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.     

Immunization with recombinant varicella-zoster virus expressing herpes simplex virus type 2 glycoprotein D reduces the severity of genital herpes in guinea pigs.

Varicella-zoster virus (VZV) is an attractive candidate for a live-virus vector for the delivery of foreign antigens. The Oka vaccine strain of VZV is safe and effective in humans, and recombinant Oka VZV (ROka) can be generated by transfecting cells with a set of overlapping cosmid DNAs. By this method, the herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) gene was inserted into an intergenic site in the unique short region of the Oka VZV genome. Expression of gD2 in cells infected with the recombinant Oka strain VZV (ROka-gD2) was confirmed by antibody staining of fixed cells and by immunoblot analysis. Immune electron microscopy demonstrated the presence of gD2 in the envelope of ROka-gD2 virions. The ability of ROka-gD2 to protect guinea pigs against HSV-2 challenge was assessed by inoculating animals with three doses of uninfected human fibroblasts, fibroblasts infected with ROka VZV, or fibroblasts infected with ROka-gD2. Neutralizing antibodies specific for HSV-2 developed in animals immunized with ROka-gD2. Forty days after the third inoculation, animals were challenged intravaginally with HSV-2. Inoculation of guinea pigs with ROka-gD2 significantly reduced the severity of primary HSV-2 infection (P < 0.001). These experiments demonstrate that the Oka strain of VZV can be used as a live virus vector to protect animals from disease with a heterologous virus.     

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.     

The varicella-zoster virus ORF66 protein induces kinase activity and is dispensable for viral replication.

Varicella-zoster virus (VZV) open reading frames (ORFs) 47 and 66 encode proteins that are homologous to a family of eukaryotic serine-threonine kinases. Prior studies showed that the VZV ORF47 protein has kinase activity in vitro and is dispensable for replication in cultured cells. To examine the role of the ORF66 protein during infection, we constructed VZV recombinants that are unable to express either the ORF66 protein (ROka 66S) or both the ORF47 and ORF66 proteins (ROka 47S/66S). VZV unable to express ORF66 grew to titers similar to those of the parental VZV (ROka) in vitro; however, VZV lacking both ORF66 and ORF47 grew to titers lower than those of ROka. Nuclear extracts from ROka 66S- or ROka 47S-infected cells showed a 48-kDa phosphoprotein(s); a phosphoprotein with a similar size was not present in nuclear extracts from ROka 47S/66S-infected cells. To determine the role of the ORF66 protein in the phosphorylation of specific VZV-encoded proteins, we immunoprecipitated known VZV phosphoproteins (ORF4, ORF62, ORF63, and ORF68 proteins) from nuclear extracts of phosphate-labeled cells infected with ROka, ROka 66S, or ROka 47S/66S. Each of the VZV phosphoproteins was phosphorylated to a similar extent in the presence or absence of either the ORF66 protein or both the ORF66 and ORF47 proteins. From these studies we conclude (i) neither ORF66 alone nor ORF66 and ORF47 in combination are essential for VZV growth in cultured cells, (ii) ORF66 either is a protein kinase or induces protein kinase activity during infection, and (iii) the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 do not require either ORF66 alone or ORF66 and ORF47 for phosphorylation in vitro.     

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.     

Regulation of varicella-zoster virus-induced cell-to-cell fusion by the endocytosis-competent glycoproteins gH and gE

The gH glycoprotein of varicella-zoster virus (VZV) is a major fusogen. The realigned short cytoplasmic tail of gH (18 amino acids) harbors a functional endocytosis motif (YNKI) that mediates internalization in both VZV-infected and transfected cells (T. J. Pasieka, L. Maresova, and C. Grose, J. Virol. 77: 4194-4202, 2003). During subsequent confocal microscopy studies of endocytosis-deficient gH mutants, we observed that cells transfected with the gH tail mutants exhibited marked fusion. Therefore, we postulated that VZV gH endocytosis served to regulate cell-to-cell fusion. Subsequent analyses of gH+gL transfection fusion assays by the Kolmogorov-Smirnov statistical test demonstrated that expression of the endocytosis-deficient gH mutants resulted in a statistically significant enhancement of cell-to-cell fusion (P < 0.0001) compared to wild-type gH. On the other hand, coexpression of VZV gE, another endocytosis-competent VZV glycoprotein, was able to temper the fusogenicity of the gH endocytosis mutants by facilitating internalization of the mutant gH protein from the cell surface. When the latter results were similarly analyzed, there was no longer any enhanced fusion by the endocytosis-deficient gH mutant protein. In summary, these studies support a role for gH endocytosis in regulating the cell surface expression of gH and thereby regulating gH-mediated fusion. The data also confirm and extend prior observations of a gE-gH interaction during viral glycoprotein trafficking in a VZV transfection system.     

Incorporation of three endocytosed varicella-zoster virus glycoproteins, gE, gH, and gB, into the virion envelope

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.     

The glycoprotein products of varicella-zoster virus gene 14 and their defective accumulation in a vaccine strain (Oka).

Many characteristics of the putative protein encoded by varicella-zoster virus (VZV) open reading fram (ORF) 14 indicate that it is a glycoprotein, which has been designated gpV. To identify the protein products of the gene, the coding sequences were placed under the control of the vaccinia virus p7.5 promoter and recombinant vaccinia viruses were constructed. Heterogeneous polypeptides with molecular weights of 95,000 to 105,000 (95K to 105K polypeptides) were expressed in cells infected by a vaccinia virus recombinant (vKIP5) containing ORF 14 from VZV Scott but were not expressed by control vaccinia viruses. These polypeptides were recognized by antibodies present in human sera that contained high levels of anti-VZV antibodies. Conversely, antisera raised in rabbits inoculated with vKIP5 reacted specifically with heterogeneous 95K to 105K polypeptides present in VZV Scott-infected but not uninfected cells; these polypeptides show a patchy plasma membrane fluorescence pattern in VZV Scott-infected cells. These same antisera neutralized VZV strain Scott infectivity in the absence of complement. Endoglycosidase F treatment of isolated gpV polypeptides and tunicamycin treatment of cells infected with the vKIP5 recombinant indicated that the polypeptides were glycosylated. Three sets of data imply that the VZV strain Oka, which has been used to produce a live attenuated virus vaccine, accumulates low levels of gpV polypeptides relative to wild-type strains: (i) blocking of antibodies in human sera with excess VZV Oka-infected cell antigen yielded residual antibodies which were reactive with the 95K to 105K gpV polypeptides expressed in cells infected by VZV strain Scott and by the vKIP5 vaccinia virus recombinant, but not with Oka-infected cell polypeptides; (ii) antisera raised to vKIP5 detected very low levels of reactive polypeptides made in VZV Oka-infected cells and neutralized VZV Oka virus much less efficiently than VZV Scott; and (iii) comparisons of the reactivity of sera from live attenuated virus vaccine vaccinees with sera derived from patients recovering from wild-type infections indicated greatly reduced levels of gpV-specific antibodies in some vaccinees.     

Development and characterization of a live varicella vaccine (Oka strain)

A live varicella vaccine (Oka strain) was developed by serial passage of the Oka strain isolated in our laboratory, in human embryonic lung cells (11 times at 34 C) and guinea pig embryo cells (12 times at 37 C). It is slightly temperature sensitive at 39 C and shows a higher ratio of infectivity in guinea pig embryo cells to infectivity in human embryo cells than wild-type strains. The DNA digest with Hpa I enzyme of the Oka strain contained one unique fragment (K), although its mobility differed only slightly from that of the corresponding fragment of wild-type strains. Studies with clinical varicella zoster virus (VZV) isolates from vaccinees indicated that tests on the ratio of infectivity in guinea pig embryo fibroblasts (GPEF) to that in human embryo fibroblasts (HuEF) and the profile of the DNA digest with Hpa I are useful for differentiation of the vaccine strain from wild-type strains. The vaccine virus showed stable immunogenicity during at least 15 further repeated passages in human diploid cells, a character which seems helpful for production of a large quantity of vaccine virus for practical use.     

Culture media optimization for Chinese hamster ovary cell growth and expression of recombinant varicella-zoster virus glycoprotein E

Herpes zoster (HZ) is caused by reactivation of varicella-zoster virus (VZV) latent in the sensory ganglia and causes severe pain, often leading to postherpetic neuralgia (PHN). Two prophylactic vaccines against HZ are currently licensed for human use, a live attenuated vaccine and a subunit vaccine containing recombinant VZV glycoprotein E (gE) as antigen. The latter has superior protective efficacy against HZ and PHN. During HZ subunit vaccine development, we obtained Chinese hamster ovary (CHO) cell clones expressing VZV gE. This study was performed to optimize culture media conditions for CHO cell growth and gE production. Using a high-throughput culture system, three CHO cell clones were cultured in microtiter plates containing 24 different basal media, and three basal media were selected. The clone with the highest gE expression was fed-batch cultured in each of the three basal media in combination with 13 different feed media. A pair of media, BalanCD CHO Growth A and EX-CELL Advanced CHO Feed 1, with the highest productivity was selected for gE production. Scale-up fed-batch cultures of the selected clone cultured in a wave bag bioreactor containing the optimized media yielded 2440 mg gE protein/L culture, a 11.5-fold increase compared to original culture conditions (batch culture in CD OptiCHO medium). The optimized media condition is used to produce VZV gE antigen for an HZ subunit vaccine, which is under phase I clinical trial. This study would provide valuable insights on culture media optimization for CHO cells expressing a recombinant vaccine antigen.

     

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.     

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.