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 gB and gE coexpression, but not gB or gE alone, leads to abundant fusion and syncytium formation equivalent to those from gH and gL coexpression

Varicella-zoster virus (VZV) is distinguished from herpes simplex virus type 1 (HSV-1) by the fact that cell-to-cell fusion and syncytium formation require only gH and gL within a transient-expression system. In the HSV system, four glycoproteins, namely, gH, gL, gB, and gD, are required to induce a similar fusogenic event. VZV lacks a gD homologous protein. In this report, the role of VZV gB as a fusogen was investigated and compared to the gH-gL complex. First of all, the VZV gH-gL experiment was repeated under a different set of conditions; namely, gH and gL were cloned into the same vaccinia virus (VV) genome. Surprisingly, the new expression system demonstrated that a recombinant VV-gH+gL construct was even more fusogenic than seen in the prior experiment with two individual expression plasmids containing gH and gL (K. M. Duus and C. Grose, J. Virol. 70:8961-8971, 1996). Recombinant VV expressing VZV gB by itself, however, effected the formation of only small syncytia. When VZV gE and gB genes were cloned into one recombinant VV genome and another fusion assay was performed, extensive syncytium formation was observed. The degree of fusion with VZV gE-gB coexpression was comparable to that observed with VZV gH-gL: in both cases, >80% of the cells in a monolayer were fused. Thus, these studies established that VZV gE-gB coexpression greatly enhanced the fusogenic properties of gB. Control experiments documented that the fusion assay required a balance between the fusogenic potential of the VZV glycoproteins and the fusion-inhibitory effect of the VV infection itself.     

Assembly and organization of glycoproteins B, C, D, and H in herpes simplex virus type 1 particles lacking individual glycoproteins: No evidence for the formation of a complex of these molecules

Glycoprotein B (gB), gC, gD, and gH:L of herpes simplex virus type 1 (HSV-1) are implicated in virus adsorption and penetration. gB, gD, and gH:L are essential for these processes, and their expression is necessary and sufficient to induce cell fusion. The current view is that these molecules act in concert as a functional complex, and cross-linking studies support this view (C. G. Handler, R. J. Eisenberg, and G. H. Cohen, J. Virol. 70:6067-6075, 1996). We examined the glycoprotein composition, with respect to gB, gC, gD, and gH, of mutant virions lacking individual glycoproteins and the sedimentation characteristics of glycoproteins extracted from these virions. The amounts of gB, gC, gD, or gH detected in virions did not alter when any one of these molecules was absent, and it therefore appears that they are incorporated into the virion independently of each other. The sedimentation characteristics of gB and gD from mutant virions were not different from those of wild-type virions. We confirmed that gB, gC, and gD could be cross-linked to each other on the virion surface but found that the absence of one glycoprotein did not alter the outcome of cross-linking reactions between the remaining molecules. The incorporation and arrangement of these glycoproteins in the virion envelope therefore appear to be independent of the individual molecular species. This is difficult to reconcile with the concept that gB, gC, gD, and gH:L are incorporated as a functional complex into the virion envelope.     

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.     

Complexes between herpes simplex virus glycoproteins gD, gB, and gH detected in cells by complementation of split enhanced green fluorescent protein

The interactions between herpes simplex virus gD and its nectin1 receptor or between gD, gB, and gH were analyzed by complementation of the N and C portions of split enhanced green fluorescent protein (EGFP) fused to the glycoproteins. The gD(N)-Nect(C) complex was readily detected; the gD(N)-gC(C) complex was undetectable, highlighting the specificity of the assay. Split EGFP complementation was detected between proteins designated gD(N)+gH(C), gD(N)+gB(C), and gH(N)+gB(C)+wtgD (gB was deleted of endocytosis motifs), both in cells transfected with two-tree glycoproteins and in syncytia. The in situ assay provides evidence that gD interacts with gH and gB independently of each other and supports a model whereby gH and gB in complex exert their activities to gD.     

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.     

Blocking antibody access to neutralizing domains on glycoproteins involved in entry as a novel mechanism of immune evasion by herpes simplex virus type 1 glycoproteins C and E

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) blocks complement activation, and glycoprotein E (gE) interferes with IgG Fc-mediated activities. While evaluating gC- and gE-mediated immune evasion in human immunodeficiency virus (HIV)-HSV-1-coinfected subjects, we noted that antibody alone was more effective at neutralizing a strain with mutations in gC and gE (gC/gE) than a wild-type (WT) virus. This result was unexpected since gC and gE are postulated to interfere with complement-mediated neutralization. We used pooled human immunoglobulin G (IgG) from HIV-negative donors to confirm the results and evaluated mechanisms of the enhanced antibody neutralization. We demonstrated that differences in antibody neutralization cannot be attributed to the concentrations of HSV-1 glycoproteins on the two viruses or to the absence of an IgG Fc receptor on the gC/gE mutant virus or to enhanced neutralization of the mutant virus by antibodies that target only gB, gD, or gH/gL, which are the glycoproteins involved in virus entry. Since sera from HIV-infected subjects and pooled human IgG contain antibodies against multiple glycoproteins, we determined whether differences in neutralization become apparent when antibodies to gB, gD, or gH/gL are used in combination. Neutralization of the gC/gE mutant was greatly increased compared that of WT virus when any two of the antibodies against gB, gD, or gH/gL were used in combination. These results suggest that gC and gE on WT virus provide a shield against neutralizing antibodies that interfere with gB-gD, gB-gH/gL, or gD-gH/gL interactions and that one function of virus neutralization is to prevent interactions between these glycoproteins.     

Oligomeric structure of glycoproteins in herpes simplex virus type 1.

A number of herpes simplex virus (HSV) glycoproteins are found in oligomeric states: glycoprotein E (gE)-gI and gH-gL form heterodimers, and both gB and gC have been detected as homodimers. We have further explored the organization of glycoproteins in the virion envelope by using both purified virions to quantitate glycoprotein amounts and proportions and chemical cross-linkers to detect oligomers. We purified gB, gC, gD, and gH from cells infected with HSV type 1 and used these as immunological standards. Glycoproteins present in sucrose gradient-purified preparations of two strains of HSV type 1, KOS and NS, were detected with antibodies to each of the purified proteins. From these data, glycoprotein molar ratios of 1:2:11:16 and 1:1:14:9 were calculated for gB/gC/gD/gH in KOS and NS, respectively. gL was also detected in virions, although we lacked a purified gL standard for quantitation. We then asked whether complexes of these glycoproteins could be identified, and if they existed as homo- or hetero-oligomers. Purified KOS was incubated at 4 degrees C with bis (sulfosuccinimidyl) suberate (BS3), an 11.4 A (1A = 0.1 mm) noncleavable, water-soluble cross-linker. Virus extracts were examined by Western blotting (immunoblotting), or immunoprecipitation followed by Western blotting, to assay for homo- and hetero-oligomers. Homodimers of gB, gC, and gD were detected, and hetero-oligomers containing gB cross-linked to gC, gC to gD, and gD to gB were also identified. gH and gL were detected as a hetero-oligomeric pair and could be cross-linked to gD or gC but not to gB. We conclude that these glycoproteins are capable of forming associations with one another. These studies suggest that glycoproteins are closely associated in virions and have the potential to function as oligomeric complexes.     

Human Anti-Varicella-Zoster Virus (VZV) Recombinant Monoclonal Antibody Produced after Zostavax Immunization Recognizes the gH/gL Complex and Neutralizes VZV Infection

Varicella-zoster virus (VZV) is a ubiquitous, highly cell-associated, and exclusively human neurotropic alphaherpesvirus. VZV infection is initiated by membrane fusion, an event dependent in part on VZV glycoproteins gH and gL. Consistent with its location on the virus envelope, the gH/gL complex is a target of neutralizing antibodies produced after virus infection. One week after immunizing a 59-year-old VZV-seropositive man with Zostavax, we sorted his circulating blood plasma blasts and amplified expressed immunoglobulin variable domain sequences by single-cell PCR. Sequence analysis identified two plasma blast clones, one of which was used to construct a recombinant monoclonal antibody (rec-RC IgG). The rec-RC IgG colocalized with VZV gE on the membranes of VZV-infected cells and neutralized VZV infection in tissue culture. Mass spectrometric analysis of proteins immunoprecipitated by rec-RC IgG identified both VZV gH and gL. Transfection experiments showed that rec-RC IgG recognized a VZV gH/gL protein complex but not individual gH or gL proteins. Overall, our recombinant monoclonal anti-VZV antibody effectively neutralizes VZV and recognizes a conformational epitope within the VZV gH/L protein complex. An unlimited supply of this antibody provides the opportunity to analyze membrane fusion events that follow virus attachment and to identify multiple epitopes on VZV-specific proteins.     

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 absence of glycoprotein gL, but not gC or gK, severely impairs pseudorabies virus neuroinvasiveness

Penetration and propagation of herpesviruses in the nervous system require the action of several glycoproteins. To assay for a function of glycoproteins gC, gK, and gL in the neuroinvasiveness of pseudorabies virus (PrV), deletion mutants lacking one of these glycoproteins and corresponding rescuants were inoculated in the nasal cavity of adult mice. We demonstrate that the lack of gL almost prevented the virus from penetrating and propagating in trigeminal, sympathetic, and parasympathetic tracks innervating the nasal cavity, while the lack of gC and gK only slowed the invasion of the nervous system. The conclusion of this and previous studies is that only gB, gD, gH, and gL are indispensable for penetration into neurons, while gB, gH, and gL (and, in some categories of neurons, also gE and gI) are necessary for transneuronal transfer in the mouse model. The deletion of other glycoprotein genes has little effect on PrV neuroinvasiveness although it may affect the dissemination of the virus.     

The Cytoplasmic Domain of Varicella-Zoster Virus Glycoprotein H Regulates Syncytia Formation and Skin Pathogenesis

The conserved herpesvirus fusion complex consists of glycoproteins gB, gH, and gL which is critical for virion envelope fusion with the cell membrane during entry. For Varicella Zoster Virus (VZV), the complex is necessary for cell-cell fusion and presumed to mediate entry. VZV causes syncytia formation via cell-cell fusion in skin and in sensory ganglia during VZV reactivation, leading to neuronal damage, a potential contributory factor for the debilitating condition of postherpetic neuralgia. The gH cytoplasmic domain (gHcyt) is linked to the regulation of gB/gH-gL-mediated cell fusion as demonstrated by increased cell fusion in vitro by an eight amino acid (aa834-841) truncation of the gHcyt. The gHcyt regulation was identified to be dependent on the physical presence of the domain, and not of specific motifs or biochemical properties as substitution of aa834-841 with V5, cMyc, and hydrophobic or hydrophilic sequences did not affect fusion. The importance of the gHcyt length was corroborated by stepwise deletions of aa834-841 causing incremental increases in cell fusion, independent of gH surface expression and endocytosis. Consistent with the fusion assay, truncating the gHcyt in the viral genome caused exaggerated syncytia formation and significant reduction in viral titers. Importantly, infection of human skin xenografts in SCID mice was severely impaired by the truncation while maintaining the gHcyt length with the V5 substitution preserved typical replication in vitro and in skin. A role for the gHcyt in modulating the functions of the gB cytoplasmic domain (gBcyt) is proposed as the gHcyt truncation substantially enhanced cell fusion in the presence of the gB[Y881F] mutation. The significant reduction in skin infection caused by hyperfusogenic mutations in either the gHcyt or gBcyt demonstrates that both domains are critical for regulating syncytia formation and failure to control cell fusion, rather than enhancing viral spread, is severely detrimental to VZV pathogenesis.     

Mutation of the YXXL endocytosis motif in the cytoplasmic tail of pseudorabies virus gE

The role of alphaherpesvirus membrane protein internalization during the course of viral infection remains a matter of speculation. To determine the role of internalization of the pseudorabies virus (PRV) gE and gI proteins, we constructed viral mutants encoding specific mutations in the cytoplasmic tail of the gE gene that inhibited internalization of the gE-gI complex. We used these mutants to assess the role of gE-gI endocytosis in incorporation of the proteins into the viral envelope and in gE-mediated spread or gE-promoted virulence. In addition, we report that another viral mutant, PRV 25, which encodes a gE protein defective in endocytosis, contains an additional, previously uncharacterized mutation in the gE gene. We compared PRV 25 to another viral mutant, PRV 107, that does not express the cytoplasmic tail of the gE protein. The gE protein encoded by PRV 107 is also defective in endocytosis. We conclude that efficient endocytosis of gE is not required for gE incorporation into virions, gE-mediated virulence, or spread of virus in the rat central nervous system. However, we do correlate the defect in endocytosis to a small-plaque phenotype in cultured cells.     

Expression of seven herpes simplex virus type 1 glycoproteins (gB, gC, gD, gE, gG, gH, and gI): comparative protection against lethal challenge in mice.

We have constructed recombinant baculoviruses individually expressing seven of the herpes simplex virus type 1 (HSV-1) glycoproteins (gB, gC, gD, gE, gG, gH, and gI). Vaccination of mice with gB, gC, gD, gE, or gI resulted in production of high neutralizing antibody titers to HSV-1 and protection against intraperitoneal and ocular challenge with lethal doses of HSV-1. This protection was statistically significant and similar to the protection provided by vaccination with live nonvirulent HSV-1 (90 to 100% survival). In contrast, vaccination with gH produced low neutralizing antibody titers and no protection against lethal HSV-1 challenge. Vaccination with gG produced no significant neutralizing antibody titer and no protection against ocular challenge. However, gG did provide modest, but statistically significant, protection against lethal intraperitoneal challenge (75% protection). Compared with the other glycoproteins, gG and gH were also inefficient in preventing the establishment of latency. Delayed-type hypersensitivity responses to HSV-1 at day 3 were highest in gG-, gH-, and gE-vaccinated mice, while on day 6 mice vaccinated with gC, gE, and gI had the highest delayed-type hypersensitivity responses. All seven glycoproteins produced lymphocyte proliferation responses, with the highest response being seen with gG. The same five glycoproteins (gB, gC, gD, gE, and gI) that induced the highest neutralization titers and protection against lethal challenge also induced some killer cell activity. The results reported here therefore suggest that in the mouse protection against lethal HSV-1 challenge and the establishment of latency correlate best with high preexisting neutralizing antibody titers, although there may also be a correlation with killer cell activity.     

Cytomegalovirus UL131-128 products promote gB conformational transition and gB-gH interaction during entry into endothelial cells

Herpesviruses use gB and gH-gL glycoproteins to execute fusion. Other virus-specific glycoproteins are required for receptor binding and fusion activation. The human cytomegalovirus (HCMV) UL131-128 proteins are essential for the infection of leukocytes, endothelial cells (ECs), and many epithelial cell lines. Here we show that UL131-128 play a role in a chain of events involving gB and gH during HCMV entry into ECs. An HCMV strain bearing the wild-type (wt) UL131-128 locus exhibited a gB transition from a protease-resistant to protease-sensitive form, a conformational change that was suppressed by a thiourea inhibitor of fusion (WY1768); in contrast, gH was susceptible to proteolysis throughout entry. Moreover, gB and gH transiently interacted, and a lipid mixing assay showed that the wt strain had carried out fusion by 60 min postinfection. However, these events were greatly altered when UL131-128-defective strains were used for infection or when there was an excess of soluble pUL128 during wt infection: the gB conformational change became WY1768 resistant, the gB-gH complex was no longer observed, and fusion was prevented. Both gB and gH in this case showed late protease resistance, related to their endocytic uptake. Our data point to the involvement of UL131-128 proteins in driving gB through a WY1768-sensitive fold transition, thus promoting a short-lived gB-gH complex and fusion; they also suggest that HCMV fuses with the EC plasma membrane and that endocytosis ensues only when the virus cannot trigger UL131-128-dependent steps.     

Role of Envelope Protein gE Endocytosis in the Pseudorabies Virus Life Cycle

Several groups have reported that certain herpesvirus envelope proteins do not remain on the surface of cells that express them but rather are internalized by endocytosis in a recycling process. The biological function of membrane protein endocytosis in the virus life cycle remains a matter of speculation and debate. In this report, we demonstrate that some, but not all, membrane proteins encoded by the alphaherpesvirus pseudorabies virus (PRV) are internalized after reaching the plasma membrane. Glycoproteins gE and gB are internalized from the plasma membrane of cells, while gI and gC are not internalized efficiently. We show for gE that the cytoplasmic domain of the protein is required for endocytosis. While the gI protein is incapable of endocytosis on its own, it can be internalized when complexed with gE. We demonstrate that endocytosis of the gE-gI complex and gB occurs early after infection of tissue culture cells but that this process stops completely after 6 h of infection, a time that correlates with significant shutoff of host protein synthesis. We also show that gE protein internalized at 4 h postinfection is not present in virions formed at a later time. We discuss the differences in PRV gE and gI endocytosis compared to that of the varicella-zoster virus homologs and the possible roles of glycoprotein endocytosis in the virus life cycle.     

Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion

Cell-cell fusion (abbreviated as cell fusion) is a characteristic pathology of medically important viruses, including varicella-zoster virus (VZV), the causative agent of chickenpox and shingles. Cell fusion is mediated by a complex of VZV glycoproteins, gB and gH-gL, and must be tightly regulated to enable skin pathogenesis based on studies with gB and gH hyperfusogenic VZV mutants. Although the function of gB and gH-gL in the regulation of cell fusion has been explored, whether host factors are directly involved in this regulation process is unknown. Here, we discovered host factors that modulated VZV gB/gH-gL mediated cell fusion via high-throughput screening of bioactive compounds with known cellular targets. Two structurally related non-antibiotic macrolides, tacrolimus and pimecrolimus, both significantly increased VZV gB/gH-gL mediated cell fusion. These compounds form a drug-protein complex with FKBP1A, which binds to calcineurin and specifically inhibits calcineurin phosphatase activity. Inhibition of calcineurin phosphatase activity also enhanced both herpes simplex virus-1 fusion complex and syncytin-1 mediated cell fusion, indicating a broad role of calcineurin in modulating this process. To characterize the role of calcineurin phosphatase activity in VZV gB/gH-gL mediated fusion, a series of biochemical, biological and infectivity assays was performed. Pimecrolimus-induced, enhanced cell fusion was significantly reduced by shRNA knockdown of FKBP1A, further supporting the role of calcineurin phosphatase activity in fusion regulation. Importantly, inhibition of calcineurin phosphatase activity during VZV infection caused exaggerated syncytia formation and suppressed virus propagation, which was consistent with the previously reported phenotypes of gB and gH hyperfusogenic VZV mutants. Seven host cell proteins that remained uniquely phosphorylated when calcineurin phosphatase activity was inhibited were identified as potential downstream factors involved in fusion regulation. These findings demonstrate that calcineurin is a critical host cell factor pivotal in the regulation of VZV induced cell fusion, which is essential for VZV pathogenesis.     

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 glycoproteins gB and gD function in a redundant fashion to promote secondary envelopment

Egress of herpes simplex virus (HSV) and other herpesviruses from cells involves extensive modification of cellular membranes and sequential envelopment and deenvelopment steps. HSV glycoproteins are important in these processes, and frequently two or more glycoproteins can largely suffice in any step. Capsids in the nucleus undergo primary envelopment at the inner nuclear membrane (INM), and then enveloped virus particles undergo deenvelopment by fusing with the outer nuclear membrane (ONM). Capsids delivered into the cytoplasm then undergo secondary envelopment, involving trans-Golgi network (TGN) membranes. The deenvelopment step involves HSV glycoproteins gB and gH/gL acting in a redundant fashion. This fusion has features common to the fusion that occurs between the virion envelope and cellular membranes when HSV enters cells, a process requiring gB, gD, and gH/gL. Whether HSV gD also participates (in a redundant fashion with gB or gH/gL) in deenvelopment has not been characterized. Secondary envelopment in the cytoplasm is known to involve HSV gD and gE/gI, also acting in a redundant fashion. Whether gB might also contribute to secondary envelopment, collaborating with gD and gE/gI, is also not clear. To address these questions, we constructed an HSV double mutant lacking gB and gD. The HSV gB(-)/gD(-) mutant exhibited no substantial defects in nuclear egress. In contrast, secondary envelopment was markedly reduced, and there were numerous unenveloped capsids that accumulated in the cytoplasm, as well as increased numbers of partially enveloped capsids and morphologically aberrant enveloped particles with thicker, oblong tegument layers. These defects were different from those observed with HSV gD(-)/gE(-)/gI(-) mutants, which accumulated capsids in large, aggregated masses in the cytoplasm. Our results suggest that HSV gB functions in secondary envelopment, apparently acting downstream of gE/gI.