cell surface receptors, this viral receptor is highly phosphorylated in cell culture. To identify the precise location of the cellular kinase-mediated phosphorylation, we generated a tailless deletion mutant and several point mutants which had altered serine and threonine residues within the cytoplasmic domain of gpI. The mutated and wild-type genes of gpI were transfected and expressed within a vaccinia virus-T7 polymerase transfection system in order to determine what effect these mutations had on the phosphorylation state of the protein in vivo and in vitro. Truncation of the cytoplasmic domain of gpI diminished the phosphorylation of gpI in vivo. Examination of the point mutants established that the major phosphorylation sequence of gpI was located between amino acids 593 and 598, a site which included four phosphorylatable serine and threonine residues. Phosphorylation analyses of the mutant and wild-type glycoproteins confirmed that gpI was a substrate for casein kinase II, with threonines 596 and 598 being critical residues. Although the mutant glycoproteins were phosphorylated by casein kinase I, protease V8 partial digestion profiles suggested that casein kinase II exerted the major effect. Thus, these mutagenesis studies demonstrated that the gpI cytoplasmic sequence Ser-Glu-Ser-Thr-Asp-Thr was phosphorylated in mammalian cells in the absence of any other herpesvirus products. Since the region defined by transfection was consistent with results obtained with in vitro phosphorylation by casein kinase II, we propose that VZV gpI is a physiologic substrate for casein kinase II. Immunofluorescence and pulse-chase experiments demonstrated that the mutant glycoproteins were processed and transported to the outer cell membrane.     

Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I.

Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, we investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [gamma-32P]ATP. The same glycoprotein was phosphorylated when [32P]GTP was substituted for [32P]ATP in the protein kinase assay. We also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. Immediately upstream from each of the casein kinase II sites was a potential casein kinase I phosphorylation site. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.     

Receptor properties of two varicella-zoster virus glycoproteins, gpI and gpIV, homologous to herpes simplex virus gE and gI.

The varicella-zoster virus (VZV) genome contains 70 reading frames (ORF), 5 of which encode the glycoproteins gpI, gpII, gpIII, gpIV, and gpV. ORF 67 and 68 lie adjacent to each other in the unique short region of the VZV genome and code for gpIV and gpI, respectively. These two genes, which are contained within the HindIII C fragment of the VZV genome, were subcloned in the correct orientation downstream from the promoter regions of the eukaryotic expression vectors pCMV5 and pBJ. After transfection, 5 to 20% of the Cos cells bound antibody specific for the given glycoprotein. In this study, it was shown that only the cells transfected with the gpI construct bound to the Fc fragment of human immunoglobulin G. Neither the transfected gpIV gene product nor the vector only bound to the Fc fragment. Thus, VZV gpI is confirmed to be the VZV-encoded Fc-binding glycoprotein. Like the wild-type form of gpI expressed in VZV-infected cells, gpI precipitated from transfected cells contained both N-linked and O-linked glycans and was heavily sialated. In addition, the transfected gpI gene product was phosphorylated both in cell culture and in protein kinase assays by mammalian casein kinases I and II. Extensive computer-assisted analyses of the VZV gpI sequence, as well as those of alphaherpesviral homolog glycoproteins, disclosed properties similar to those of other cell surface receptors; these included (i) exocytoplasmic regions rich in cysteine residues, (ii) membrane-proximal regions with potential O-linked glycosylation sites, and (iii) cytoplasmic domains with consensus phosphorylation sites.     

Use of lambda gt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins.

A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambda gt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambda gt11 vector, the cloned proteins were expressed in Escherichia coli as beta-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of [14C]glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX; gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.     

Neutralizing antibodies induced by recombinant vaccinia virus expressing varicella-zoster virus gpIV.

Monoclonal antibodies generated against varicella-zoster virus (VZV) glycoprotein I (gpI) also recognize VZV gpIV (A. Vafai, Z. Wroblewska, R. Mahalingam, G. Cabirac, M. Wellish, M. Cisco, and D. Gilden, J. Virol. 62:2544-2551, 1988). To determine whether the virus-neutralizing activity of these antibodies belongs to gpI, gpIV, or both, the open reading frame encoding gpIV was inserted into the vaccinia virus genome. Immunoprecipitation of recombinant vaccinia virus-infected cells with anti-gpIV monoclonal antibody yielded synthesis and processing of gpIV similar to those expressed in VZV-infected cells. Antibodies raised against VVgpIV in a rabbit recognized both native gpI and gpIV and neutralized VZV infectivity. In addition, antibodies raised against recombinant vaccinia virus carrying VZV gpI neutralized VZV infection. These results indicate a structural relationship between VZV gpI and gpIV and show that gpI and gpIV each induce virus-neutralizing antibody.     

Complex Formation Facilitates Endocytosis of the Varicella-Zoster Virus gE:gI Fc Receptor

Open reading frames within the unique short segment of alphaherpesvirus genomes participate in egress and cell-to-cell spread. The case of varicella-zoster virus (VZV) is of particular interest not only because the virus is highly cell associated but also because its most prominent cell surface protein, gE, bears semblance to the mammalian Fc receptor FcγRII. A previous study demonstrated that when expressed alone in cells, VZV gE was endocytosed from the cell surface through a tyrosine localization motif in its cytoplasmic tail (J. K. Olson and C. Grose, J. Virol. 71:4042–4054, 1997). Since VZV gE is normally found in association with gI in the infected cell, the present study was directed at defining the trafficking of the VZV gE:gI protein complex. First, VZV gI underwent endocytosis and recycling when it was expressed alone in cells, and interestingly, VZV gI contained a methionine-leucine internalization motif in its cytoplasmic tail. Second, VZV gI was found by confocal microscopy to colocalize with VZV gE during endocytosis and recycling in cells. Third, by a quantitative internalization assay, VZV gE:gI was shown to undergo endocytosis more efficiently (steady state, 55 to 60%) than either gE alone (steady state, ~32%) or gI alone (steady state, ~45%). Further, examination of endocytosis-deficient mutant proteins demonstrated that VZV gI exerted a more pronounced effect than gE on internalization of the complex. Most importantly, therefore, these studies suggest that VZV gI behaves as an accessory component by facilitating the endocytosis of the major constituent gE and thereby modulating the trafficking of the entire cell surface gE:gI Fc receptor complex.     

The extracellular domain of herpes simplex virus gE is sufficient for accumulation at cell junctions but not for cell-to-cell spread

Herpes simplex virus (HSV) expresses a number of membrane glycoproteins, including gB, gD, and gH/gL, that function in both entry of virus particles and movement of virus from an infected cell to an uninfected cell (cell-to-cell spread). However, a complex of HSV glycoproteins gE and gI (gE/gI) is required for efficient cell-to-cell spread, especially between cells that form extensive cell junctions, yet it is not necessary for entry of extracellular virions. We previously showed that gE/gI has the capacity to localize specifically to cell junctions; the glycoprotein complex was found at lateral surfaces of cells in contact with other cells but not at those lateral surfaces not forming junctions or at apical surfaces. By virtue of these properties, gE/gI is an important molecular handle on the poorly understood process of cell-to-cell spread. Here, we show that the cytoplasmic domain of gE is important for the proper delivery of gE/gI to lateral surfaces of cells. Without this domain, gE/gI is found on the apical surface of epithelial cells, and more uniformly in the cytoplasm, although incorporation into the virion envelope is unaffected. However, even without proper trafficking signals, a substantial fraction of gE/gI retained the capacity to accumulate at cell junctions. Therefore, the extracellular domain of gE can mediate accumulation of gE/gI at cell junctions, if the glycoprotein can be delivered there, probably through interactions with ligands on the opposing cell. The role of phosphorylation of the cytoplasmic domain of gE was also studied. A second mutant HSV type 1 was constructed in which three serine residues that form a casein kinase II phosphorylation site were changed to alanine residues, reducing phosphorylation by 70 to 80%. This mutation did not affect accumulation at cell junctions or 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.     

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.     

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.     

Specific lysis of targets expressing varicella-zoster virus gpI or gpIV by CD4+ human T-cell clones.

Varicella-zoster virus (VZV)-specific CD4-positive T cells are known to lyse targets expressing VZV antigen, but little is known of the glycoprotein specificity or phenotype of these cells. To test the ability of T cells to distinguish between gpI and gpIV (which share an antibody-defined epitope), we prepared clones from blood from four healthy individuals by limiting dilution. Among 68 T-cell clones from four donors which were VZV specific in tests of proliferation, 30 lysed autologous Epstein-Barr virus-transformed lymphoblasts which had been superinfected with a recombinant vaccinia virus which included the whole VZV gpI sequence. These clones were characterized as major histocompatibility complex class II restricted by inhibition of their cytotoxicity with HLA-DR and CD4 monoclonal antibodies. Twenty-one clones lysed targets expressing gpIV. Fifteen of these clones lysed targets expressing gpI and gpIV. Four clones with gpI-gpIV specificity were examined in detail, and their dual specificity was confirmed by cold target inhibition. These four clones failed to kill target cells infected with a mutant gpIV recombinant vaccinia virus from which amino acid residues 212 to 354 had been deleted. This region includes one of the two gpIV decapeptides which have 50% homology with amino acids 111 to 121 of gpI. Our data confirm that T-cell-receptor-associated structures are required for specific lysis of VZV targets and indicate that (i) gpI-specific CD4 cytotoxic T cells outnumber gpIV-specific T cells in blood and (ii) 50% of gpI-specific T-cell clones also lyse gpIV-expressing targets.     

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 Fc receptor gE glycoprotein: serine/threonine and tyrosine phosphorylation of monomeric and dimeric forms.

Varicella-zoster virus (VZV) glycoprotein gE is the predominant viral cell surface molecule; it behaves as an Fc receptor for immunoglobulin G, but its central function may be more closely related to viral egress and cell-to-cell spread. To further analyze the receptor properties of VZV gE, the gE gene (also called open reading frame 68) was expressed by a baculovirus vector in insect cells. The recombinant baculovirus gE product had a molecular mass of 64 kDa, smaller than the previously documented 98 kDa of mature gE expressed in mammalian cells. The major reason for the lowered molecular mass was diminished glycosylation. In addition to the 64-kDa form, a larger (130-kDa) form was observed in insect cells and represented dimerized 64-kDa molecules. Both the monomeric and dimeric gE forms were highly phosphorylated in insect cells. Protein kinase assays conducted in vitro with [gamma-32P]ATP and [gamma-32P]GTP indicated that endogenous casein kinase II was phosphorylating monomeric gE, while the dimeric gE form was phosphorylated by another kinase which did not utilize [gamma-32P]GTP. When immobilized recombinant gE molecules were probed with a monoclonal antibody which specifically recognizes a phosphotyrosine linkage, the gE dimer was found to be tyrosine phosphorylated whereas the monomer was not similarly modified. When recombinant gE produced in HeLa cells was probed with the same antiphosphotyrosine antibody, a dimeric gE form at 130 kDa was detected on the cell surface. These results suggested that VZV gE closely resembled other cell surface receptors, being modified on its various forms by both serine/threonine and tyrosine protein kinases. In this case, tyrosine phosphorylation occurred on a previously unrecognized and underglycosylated VZV gE dimeric product.     

A functional YNKI motif in the short cytoplasmic tail of varicella-zoster virus glycoprotein gH mediates clathrin-dependent and antibody-independent endocytosis

The trafficking of varicella-zoster virus (VZV) gH was investigated under both infection and transfection conditions. In initial endocytosis assays performed in infected cells, the three glycoproteins gE, gI, and gB served as positive controls for internalization from the plasma membrane. Subsequently, we discovered that gH in VZV-infected cells was also internalized and followed a similar trafficking pattern. This observation was unexpected because all herpesvirus gH homologues have short endodomains not known to contain trafficking motifs. Further investigation demonstrated that VZV gH, when expressed alone with its chaperone gL, was capable of endocytosis in a clathrin-dependent manner, independent of gE, gI, or gB. Upon inspection of the short gH cytoplasmic tail, we discovered a putative tyrosine-based endocytosis motif (YNKI). When the tyrosine was replaced with an alanine, endocytosis of gH was blocked. Utilizing an endocytosis assay dependent on biotin labeling, we further documented that endocytosis of VZV gH was antibody independent. In control experiments, we showed that gE, gI, and gB also internalized in an antibody-independent manner. Alignment analysis of the VZV gH cytoplasmic tail to other herpesvirus gH homologues revealed two important findings: (i) herpes simplex virus type 1 and 2 homologues lacked an endocytosis motif, while all other alphaherpesvirus gH homologues contained a potential motif, and (ii) the VZV gH and simian varicella virus gH cytoplasmic tails were likely longer in length (18 amino acids) than predicted in the original sequence analyses (12 and 16 amino acids, respectively). The longer tails provided the proper context for a functional endocytosis motif.     

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.