The herpes simplex virus 1 IgG fc receptor blocks antibody-mediated complement activation and antibody-dependent cellular cytotoxicity in vivo

Herpes simplex virus 1 (HSV-1) glycoprotein E (gE) mediates cell-to-cell spread and functions as an IgG Fc receptor (FcγR) that blocks the Fc domain of antibody targeting the virus or infected cell. Efforts to assess the functions of the HSV-1 FcγR in vivo have been hampered by difficulties in preparing an FcγR-negative strain that is relatively intact for spread. Here we report the FcγR and spread phenotypes of NS-gE264, which is a mutant strain that has four amino acids inserted after gE residue 264. The virus is defective in IgG Fc binding yet causes zosteriform disease in the mouse flank model that is only minimally reduced compared with wild-type and the rescue strains. The presence of zosteriform disease suggests that NS-gE264 spread functions are well maintained. The HSV-1 FcγR binds the Fc domain of human, but not murine IgG; therefore, to assess FcγR functions in vivo, mice were passively immunized with human IgG antibody to HSV. When antibody was inoculated intraperitoneally 20 h prior to infection or shortly after virus reached the dorsal root ganglia, disease severity was significantly reduced in mice infected with NS-gE264, but not in mice infected with wild-type or rescue virus. Studies of C3 knockout mice and natural killer cell-depleted mice demonstrated that the HSV-1 FcγR blocked both IgG Fc-mediated complement activation and antibody-dependent cellular cytotoxicity. Therefore, the HSV-1 FcγR promotes immune evasion from IgG Fc-mediated activities and likely contributes to virulence at times when antibody is present, such as during recurrent infections.     

Herpes simplex virus type 1 evades the effects of antibody and complement in vivo

Herpes simplex virus type 1 (HSV-1) encodes a complement-interacting glycoprotein, gC, and an immunoglobulin G (IgG) Fc binding glycoprotein, gE, that mediate immune evasion by affecting multiple aspects of innate and acquired immunity, including interfering with complement components C1q, C3, C5, and properdin and blocking antibody-dependent cellular cytotoxicity. Previous studies evaluated the individual contributions of gC and gE to immune evasion. Experiments in a murine model that examines the combined effects of gC and gE immune evasion on pathogenesis are now reported. Virulence of wild-type HSV-1 is compared with mutant viruses defective in gC-mediated C3 binding, gE-mediated IgG Fc binding, or both immune evasion activities. Eliminating both activities greatly increased susceptibility of HSV-1 to antibody and complement neutralization in vitro and markedly reduced virulence in vivo as measured by disease scores, virus titers, and mortality. Studies with C3 knockout mice indicated that other activities attributed to these glycoproteins, such as gC-mediated virus attachment to heparan sulfate or gE-mediated cell-to-cell spread, do not account for the reduced virulence of mutant viruses. The results support the importance of gC and gE immune evasion in vivo and suggest potential new targets for prevention and treatment of HSV disease.     

Herpes simplex virus type 1 glycoprotein e is required for axonal localization of capsid, tegument, and membrane glycoproteins

Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) promotes cell-to-cell spread at basolateral surfaces of epithelial cells, but its activity in neurons is less clear. We used the mouse retina infection model and neuronal cell cultures to define the spread phenotype of gE mutant viruses. Wild-type (WT) and gE-null (NS-gEnull) viruses both infected retina ganglion cell neurons; however, NS-gEnull viral antigens failed to reach the optic nerve, which indicates a defect in axonal localization. We evaluated two Fc receptor-negative gE mutant viruses containing four amino acid inserts in the gE ectodomain. One mutant virus failed to spread from the retina into the optic nerve, while the other spread normally. Therefore, the gE ectodomain is involved in axonal localization, and the Fc receptor and neuronal spread are mediated by overlapping but distinct gE domains. In the retina infection model, virus can travel to the brain via the optic nerve from presynaptic to postsynaptic neurons (anterograde direction) or via nerves that innervate the iris and ciliary body from postsynaptic to presynaptic neurons (retrograde direction). WT virus infected the brain by anterograde and retrograde routes, whereas NS-gEnull virus failed to travel by either pathway. The site of the defect in retrograde spread remains to be determined; however, infection of rat superior cervical ganglia neurons in vitro indicates that gE is required to target virion components to the axon initial segment. The requirement for gE in axonal targeting and retrograde spread highlights intriguing similarities and differences between HSV-1 and pseudorabies virus gE.     

In Vivo Immune Evasion Mediated by the Herpes Simplex Virus Type 1 Immunoglobulin G Fc Receptor

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcγR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcγR activity without affecting other gE functions, we constructed a mutant virus, NS-gE₃₃₉, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcγRs. NS-gE₃₃₉ expresses gE and gI, is FcγR⁻, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcγR does not bind murine IgG; therefore, the absence of an FcγR should not affect virulence in mice. NS-gE₃₃₉ causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcγR⁻ mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcγR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE₃₃₉-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcγR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.     

Herpes simplex virus glycoproteins E and I facilitate cell-to-cell spread in vivo and across junctions of cultured cells.

Herpes simplex virus (HSV) glycoproteins E and I (gE and gI) can act as a receptor for the Fc domain of immunoglobulin G (IgG). To examine the role of HSV IgG Fc receptor in viral pathogenesis, rabbits and mice were infected by the corneal route with HSV gE- or gI- mutants. Wild-type HSV-1 produced large dendritic lesions in the corneal epithelium and subsequent stromal disease leading to viral encephalitis, whereas gE- and gI- mutant viruses produced microscopic punctate or small dendritic lesions in the epithelium and no corneal disease or encephalitis. These differences were not related to the ability of the gE-gI oligomer to bind IgG because the differences were observed before the appearance of anti-HSV IgG and in mice, in which IgG binds to the Fc receptor poorly or not at all. Mutant viruses produced small plaques on monolayers of normal human fibroblasts and epithelial cells. Replication of gE- and gI- mutant viruses in human fibroblasts were normal, and the rates of entry of mutant and wild-type viruses into fibroblasts were similar; however, spread of gE- and gI- mutant viruses from cell to cell was significantly slower than that of wild-type HSV-1. In experiments in which fibroblast monolayers were infected with low multiplicities of virus and multiple rounds of infection occurred, the presence of neutralizing antibodies in the culture medium caused the yields of mutant viruses to drop dramatically, whereas there was a lesser effect on the production of wild-type HSV. It appears that cell-to-cell transmission of wild-type HSV-1 occurs by at least two mechanisms: (i) release of virus from cells and entry of extracellular virus into a neighboring cell and (ii) transfer of virus across cell junctions in a manner resistant to neutralizing antibodies. Our results suggest that gE- and gI- mutants are defective in the latter mechanism of spread, suggesting the possibility that the gE-gI complex facilitates virus transfer across cell junctions, a mode of spread which may predominate in some tissues. It is ironic that the gE-gI complex, usually considered an IgG Fc receptor, may, through its ability to mediate cell-to-cell spread, actually protect HSV from IgG in a manner different than previously thought.     

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.     

Herpes simplex virus type 1 encodes two Fc receptors which have different binding characteristics for monomeric immunoglobulin G (IgG) and IgG complexes.

Two herpes simplex virus type 1 glycoproteins, gE and gI, have been shown to form a complex that binds the Fc domain of immunoglobulin G (IgG). We demonstrate that this complex is required for the binding of monomeric nonimmune IgG but that gE alone is sufficient for binding polymeric IgG in the form of IgG complexes. Evidence that gE but not gI is required for binding IgG complexes is as follows. IgG complexes bound equally well to cells infected with gI-negative mutants or with wild-type virus, whereas cells infected with gE-negative mutants did not bind IgG complexes. Furthermore, L cells transiently transfected to express gE bound IgG complexes. Additional evidence that gI fails to augment binding of IgG complexes comes from experiments in which the gI gene was inducibly expressed in cells after infection. Inducible gI expression failed to increase binding of IgG complexes to infected cells in comparison with cells not capable of inducible gI expression. In contrast, expression of both gE and gI was necessary for binding of monomeric IgG, as demonstrated by flow cytometry using cells infected with gE-negative and gI-negative mutants. These observations demonstrate that herpes simplex virus type 1 Fc receptors (FcRs) have different binding characteristics for monomeric IgG and IgG complexes. Furthermore, it appears that gE is the FcR for IgG complexes and that gE and gI form the FcR for monomeric IgG.     

Herpes simplex virus immunoglobulin G Fc receptor activity depends on a complex of two viral glycoproteins, gE and gI.

Evidence was recently presented that herpes simplex virus type 1 (HSV-1) immunoglobulin G (IgG) Fc receptors are composed of a complex containing a previously described glycoprotein, gE, and a novel virus-induced polypeptide, provisionally named g70 (D. C. Johnson and V. Feenstra, J. Virol. 61:2208-2216, 1987). Using a monoclonal antibody designated 3104, which recognizes g70, in conjunction with antipeptide sera and virus mutants unable to express g70 or gE, we have mapped the gene encoding g70 to the US7 open reading frame of HSV-1 adjacent to the gE gene. Therefore, g70 appears to be identical to a recently described polypeptide which was named gI (R. Longnecker, S. Chatterjee, R. J. Whitley, and B. Roizman, Proc. Natl. Acad. Sci. USA 84:147-151, 1987). Under mildly denaturing conditions, monoclonal antibody 3104 precipitated both gI and gE from extracts of HSV-1-infected cells. In addition, rabbit IgG precipitated the gE-gI complex from extracts of cells transfected with a fragment of HSV-1 DNA containing the gI, gE, and US9 genes. Cells infected with mutant viruses which were unable to express gE or gI did not bind radiolabeled IgG; however, cells coinfected with two viruses, one unable to express gE and the other unable to express gI, bound levels of IgG approaching those observed with wild-type viruses. These results further support the hypothesis that gE and gI form a complex which binds IgG by the Fc domain and that neither polypeptide alone can bind IgG.     

Characterization of the interaction between the herpes simplex virus type I Fc receptor and immunoglobulin G

Herpes simplex virus type I (HSV-1) virions and HSV-1-infected cells bind to human immunoglobulin G (hIgG) via its Fc region. A complex of two surface glycoproteins encoded by HSV-1, gE and gI, is responsible for Fc binding. We have co-expressed soluble truncated forms of gE and gI in Chinese hamster ovary cells. Soluble gE-gI complexes can be purified from transfected cell supernatants using a purification scheme that is based upon the Fc receptor function of gE-gI. Using gel filtration and analytical ultracentrifugation, we determined that soluble gE-gI is a heterodimer composed of one molecule of gE and one molecule of gI and that gE-gI heterodimers bind hIgG with a 1:1 stoichiometry. Biosensor-based studies of the binding of wild type or mutant IgG proteins to soluble gE-gI indicate that histidine 435 at the CH2-CH3 domain interface of IgG is a critical residue for IgG binding to gE-gI. We observe many similarities between the characteristics of IgG binding by gE-gI and by rheumatoid factors and bacterial Fc receptors such as Staphylococcus aureus protein A. These observations support a model for the origin of some rheumatoid factors, in which they represent anti-idiotypic antibodies directed against antibodies to bacterial and viral Fc receptors.     

Characterization of domains of herpes simplex virus type 1 glycoprotein E involved in Fc binding activity for immunoglobulin G aggregates.

Herpes simplex virus type 1 glycoproteins gE and gI form receptors for the Fc domain of immunoglobulin G (IgG) which are expressed on the surface of infected cells and on the virion envelope and which protect the virus from immune attack. Glycoprotein gE-1 is a low-affinity Fc receptor (FcR) that binds IgG aggregates, while gE-1 and gI-1 form a complex which serves as a higher-affinity FcR capable of binding IgG monomers. In this study, we describe two approaches used to map an Fc binding domain on gE-1 for IgG aggregates. First, we constructed nine plasmids encoding gE-1/gD-1 fusions proteins, each containing a large gE-1 peptide inserted into the ectodomain of gD-1. Fusion proteins were tested for FcR activity with IgG-sensitized erythrocytes in a rosetting assay. Three of the fusion proteins containing overlapping gE-1 peptides demonstrated FcR activity; the smallest peptide that retained Fc binding activity includes gE-1 amino acids 183 to 402. These results indicate that an Fc binding domain is located between gE-1 amino acids 183 and 402. To more precisely map the Fc binding domain, we tested a panel of 21 gE-1 linker insertion mutants. Ten mutants with insertions between gE-1 amino acids 235 and 380 failed to bind IgG-sensitized erythrocytes, while each of the remaining mutants demonstrated wild-type Fc binding activity. Taken together, these results indicate that the region of gE-1 between amino acids 235 and 380 forms an FcR domain. A computer-assisted analysis of the amino acid sequence of gE-1 demonstrates an immunoglobulin-like domain contained within this region (residues 322 to 359) which shares homology with mammalian FcRs.     

The extracellular domain of herpes simplex virus gE is indispensable for efficient cell-to-cell spread: evidence for gE/gI receptors

Herpes simplex virus (HSV) spreads rapidly and efficiently within epithelial and neuronal tissues. The HSV glycoprotein heterodimer gE/gI plays a critical role in promoting cell-to-cell spread but does not obviously function during entry of extracellular virus into cells. Thus, gE/gI is an important molecular handle on the poorly understood process of cell-to-cell spread. There was previous evidence that the large extracellular (ET) domains of gE/gI might be important in cell-to-cell spread. First, gE/gI extensively accumulates at cell junctions, consistent with being tethered there. Second, expression of gE/gI in trans interfered with HSV spread between epithelial cells. To directly test whether the gE ET domain was necessary for gE/gI to promote virus spread, a panel of gE mutants with small insertions in the ET domain was constructed. Cell-to-cell spread was reduced when insertions were made within either of two regions, residues 256 to 291 or 348 to 380. There was a strong correlation between loss of cell-to-cell spread function and binding of immunoglobulin. gE ET domain mutants 277, 291, and 348 bound gI, produced mature forms of gE that reached the cell surface, and were incorporated into virions yet produced plaques similar to gE null mutants. Moreover, all three mutants were highly restricted in spread within the corneal epithelium, in the case of mutant 277 to only 4 to 6% of the number of cells compared with wild-type HSV. Therefore, the ET domain of gE is indispensable for efficient cell-to-cell spread. These observations are consistent with our working hypothesis that gE/gI can bind extracellular ligands, so-called gE/gI receptors that are concentrated at epithelial cell junctions. This fits with similarities in structure and function of gE/gI and gD, which is a receptor binding protein.     

Immunization strategies to block the herpes simplex virus type 1 immunoglobulin G Fc receptor

Herpes simplex virus type 1 (HSV-1) glycoprotein gE functions as an immunoglobulin G (IgG) Fc receptor (FcgammaR) that promotes immune evasion. When an IgG antibody binds by the F(ab')(2) domain to an HSV antigen, the Fc domain of some of the same antibody molecules binds to the FcgammaR, which blocks Fc-mediated functions. gE is a type 1 membrane glycoprotein with a large ectodomain that is expressed on the virion envelope and infected-cell surface. Our goal was to determine if immunizing with gE protein fragments could produce antibodies that bind by the F(ab')(2) domain to gE and block the FcgammaR, as measured by competitively inhibiting nonimmune human IgG binding to the FcgammaR. Three gE peptides were constructed in baculovirus spanning almost the entire ectodomain and used to immunize mice and rabbits. Two fragments were highly effective at producing antibodies that bind by the F(ab')(2) domain and block the FcgammaR. The most potent of these two antibodies was far more effective at blocking the FcgammaR than antibodies that are only capable of binding by the Fc domains to the FcgammaR, including anti-gC, anti-gD, and nonimmune IgG. These results suggest that immunizing with gE fragments has potential for preventing immune evasion by blocking activities mediated by the HSV-1 FcgammaR.     

Glycoproteins E and I facilitate neuron-to-neuron spread of herpes simplex virus.

Two herpes simplex virus (HSV) glycoproteins E and I (gE and gI) form a heterooligomer which acts as an Fc receptor and also facilitates cell-to-cell spread of virus in epithelial tissues and between certain cultured cells. By contrast, gE-gI is not required for infection of cells by extracellular virus. HSV glycoproteins gD and gJ are encoded by neighboring genes, and gD is required for both virus entry into cells and cell-to-cell spread, whereas gJ has not been shown to influence these processes. Since HSV infects neurons and apparently spreads across synaptic junctions, it was of interest to determine whether gD, gE, gI and gJ are also important for interneuronal transfer of virus. We tested the roles of these glycoproteins in neuron-to-neuron transmission of HSV type 1 (HSV-1) by injecting mutant viruses unable to express these glycoproteins into the vitreous body of the rat eye. The spread of virus infection was measured in neuron-rich layers of the retina and in the major retinorecipient areas of the brain. Wild-type HSV-1 and a gJ- mutant spread rapidly between synaptically linked retinal neurons and efficiently infected major retinorecipient areas of the brain. gD mutants, derived from complementing cells, infected only a few neurons and did not spread in the retina or brain. Mutants unable to express gE or gI were markedly restricted in their ability to spread within the retina, produced 10-fold-less virus in the retina, and spread inefficiently to the brain. Furthermore, when compared with wild-type HSV-1, gE- and gI- mutants spread inefficiently from cell to cell in cultures of neurons derived from rat trigeminal ganglia. Together, our results suggest that the gE-gI heterooligomer is required for efficient neuron-to-neuron transmission through synaptically linked neuronal pathways.     

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.     

Induction of immunoglobulin G Fc receptors by recombinant vaccinia viruses expressing glycoproteins E and I of herpes simplex virus type 1.

Glycoprotein E (gE) of herpes simplex virus type 1 (HSV-1) will bind immunoglobulin G (IgG) (Fc) affinity columns (R. B. Bauke and P. G. Spear, J. Virol. 32:779-789, 1979), but recent evidence suggests that the HSV-1 Fc receptor is composed of a complex of gE and glycoprotein I (gI) and that both gI and gE are required for Fc receptor activity (D. C. Johnson and V. Feenstra, J. Virol. 61:2208-2216, 1987; D. C. Johnson, M. C. Frame, M. W. Ligas, A. M. Cross, and N. D. Stow, J. Virol. 62:1347-1354, 1988). We have expressed gE and gI, either alone or in combination, on the surface of HeLa cells by using recombinant vaccinia viruses and have measured Fc receptor activity by Fc-rosetting or IgG-binding assays. Expression of gE alone resulted in the induction of Fc receptor activity, while expression of gI alone gave no detectable Fc binding. Coexpression of gE and gI resulted in higher levels of IgG binding than did expression of gE alone, despite the fact that under conditions of coexpression, the levels of surface gE were reduced. We propose that gE and gI together form a receptor of higher affinity than gE alone and that HSV-1 therefore has the potential to induce two Fc receptors of different affinities.     

pH dependence and stoichiometry of binding to the Fc region of IgG by the herpes simplex virus Fc receptor gE-gI

Herpes simplex virus type 1 encodes two glycoproteins, gE and gI, that form a heterodimer on the surface of virions and infected cells. The gE-gI heterodimer has been implicated in cell-to-cell spread of virus and is a receptor for the Fc fragment of IgG. Previous studies localized the gE-gI-binding site on human IgG to a region near the interface between the C(H)2 and C(H)3 domains of Fc, which also serves as the binding site for bacterial and mammalian Fc receptors. Although there are two potential gE-gI-binding sites per Fc homodimer, only one gE-gI heterodimer binds per IgG in gel filtration experiments. Here we report production of recombinant human Fc molecules that contain zero, one, or two potential gE-gI-binding sites and use them in analytical ultracentrifugation experiments to show that two gE-gI heterodimers can bind to each Fc. Further characterization of the gE-gI interaction with Fc reveals a sharp pH dependence of binding, with K(D) values of approximately 340 and approximately 930 nm for the first and second binding events, respectively, at the slightly basic pH of the cell surface (pH 7.4), but undetectable binding at pH 6.0. This strongly pH-dependent interaction suggests a physiological role for gE-gI dissociation from IgG within acidic intracellular compartments, consistent with a mechanism whereby herpes simplex virus promotes intracellular degradation of anti-viral antibodies.     

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.     

Herpes simplex virus-specific CD8+ T cells can clear established lytic infections from skin and nerves and can partially limit the early spread of virus after cutaneous inoculation

HSV infects skin or mucosal epithelium as well as entering the sensory nerves and ganglia. We have used TCR-transgenic T cells specific for the immunodominant class I-restricted determinant from HSV glycoprotein B (gB) combined with a flank zosteriform model of infection to examine the ability of CD8+ T cells to deal with infection. During the course of zosteriform disease, virus rapidly spreads from the primary inoculation site in the skin to sensory dorsal root ganglia and subsequently reappears in the distal flank. Virus begins to be cleared from all sites about 5 days after infection when gB-specific CD8+ T cells first appear within infected tissues. Although activated gB-specific effectors can partially limit virus egress from the skin, they do so only at the earliest times after infection and are ineffective at halting the progression of zosteriform disease once virus has left the inoculation site. In contrast, these same T cells can completely clear ongoing lytic replication if transferred into infected immunocompromised RAG-1-/- mice. Therefore, we propose that the role of CD8+ T cells during the normal course of disease is to clear replicating virus after infection is well established rather than limit the initial spread of HSV from the primary site of inoculation.     

Live attenuated herpes simplex virus 2 glycoprotein E deletion mutant as a vaccine candidate defective in neuronal spread

A herpes simplex virus 2 (HSV-2) glycoprotein E deletion mutant (gE2-del virus) was evaluated as a replication-competent, attenuated live virus vaccine candidate. The gE2-del virus is defective in epithelial cell-to-axon spread and in anterograde transport from the neuron cell body to the axon terminus. In BALB/c and SCID mice, the gE2-del virus caused no death or disease after vaginal, intravascular, or intramuscular inoculation and was 5 orders of magnitude less virulent than wild-type virus when inoculated directly into the brain. No infectious gE2-del virus was recovered from dorsal root ganglia (DRG) after multiple routes of inoculation; however, gE2-del DNA was detected by PCR in lumbosacral DRG at a low copy number in some mice. Importantly, no recurrent vaginal shedding of gE2-del DNA was detected in immunized guinea pigs. Intramuscular immunization outperformed subcutaneous immunization in all parameters evaluated, although individual differences were not significant, and two intramuscular immunizations were more protective than one. Immunized animals had reduced vaginal disease, vaginal titers, DRG infection, recurrent genital lesions, and recurrent vaginal shedding of HSV-2 DNA; however, protection was incomplete. A combined modality immunization using live virus and HSV-2 glycoprotein C and D subunit antigens in guinea pigs did not totally eliminate recurrent lesions or recurrent vaginal shedding of HSV-2 DNA. The gE2-del virus used as an immunotherapeutic vaccine in previously HSV-2-infected guinea pigs greatly reduced the frequency of recurrent genital lesions. Therefore, the gE2-del virus is safe, other than when injected at high titer into the brain, and is efficacious as a prophylactic and immunotherapeutic vaccine.     

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

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