Comparative usage of herpesvirus entry mediator A and nectin-1 by laboratory strains and clinical isolates of herpes simplex virus

The herpesvirus entry mediator A (HVEM/HveA) and nectin-1 (HveC/CD111) are two major receptors for herpes simplex virus (HSV). Although structurally unrelated, both receptors can independently mediate entry of wild-type (wt) HSV-1 and HSV-2 by interacting with the viral envelope glycoprotein D (gD). Laboratory strains with defined mutations in gD (e.g. rid1) do not use HVEM but use nectin-2 (HveB/CD112) for entry. The relative usage of HVEM and nectin-1 during HSV infection in vivo is not known. In the absence of a defined in vivo model, we used in vitro approaches to address this question. First, we screened HSV clinical isolates from various origins for receptor tropism and found that all used both HVEM and nectin-1. Second, we determined the numbers of surface receptors on various susceptible and resistant cell lines as well as on primary fibroblasts derived from an individual with cleft lip/palate ectodermal dysplasia (CLPED1). Although CLPED1 cells can only express a defective form of nectin-1, they allowed entry of wild type and mutant HSV strains by usage of either HVEM or nectin-2. Finally, we compared the ability of HVEM and nectin-1 to mediate entry when expressed at varying cell surface densities. Both receptors showed a direct relationship between the number of receptors and HSV susceptibility. Direct comparison of receptors suggests that nectin-1 is more efficient at promoting entry than HVEM. Overall, our data suggest that both receptors play a role during HSV infection in vivo and that both are highly efficient even at low levels of expression.     

Early detection of endogenous retroviruses in chemically induced mouse cells

Two cell surface proteins (nectin-1/HveC and nectin-2/HveB) shown previously to serve as receptors for the entry of herpes simplex virus 1 (HSV-1) wild-type and/or mutant strains were found to serve also as receptors for HSV-1-induced cell fusion. Transfection with genomic DNA from a syncytial HSV-1 strain encoding wild-type gD resulted in fusion of Chinese hamster ovary (CHO) cells expressing nectin-1 but not of cells expressing nectin-2. In contrast, transfection with DNA from a related HSV-1 strain encoding the mutant Rid1 form of gD resulted in fusion of CHO cells expressing either receptor but not of control cells. These results are consistent with the ability of each receptor to mediate entry of viruses expressing wild-type or Rid1 gD and with results obtained previously with HVEM (HveA), a third HSV-l entry receptor. Undersulfation of GAGs in receptor-expressing cell lines predictably reduced susceptibility to HSV-l infection. In contrast, susceptibility to cell fusion mediated by HVEM or nectin-1 was not reduced. Undersulfation of GAGs partially inhibited cell fusion mediated by nectin-2. We conclude that HSV-1-induced cell fusion requires a gD-binding entry receptor, that ability of an HSV-1 strain to use HVEM, nectin-2 or nectin-1 for cell fusion depends on the allele of gD expressed and that GAGs may influence cell fusion, dependent on the gD-binding receptor used, but are less important for cell fusion mediated by HVEM, nectin-2 or nectin-l than for viral entry.     

Spinoculation of heparan sulfate deficient cells enhances HSV-1 entry, but does not abolish the need for essential glycoproteins in viral fusion

Cell surface heparan sulfate functions as a co-receptor in HSV-1 entry. In order to study its significance in context with specific gD receptors (nectin-1, HVEM, and 3-O-sulfated heparan sulfate) a low speed centrifugation based virus inoculation (spinoculation) method was used. The experiments were performed at 1200 x g using glycosylaminoglycan positive (GAG+) or deficient (GAG-) cells expressing gD receptors. Clearly, spinoculation of GAG- nectin-1 or HVEM cells enhanced significantly viral entry compared to similar but unspun cells. The enhanced entry was due to increased virus deposition at the cell surface and not due to pelleting of the virus. Among the gD receptors, spinoculated GAG- HVEM cells showed restoration of HSV-1 entry compared to unspinoculated GAG+ HVEM cells. In contrast, spinoculated GAG- nectin-1 cells showed less entry than unspinoculated GAG+ nectin-1 cells. GAG- 3-O-sulfotransferase-expressing cells or heparinase treated GAG+ 3-O-sulfated heparan sulfate cells, in contrast, remained resistant to entry even after spinoculation. To investigate further, any potential effects of centrifugation on membrane fusion, a virus-free cell fusion assay was performed. Clearly, spinning had no effects on cell fusion, nor could it replace the need for all four essential glycoproteins. Taken together these results suggest that heparan sulfate plays a role of an attachment receptor, which could be substituted by spinoculation. This effect, however, varies with the gD receptor used, which in turn, could be used as a means for identifying gD receptor usage for entry into a cell type.     

Glycoprotein D homologs in herpes simplex virus type 1, pseudorabies virus, and bovine herpes virus type 1 bind directly to human HveC(nectin-1) with different affinities

Distinct subsets of human receptors for alphaherpesviruses mediate the entry of herpes simplex virus (HSV), pseudorabies virus (PrV), or bovine herpes virus type 1 (BHV-1) into cells. Glycoprotein D (gD) is essential for receptor-mediated entry of all three viruses into cells. However, the gD homologs of these viruses share only 22-33% amino acid identity. Several entry receptors for HSV have been identified. Two of these, HveA (HVEM) and HveC (nectin-1), mediate entry of most HSV-1 and HSV-2 strains and are bound directly by HSV gD. A third receptor, HveB (nectin-2), mediates entry of HSV-2 and only a limited number of HSV-1 strains. HveB and HveC can also serve as entry receptors for PrV, whereas only HveC can serve this function for BHV-1. We show here that gD from PrV and BHV-1 binds directly to the human receptors that mediate PrV and BHV-1 entry. We expressed soluble forms of PrV gD and BHV-1 gD using recombinant baculoviruses and purified each protein. Using ELISA, we detected direct binding of PrV gD to HveB and HveC and direct binding of BHV-1 gD to HveC. Biosensor analysis revealed that PrV gD had a 10-fold higher affinity than HSV-1 gD for human HveC. In contrast, the binding of BHV-1 gD to HveC was weak. PrV gD and HSV-1 gD competed for binding to the V domain of HveC and both inhibited entry of the homologous and heterologous viruses. These data suggest that the two forms of gD bind to a common region on human HveC despite their low amino acid similarity. Based on affinities for human HveC, we predict a porcine HveC homolog may be important for PrV infection in its natural host, whereas a BHV-1 infection in its natural host may be mediated by a receptor other than a bovine HveC homolog.     

The herpes simplex virus receptor nectin-1 is down-regulated after trans-interaction with glycoprotein D

During herpes simplex virus (HSV) entry, membrane fusion occurs either on the cell surface or after virus endocytosis. In both cases, binding of glycoprotein D (gD) to a receptor such as nectin-1 or HVEM is required. In this study, we co-cultured cells expressing gD with nectin-1 expressing cells to investigate the effects of gD on nectin-1 at cell contacts. After overnight co-cultures with gD expressing cells, there was a down-regulation of nectin-1 in B78H1-C10, SY5Y, A431 and HeLa cells, which HSV enters by endocytosis. In contrast, on Vero cells, which HSV enters at the plasma membrane, nectin-1 was not down-regulated. Further analysis of B78H1-derived cells showed that nectin-1 down-regulation corresponds to the ability of gD to bind nectin-1 and is achieved by internalization and low-pH-dependent degradation of nectin-1. Moreover, gD is necessary for virion internalization in B78H1 cells expressing nectin-1. These data suggest that the determinants of gD-mediated internalization of nectin-1 may direct HSV to an endocytic pathway during entry.     

The nectin-1alpha transmembrane domain, but not the cytoplasmic tail, influences cell fusion induced by HSV-1 glycoproteins

Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1alpha involved in cell fusion, we measured the ability of nectin-1alpha/nectin-2alpha chimeras, nectin-1alpha/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1alpha to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1alpha cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane-spanning domains of nectin-1alpha and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1alpha interaction in fusion.     

Glycoprotein D actively induces rapid internalization of two nectin-1 isoforms during herpes simplex virus entry

Entry of herpes simplex virus (HSV) occurs either by fusion at the plasma membrane or by endocytosis and fusion with an endosome. Binding of glycoprotein D (gD) to a receptor such as nectin-1 is essential in both cases. We show that virion gD triggered the rapid down-regulation of nectin-1 with kinetics similar to those of virus entry. In contrast, nectin-1 was not constitutively recycled from the surface of uninfected cells. Both the nectin-1α and β isoforms were internalized in response to gD despite having different cytoplasmic tails. However, deletion of the nectin-1 cytoplasmic tail slowed down-regulation of nectin-1 and internalization of virions. These data suggest that nectin-1 interaction with a cytoplasmic protein is not required for its down-regulation. Overall, this study shows that gD binding actively induces the rapid internalization of various forms of nectin-1. We suggest that HSV activates a nectin-1 internalization pathway to use for endocytic entry.     

Structure-function analysis of herpes simplex virus glycoprotein B with fusion-from-without activity

Fusion-from-without (FFWO) is the rapid induction of cell fusion by virions in the absence of viral protein synthesis. The combination of two amino acid mutations in envelope glycoprotein B (gB), one in the ectodomain and one in the cytoplasmic tail, can confer FFWO activity to wild type herpes simplex virus (HSV). In this report, we analyzed the entry and cell fusion phenotypes of HSV that contains FFWO gB, with emphasis on the cellular receptors for HSV, nectin-1, nectin-2 and HVEM. The ability of an HSV strain with FFWO gB to efficiently mediate FFWO via a specific gD-receptor correlated with its ability to mediate viral entry by that receptor. A FFWO form of gB was not sufficient to switch the entry of HSV from a pH-dependent, endocytic pathway to a direct fusion, pH-independent pathway. The conformation of gB with FFWO activity was not globally altered relative to wild type. However, distinct monoclonal antibodies had reduced reactivity with FFWO gB, suggesting an altered antigenic structure relative to wild type. FFWO was blocked by preincubation of virions with neutralizing antibodies to gB or gD. Together with previous studies, the results indicate that the roles of gB in FFWO and in virus-cell fusion during entry are related but not identical. This study also suggests that the FFWO function of gB is not a specific determinant for the selection of HSV entry pathway and that antigenic differences in FFWO gB may reflect its enhanced fusion activity.     

Use of chimeric nectin-1(HveC)-related receptors to demonstrate that ability to bind alphaherpesvirus gD is not necessarily sufficient for viral entry.

Human nectin-1 (HveC, Prr1), a member of the immunoglobulin superfamily and a receptor for the entry of herpes simplex viruses 1 and 2 (HSV-1, HSV-2), pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1), binds to viral gD. For HSV-1, HSV-2, and PRV, the gD-binding region of nectin-1 has been localized to the N-terminal V-like domain. To determine whether the two C-like domains of nectin-1 influenced gD binding and entry activity, genes encoding chimeric proteins were constructed. Portions of nectin-1 were replaced with homologous regions from nectin-2 (HveB, Prr2), a related protein with ability to mediate the entry of PRV, HSV-2, and Rid mutants of HSV-1, but not HSV-1 or BHV-1. Also, one or more domains of nectin-1 were fused to the two membrane-proximal Ig domains of CD4, a protein with no herpesvirus entry or gD-binding activity. The chimeric proteins were expressed in Chinese hamster ovary cells, which normally lack alphaherpesvirus entry receptors, and detected on the cell surface by one or more anti-nectin-1 monoclonal antibodies. One chimeric protein (nectin-1 amino acids 1-124 fused to CD4) failed to bind to soluble forms of HSV-1, HSV-2, PRV, and BHV-1 gD and, as expected, also failed to mediate entry of the viruses from which these gDs were derived. The other chimeric receptors bound all forms of gD. Some mediated the entry of all the viruses tested but others mediated entry of some but not all the viruses. We conclude that binding of gD to the nectin-1 V domain is not sufficient for entry activity, that there are structural requirements for entry activity independent of gD binding, and that these requirements are different for the several alphaherpesviruses that can use nectin-1 as a receptor.     

Evaluation of the antiviral potential of the soluble forms of glycoprotein D receptors on ocular herpes caused by HSV-1 and HSV-2 infections in a transgenic mouse model

Ocular herpes, caused by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections, remains an important corneal disease, which may result in loss of vision. Because the frequency of acyclovir resistance in HSV has increased, novel antiviral agents are needed for therapeutic approaches to ocular herpes. Several studies have demonstrated that fusion proteins containing entire ectodomain of HSV glycoprotein D receptors, including herpesvirus entry mediator A (HVEM), nectin-1 and nectin-2, and the Fc portion of human IgG (HVEMIg, nectin-1Ig, and nectin-2Ig, respectively), can exert antiviral effects in vitro and in vivo. Here, to evaluate the antiviral potential of HVEMIg, nectin-1Ig, and nectin-2Ig against ocular infections with HSV, transgenic mice expressing these fusion proteins were ocularly inoculated with HSV-1 and HSV-2. Transgenic mouse lines expressing HVEMIg and nectin-1Ig showed marked resistance to ocular herpes; on the other hand, mouse lines expressing nectin-2Ig did not. Furthermore, to investigate the therapeutic effects of nectin-1Ig, which can neutralize HSVs in vitro against ocular disease, transgenic mouse serum containing nectin-1Ig was dropped into the eyes of wild-type mice after HSV infection. Reduction of severe symptoms could be observed in mice treated with nectin-1Ig serum. These results warrant further study of soluble HVEM and nectin-1 products as preventive and therapeutic agents against ocular herpes caused by HSV-1 and HSV-2 infections, especially nectin-1Ig as a new eye drop.     

Fusion activity of lipid-anchored envelope glycoproteins of herpes simplex virus type 1

Expression of the herpes simplex virus type 1 (HSV-1) glycoproteins gB, gD, gH, and gL is necessary and sufficient to cause cell fusion. To identify the requirements for a membrane-spanning domain in HSV-1 glycoprotein-induced cell fusion, we created gB, gD, and gH mutants with transmembrane and cytoplasmic domains replaced by a glycosylphosphatidylinositol (gpi)-addition sequence. The corresponding gBgpi, gDgpi, and gHgpi proteins were expressed with wild-type efficiency at the cell surface and were linked to the plasma membrane via a gpi anchor. The gDgpi mutant promoted cell fusion near wild-type gD levels when co-expressed with gB, gH, and gL in a cell-mixing fusion assay, indicating that the gD transmembrane and cytoplasmic domains were not required for fusion activity. A plasma membrane link was required for fusion because a gD mutant lacking a transmembrane and cytoplasmic domain was nonfunctional for fusion. The gDgpi mutant was also able to cooperate with wild-type gB, gH, and gL to form syncytia, albeit at a size smaller than those formed in the wild-type situation. The gBgpi and gHgpi mutants were unable to promote fusion when expressed with the other wild-type viral glycoproteins, highlighting the requirement of the specific transmembrane and cytoplasmic domains for gB and gH function.     

The requirements for herpes simplex virus type 1 cell-cell spread via nectin-1 parallel those for virus entry

Herpes simplex virus type 1 (HSV-1) spreads from an infected cell to an uninfected cell by virus entry, virus-induced cell fusion, and cell-cell spread. The three forms of virus spread require the viral proteins gB, gD, and gH-gL, as well as a cellular gD receptor. The mutual requirement for the fusion glycoproteins and gD receptor suggests that virus entry, cell fusion, and cell-cell spread occur by a similar mechanism. The goals of this study were to examine the role of the nectin-1alpha transmembrane domain and cytoplasmic tail in cell-cell spread and to obtain a better understanding of the receptor-dependent events occurring at the plasma membrane during cell-cell spread. We determined that an intact nectin-1alpha V-like domain was required for cell-cell spread, while a membrane-spanning domain and cytoplasmic tail were not. Chimeric forms of nectin-1 that were non-functional for virus entry did not mediate cell-cell spread regardless of whether they could mediate cell fusion. Also, cell-cell spread of syncytial isolates was dependent upon nectin-1alpha expression and occurred through a nectin-1-dependent mechanism. Taken together, our results indicate that nectin-1-dependent events occurring at the plasma membrane during cell-cell spread were equivalent to those for virus entry.     

Characterization of soluble glycoprotein D-mediated herpes simplex virus type 1 infection

Herpes simplex virus type 1 (HSV-1) entry into permissive cells involves attachment to cell-surface glycosaminoglycans (GAGs) and fusion of the virus envelope with the cell membrane triggered by the binding of glycoprotein D (gD) to cognate receptors. In this study, we characterized the observation that soluble forms of the gD ectodomain (sgD) can mediate entry of gD-deficient HSV-1. We examined the efficiency and receptor specificity of this activity and used sequential incubation protocols to determine the order and stability of the initial interactions required for entry. Surprisingly, virus binding to GAGs did not increase the efficiency of sgD-mediated entry and gD-deficient virus was capable of attaching to GAG-deficient cells in the absence of sgD. These observations suggested a novel binding interaction that may play a role in normal HSV infection.     

Kinetic analysis of glycoprotein C of herpes simplex virus types 1 and 2 binding to heparin,heparan sulfate,and complement component C3b

Glycoprotein C (gC) from herpes simplex virus (HSV) facilitates virus entry by attaching the virion to host cell-surface heparan sulfate (HS). Although gC from HSV-1 (gC1) and from HSV-2 (gC2) bind to heparin, gC2 is believed to play a less significant role than gC1 in attachment of virus to cells. This attachment step is followed by the binding of gD to one of several cellular receptors. gC also plays an important role in immune evasion by binding to the C3b fragment of the third component of the host complement system. Yet, although both gC1 and gC2 protect HSV against complement-mediated neutralization, only gC on HSV-1-infected cells acts as a receptor for C3b. We used optical biosensor technology to quantitate the affinities (K(D)) and the stabilities (k(off)) between both serotypes of gC with heparin, HS, and C3b to address three questions concerning gC interactions. First, can differences in affinity or stability account for differences between the contributions of HSV-1 and HSV-2 gC in attachment? Our data show that the gC2-HS complex is highly unstable (k(off) = 0.2 s(-1)) compared to the gC1-HS complex (k(off) = 0.003 s(-1)), suggesting why gC2 may not play an important role in attachment of virus to cells as does gC1. Second, does gC2 have a lower affinity for C3b than does gC1, thereby explaining the lack of C3b-receptor activity on HSV-2 infected cells? Surprisingly, gC2 had a 10-fold higher affinity for C3b compared to gC1, so this functional difference in serotypes cannot be accounted for by affinity. Third, do differences in gC-HS and gD-receptor affinities support a model of HSV entry in which the gC-HS interaction is of lower affinity than the gD-receptor interaction? Our biosensor results indicate that gC has a higher affinity for HS than gD does for cellular receptors HveA (HVEM) and HveC (nectin-1).     

Different receptors binding to distinct interfaces on herpes simplex virus gD can trigger events leading to cell fusion and viral entry

One of the herpes simplex virus envelope glycoproteins, designated gD, is the principal determinant of cell recognition for viral entry. Other viral glycoproteins, gB, gH and gL, cooperate with gD to mediate the membrane fusion that is required for viral entry and cell fusion. Membrane fusion is triggered by the binding of gD to one of its receptors. These receptors belong to three different classes of cell surface molecules. This review summarizes recent findings on the structure and function of gD. The results presented indicate that gD may assume more than one conformation, one in the absence of receptor, another when gD is bound to the herpesvirus entry mediator, a member of the TNF receptor family, and a third when gD is bound to nectin-1, a cell adhesion molecule in the immunoglobulin superfamily. Finally, information and ideas are presented about a membrane-proximal region of gD that is required for membrane fusion, but not for receptor binding, and that may have a role in activating the fusogenic activity of gB, gH and gL.     

Cell fusion induced by herpes simplex virus glycoproteins gB, gD, and gH-gL requires a gD receptor but not necessarily heparan sulfate

To characterize cellular factors required for herpes simplex virus type 1 (HSV-1)-induced cell fusion, we used an efficient and quantitative assay relying on expression of HSV-1 glycoproteins in transfected cells. We showed the following: (1) Cell fusion depended not only on expression of four viral glycoproteins (gB, gD, and gH-gL), as previously shown, but also on expression of cell surface entry receptors specific for gD. (2) Cell fusion required expression of all four glycoproteins in the same cell. (3) Heparan sulfate was not required for cell fusion. (4) Coexpression of receptor with the four glycoproteins in the same cell reduced fusion activity, indicating that interaction of gD and receptor can limit polykaryocyte formation. Overall, the viral and cellular determinants of HSV-1-induced cell fusion are similar to those for viral entry, except that HSV-1 entry is significantly enhanced by binding of virus to cell surface heparan sulfate.     

Enhanced resistance to herpes simplex virus type 1 infection in transgenic mice expressing a soluble form of herpesvirus entry mediator

Herpesvirus entry mediator (HVEM) is a member of the tumor necrosis factor (TNF) receptor family used as a cellular receptor by virion glycoprotein D (gD) of herpes simplex virus (HSV). Both human and mouse forms of HVEM can mediate entry of HSV-1 but have no entry activity for pseudorabies virus (PRV). To assess the antiviral potential of HVEM in vivo, three transgenic mouse lines expressing a soluble form of HVEM (HVEMIg) consisting of an extracellular domain of murine HVEM and the Fc portion of human IgG1 were generated. All of the transgenic mouse lines showed marked resistance to HSV-1 infection when the mice were challenged intraperitoneally with HSV-1, but not to PRV infection. The present results demonstrate that HVEMIg is able to exert a significant antiviral effect against HSV-1 infection in vivo.     

Herpes simplex virus entry is associated with tyrosine phosphorylation of cellular proteins

The initial step in herpes simplex virus (HSV) entry is binding of virion glycoprotein (g)C and/or gB to cell surface heparan sulfate. After this initial attachment, gD interacts with cell surface receptor or receptors, and the virion envelope fuses with the cell membrane. Fusion requires viral glycoproteins gB, gD, gL, and gH, but the cellular factors that participate in or the pathways activated by viral entry have not been defined. To determine whether signal transduction pathways are triggered by viral-cell fusion, we examined the association of viral entry with tyrosine phosphorylation of cellular proteins. Using immunoprecipitation and Western blotting, we found that at least three cytoplasmic host cell proteins, designated p80, p104, and p140, become tyrosine phosphorylated within 5-10 min after exposure to HSV-1 or HSV-2. However, no phosphorylation is detected when cells are exposed to a mutant virus deleted in gL that binds but fails to penetrate. Phosphorylation is restored when the gL-deletion virus is grown on a complementing cell line. Viral entry and the phosphorylation of p80, p104, and p140 are inhibited when cells are infected with virus in the presence of protein tyrosine kinase inhibitors. Taken together, these studies suggest that tyrosine phosphorylation of host cellular proteins is triggered by viral entry.     

A role for heparan sulfate 3-O-sulfotransferase isoform 2 in herpes simplex virus type 1 entry and spread

Heparan sulfate (HS) 3-O-sulfotransferase isoform-2 (3-OST-2), which belongs to a family of enzymes capable of generating herpes simplex virus type-1 (HSV-1) entry and spread receptors, is predominantly expressed in human brain. Despite its unique expression pattern, the ability of 3-OST-2 to mediate HSV-1 entry and cell-to-cell fusion is not known. Our results demonstrate that expression of 3-OST-2 can render Chinese hamster ovary K1 (CHO-K1) cells susceptible to entry of wild-type and mutant strains of HSV-1. Evidence for generation of gD receptors by 3-OST-2 were suggested by gD-mediated interference assay and the ability of 3-OST-2-expressing CHO-K1 cells to preferentially bind HSV-1 gD, which could be reversed by prior treatment of cells with HS lyases (heparinases II/III). In addition, 3-OST-2-expressing CHO-K1 cells acquired the ability to fuse with cells-expressing HSV-1 glycoproteins, a phenomenon that mimics a way of viral spread in vivo. Demonstrating specificity, the cell fusion was inhibited by soluble 3-O-sulfated forms of HS, but not unmodified HS. Taken together, our results raise the possibility of a role of 3-OST-2 in the spread of HSV-1 infection in the brain.