Varicella-zoster virus glycoprotein gpI/gpIV receptor: expression, complex formation, and antigenicity within the vaccinia virus-T7 RNA polymerase transfection system.
ABSTRACT: 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.
Project description: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.
Project description:Varicella-zoster virus (VZV) glycoprotein gpIV, to be renamed VZV gI, forms a heterodimer with glycoprotein gpI (gE) which functions as an Fc receptor in virus-infected cells. Like VZV gpI (gE), this viral glycoprotein is phosphorylated in cell culture during biosynthesis. In this report, we investigated the nature and specificity of the phosphorylation event involving VZV gpIV (gI). Phosphoamino acid analysis indicated that gpIV (gI) was modified mainly on serine residues. To identify the precise location of the phosphorylation site on the 64-kDa protein, a step-by-step mutagenesis procedures was followed. Initially a tailless mutant was generated, and this truncated product was no longer phosphorylated. Thereafter, point mutations were made within the cytoplasmic tail of gpIV (gI) at potential phosphorylation sites. The phosphorylation site was localized to the following sequence: Ser-Pro-Pro (amino acids 343 to 345). Examination of the point mutants established that serine 343 in the cytoplasmic tail was the major phosphoacceptor. In addition, we found that the prolines located immediately to the C terminus of serine 343 were an integral part of the kinase recognition sequence. This site was located immediately N terminal to a predicted beta-turn secondary structure. By comparison with known substrate consensus sequences for various protein kinases, these data suggested that the phosphorylation of VZV gpIV (gI) was catalyzed by a proline-directed protein kinase. Computer homology analysis of other alphaherpesviruses demonstrated that a similar potential phosphorylation site was highly conserved in the cytoplasmic tails of herpes simplex virus type 1 gI, equine herpesvirus type 1 gI, and pseudorabies virus gp63.
Project description:The cytoplasmic tails of all three major varicella-zoster virus (VZV) glycoproteins, gE, gH, and gB, harbor functional tyrosine-based endocytosis motifs that mediate internalization. The aim of the present study was to examine whether endocytosis from the plasma membrane is a cellular route by which VZV glycoproteins are delivered to the final envelopment compartment. In this study, we demonstrated that internalization of the glycoproteins occurred in the first 24 h postinfection but was reduced later in infection. Using surface biotinylation of VZV-infected cells followed by a glutathione cleavage assay, we showed that endocytosis was independent of antibody binding to gE, gH, and gB. Subsequently, with this assay, we demonstrated that biotinylated gE, gH, and gB retrieved from the cell surface were incorporated into nascent virus particles isolated after density gradient sedimentation. To confirm and extend this finding, we repeated the above sedimentation step and specifically detected envelopes decorated with Streptavidin-conjugated gold beads on a majority of complete virions through examination by transmission electron microscopy. In addition, a gE-gI complex and a gE-gH complex were found on the virions. Therefore, the above studies established that VZV subsumed a postendocytosis trafficking pathway as one mechanism by which to deliver viral glycoproteins to the site of virion assembly in the cytoplasm. Furthermore, since a recombinant VZV genome lacking only endocytosis-competent gE cannot replicate, these results supported the conclusion that the endocytosis-envelopment pathway is an essential component of the VZV life cycle.
Project description: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.
Project description: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, ?gI 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 ?105-125 (with residues 105 to 125 deleted) viruses had small-plaque phenotypes with reduced replication kinetics in vitro similar to those of the ?gI virus. The ?105-125 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 ?105-125 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.
Project description:Varicella-zoster virus (VZV) is a highly contagious agent of varicella and herpes zoster. Varicella can be lethal to immunocompromised patients, babies, HIV patients and other adults with impaired immunity. Serological evaluation of immunity to VZV will help determine which individuals are susceptible and evaluate vaccine effectiveness. A collection of 110 monoclonal antibodies (mAbs) were obtained by immunization of mice with membrane proteins or cell-free virus. The mAbs were well characterized, and a competitive sandwich ELISA (capture mAb: 8H6; labelling mAb: 1B11) was established to determine neutralizing antibodies in human serum with reference to the FAMA test. A total of 920 human sera were evaluated. The competitive sandwich ELISA showed a sensitivity of 95.6%, specificity of 99.77% and coincidence of 97.61% compared with the fluorescent-antibody-to-membrane-antigen (FAMA) test. The capture mAb 8H6 was characterized as a specific mAb for VZV ORF9, a membrane-associated tegument protein that interacts with glycoprotein E (gE), glycoprotein B (gB) and glycoprotein C (gC). The labelling mAb 1B11 was characterized as a complement-dependent neutralizing mAb specific for the immune-dominant epitope located on gE, not on other VZV glycoproteins. The established competitive sandwich ELISA could be used as a rapid and high-throughput method for evaluating immunity to VZV.
Project description:Herpes zoster (HZ) (shingles) is the clinical manifestation of varicella-zoster virus (VZV) reactivation. HZ typically develops as people age, due to decreased cell-mediated immunity. However, the importance of antibodies for immunity against HZ prevention remains to be understood. The goal of this study was to examine the breadth and functionality of VZV-specific antibodies after vaccination with a live attenuated HZ vaccine (Zostavax). Direct enumeration of VZV-specific antibody-secreting cells (ASCs) via enzyme-linked immunosorbent spot assay (ELISPOT assay) showed that Zostavax can induce both IgG and IgA ASCs 7 days after vaccination but not IgM ASCs. The VZV-specific ASCs range from 33 to 55% of the total IgG ASCs. Twenty-five human VZV-specific monoclonal antibodies (MAbs) were cloned and characterized from single-cell-sorted ASCs of five subjects (>60 years old) who received Zostavax. These MAbs had an average of ∼20 somatic hypermutations per VH gene, similar to those seen after seasonal influenza vaccination. Fifteen of the 25 MAbs were gE specific, whereas the remaining MAbs were gB, gH, or gI specific. The most potent neutralizing antibodies were gH specific and were also able to inhibit cell-to-cell spread of the virus in vitro Most gE-specific MAbs were able to neutralize VZV, but they required the presence of complement and were unable to block cell-to-cell spread. These data indicate that Zostavax induces a memory B cell recall response characterized by anti-gE > anti-gI > anti-gB > anti-gH antibodies. While antibodies to gH could be involved in limiting the spread of VZV upon reactivation, the contribution of anti-gE antibodies toward protective immunity after Zostavax needs further evaluation.IMPORTANCE Varicella-zoster virus (VZV) is the causative agent of chickenpox and shingles. Following infection with VZV, the virus becomes latent and resides in nerve cells. Age-related declines in immunity/immunosuppression can result in reactivation of this latent virus, causing shingles. It has been shown that waning T cell immunity correlates with an increased incidence of VZV reactivation. Interestingly, serum with high levels of VZV-specific antibodies (VariZIG; IV immunoglobulin) has been administered to high-risk populations, e.g., immunocompromised children, newborns, and pregnant women, after exposure to VZV and has shown some protection against chickenpox. However, the relative contribution of antibodies against individual surface glycoproteins toward protection from shingles in elderly/immunocompromised individuals has not been established. Here, we examined the breadth and functionality of VZV-specific antibodies after vaccination with the live attenuated VZV vaccine Zostavax in humans. This study will add to our understanding of the role of antibodies in protection against shingles.
Project description:The highly conserved herpesvirus glycoprotein complex, gB/gH-gL, mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multi-nucleated cells, or syncytia, during the infection of human tissues but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. The gBcyt regulation is necessary for VZV pathogenesis as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt regulated fusion was investigated by comparing melanoma cells infected with wild type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytia formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization and rapid displacement of nuclei to dense central structures when compared to pOka using live cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using RNA-seq to identify viral and cellular responses induced when the gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and glycoproteins, gC, gE and gI, was significantly reduced at 36 hours post infection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate the gBcyt in the regulation gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection. Overall design: Biological duplicates from 3 time points (12, 24 and 36 hours post infection) of uninfected MeWo cells or MeWo cells infected with varicella-zoster virus strain pOka or mutants gB[Y881F], gB[Y920F] or gB[Y881/920F]
Project description:Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for VZV replication. To further analyze the functions of gE in VZV replication, a full deletion and point mutations were made in the 62-amino-acid (aa) C-terminal domain. Targeted mutations were introduced in YAGL (aa 582 to 585), which mediates gE endocytosis, AYRV (aa 568 to 571), which targets gE to the trans-Golgi network (TGN), and SSTT, an "acid cluster" comprising a phosphorylation motif (aa 588 to 601). Substitutions Y582G in YAGL, Y569A in AYRV, and S593A, S595A, T596A, and T598A in SSTT were introduced into the viral genome by using VZV cosmids. These experiments demonstrated a hierarchy in the contributions of these C-terminal motifs to VZV replication and virulence. Deletion of the gE C terminus and mutation of YAGL were lethal for VZV replication in vitro. Mutations of AYRV and SSTT were compatible with recovery of VZV, but the AYRV mutation resulted in rapid virus spread in vitro and the SSTT mutation resulted in higher virus titers than were observed for the parental rOka strain. When the rOka-gE-AYRV and rOka-gE-SSTT mutants were evaluated in skin and T-cell xenografts in SCIDhu mice, interference with TGN targeting was associated with substantial attenuation, especially in skin, whereas the SSTT mutation did not alter VZV infectivity in vivo. These results provide the first information about how targeted mutations of this essential VZV glycoprotein affect viral replication in vitro and VZV virulence in dermal and epidermal cells and T cells within intact tissue microenvironments in vivo.
Project description: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.