Herpes simplex virus type 1 entry through a cascade of virus-cell interactions requires different roles of gD and gH in penetration.

We examined the entry process of herpes simplex virus type 1 (HSV-1) by using infectious virus and previously characterized noninfectious viruses that can bind to cells but cannot penetrate as a result of inactivation of essential viral glycoprotein D (gD) or H (gH). After contact of infectious virus with the cell plasma membrane, discernible changes of the envelope and tegument could be seen by electron microscopy. Noninfectious virions were arrested at distinct steps in interactions with cells. Viruses inactivated by anti-gD neutralizing antibodies attached to cells but were arrested prior to initiation of a visible fusion bridge between the virus and cell. As judged from its increased sensitivity to elution, virus lacking gD was less stably bound to cells than was virus containing gD. Moreover, soluble gD could substantially reduce virus attachment when added to cells prior to or with the addition of virus. Virus inactivated by anti-gH neutralizing antibodies attached and could form a fusion bridge but did not show expansion of the fusion bridge or extensive rearrangement of the envelope and tegument. We propose a model for infectious entry of HSV-1 by a series of interactions between the virion envelope and the cell plasma membrane that trigger virion disassembly, membrane fusion, and capsid penetration. In this entry process, gD mediates a stable attachment that is likely required for penetration, and gH seems to participate in fusion initiation or expansion.     

Cellular and viral requirements for rapid endocytic entry of herpes simplex virus

It was recently demonstrated that herpes simplex virus (HSV) successfully infects Chinese hamster ovary (CHO) cells expressing glycoprotein D (gD) receptors and HeLa cells by an endocytic mechanism (A. V. Nicola, A. M. McEvoy, and S. E. Straus, J. Virol. 77:5324-5332, 2003). Here we define cellular and viral requirements of this pathway. Uptake of intact, enveloped HSV from the cell surface into endocytic vesicles was rapid (t(1/2) of 8 to 9 min) and independent of the known cell surface gD receptors. Following uptake from the surface, recovery of intracellular, infectious virions increased steadily up to 20 min postinfection (p.i.), which corresponds to accumulation of enveloped virus in intracellular compartments. There was a sharp decline in recovery by 30 min p.i., suggesting loss of the virus envelope as a result of capsid penetration from endocytic organelles into the cytosol. In the absence of gD receptors, endocytosed virions did not successfully penetrate into the cytosol but were instead transported to lysosomes for degradation. Inhibitors of phosphatidylinositol (PI) 3-kinase, such as wortmannin, blocked transport of incoming HSV to the nuclear periphery and virus-induced gene expression but had no effect on virus binding or uptake. This suggests a role for PI 3-kinase activity in trafficking of HSV through the cytosol. Viruses that lack viral glycoproteins gB, gD, or gH-gL were defective in transport to the nucleus and had reduced infectivity. Thus, similar to entry via direct penetration at the cell surface, HSV entry into cells by wortmannin-sensitive endocytosis is efficient, involves rapid cellular uptake of viral particles, and requires gB, gD, and gH-gL.     

Distinct cellular responses differentiating alcohol- and hepatitis C virus-induced liver cirrhosis

Background

Herpes simplex virus (HSV) can utilize multiple pathways to enter host cells. The factors that determine which route is taken are not clear. Chinese hamster ovary (CHO) cells that express glycoprotein D (gD)-binding receptors are model cells that support a pH-dependent, endocytic entry pathway for all HSV strains tested to date. Fusion-from-without (FFWO) is the induction of target cell fusion by addition of intact virions to cell monolayers in the absence of viral protein expression. The receptor requirements for HSV-induced FFWO are not known. We used the syncytial HSV-1 strain ANG path as a tool to evaluate the complex interplay between receptor usage, membrane fusion, and selection of entry pathway.

Results

Inhibitors of endocytosis and endosome acidification blocked ANG path entry into CHO cells expressing nectin-1 receptors, but not CHO-nectin-2 cells. Thus, under these conditions, nectin-2 mediates pH-independent entry at the plasma membrane. In addition, CHO-nectin-2 cells supported pH-dependent, endocytic entry of different strains of HSV-1, including rid1 and HFEM. The kinetics of ANG path entry was rapid (t_1/2 of 5–10 min) regardless of entry route. However, HSV-1 ANG path entry by fusion with the CHO-nectin-2 cell plasma membrane was more efficient and resulted in larger syncytia. ANG path virions added to the surface of CHO-nectin-2 cells, but not receptor-negative CHO cells or CHO-nectin-1 cells, induced rapid FFWO.

Conclusion

HSV-1 ANG path can enter CHO cells by either endocytic or non-endocytic pathways depending on whether nectin-1 or nectin-2 is present. In addition to these cellular receptors, one or more viral determinants is important for the selection of entry pathway. HSV-induced FFWO depends on the presence of an appropriate gD-receptor in the target membrane. Nectin-1 and nectin-2 target ANG path to divergent cellular pathways, and these receptors may have different roles in triggering viral membrane fusion.     

Herpes simplex virus nucleocapsids mature to progeny virions by an envelopment --> deenvelopment --> reenvelopment pathway

Herpes simplex virus (HSV) nucleocapsids acquire an envelope by budding through the inner nuclear membrane, but it is uncertain whether this envelope is retained during virus maturation and egress or whether mature progeny virions are derived by deenvelopment at the outer nuclear membrane followed by reenvelopment in a cytoplasmic compartment. To resolve this issue, we used immunogold electron microscopy to examine the distribution of glycoprotein D (gD) in cells infected with HSV-1 encoding a wild-type gD or a gD which is retrieved to the endoplasmic reticulum (ER). In cells infected with wild-type HSV-1, extracellular virions and virions in the perinuclear space bound approximately equal amounts of gD antibody. In cells infected with HSV-1 encoding an ER-retrieved gD, the inner and outer nuclear membranes were heavily gold labeled, as were perinuclear enveloped virions. Extracellular virions exhibited very little gold decoration (10- to 30-fold less than perinuclear virions). We conclude that the envelope of perinuclear virions must be lost during maturation and egress and that mature progeny virions must acquire an envelope from a post-ER cytoplasmic compartment. We noted also that gD appears to be excluded from the plasma membrane in cells infected with wild-type virus.     

Herpes simplex virus glycoproteins gD and gE/gI serve essential but redundant functions during acquisition of the virion envelope in the cytoplasm

The late stages of assembly of herpes simplex virus (HSV) and other herpesviruses are not well understood. Acquisition of the final virion envelope apparently involves interactions between viral nucleocapsids coated with tegument proteins and the cytoplasmic domains of membrane glycoproteins. This promotes budding of virus particles into cytoplasmic vesicles derived from the trans-Golgi network or endosomes. The identities of viral membrane glycoproteins and tegument proteins involved in these processes are not well known. Here, we report that HSV mutants lacking two viral glycoproteins, gD and gE, accumulated large numbers of unenveloped nucleocapsids in the cytoplasm. These aggregated capsids were immersed in an electron-dense layer that appeared to be tegument. Few or no enveloped virions were observed. More subtle defects were observed with an HSV unable to express gD and gI. A triple mutant lacking gD, gE, and gI exhibited more severe defects in envelopment. We concluded that HSV gD and the gE/gI heterodimeric complex act in a redundant fashion to anchor the virion envelope onto tegument-coated capsids. In the absence of either one of these HSV glycoproteins, envelopment proceeds; however, without both gD and gE, or gE/gI, there is profound inhibition of cytoplasmic envelopment.     

Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM

HVEM (for herpesvirus entry mediator) is a member of the tumor necrosis factor receptor superfamily and mediates entry of many strains of herpes simplex virus (HSV) into normally nonpermissive Chinese hamster ovary (CHO) cells. We used sucrose density centrifugation to demonstrate that purified HSV-1 KOS virions bind directly to a soluble, truncated form of HVEM (HVEMt) in the absence of any other cell-associated components. Therefore, HVEM mediates HSV entry by serving as a receptor for the virus. We previously showed that soluble, truncated forms of HSV glycoprotein D (gDt) bind to HVEMt in vitro. Here we show that antibodies specific for gD, but not the other entry glycoproteins gB, gC, or the gH/gL complex, completely block HSV binding to HVEM. Thus, virion gD is the principal mediator of HSV binding to HVEM. To map sites on virion gD which are necessary for its interaction with HVEM, we preincubated virions with gD-specific monoclonal antibodies (MAbs). MAbs that recognize antigenic sites Ib and VII of gD were the only MAbs which blocked the HSV-HVEM interaction. MAbs from these two groups failed to coprecipitate HVEMt in the presence of soluble gDt, whereas the other anti-gD MAbs coprecipitated HVEMt and gDt. Previous mapping data indicated that site VII includes amino acids 11 to 19 and site Ib includes 222 to 252. The current experiments indicate that these sites contain residues important for HSV binding to HVEM. Group Ib and VII MAbs also blocked HSV entry into HVEM-expressing CHO cells. These results suggest that the mechanism of neutralization by these MAbs is via interference with the interaction between gD in the virus and HVEM on the cell. Group Ia and II MAbs failed to block HSV binding to HVEM yet still neutralized HVEM-mediated entry, suggesting that these MAbs block entry at a step other than HVEM binding.     

Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors.

Herpes simplex virus (HSV) glycoprotein D (gD) plays an essential role in the entry of virus into cells. HSV mutants unable to express gD were constructed. The mutants can be propagated on VD60 cells, which supply the viruses with gD; however, virus particles lacking gD were produced in mutant-infected Vero cells. Virus particles with or without gD adsorbed to a large number (greater than 4 x 10(4] of sites on the cell surface; however, virions lacking gD did not enter cells. Cells pretreated with UV-inactivated virions containing gD (approximately 5 x 10(3) particles per cell) were resistant to infection with HSV type 1 (HSV-1) and HSV-2. In contrast, cells pretreated with UV-inactivated virions lacking gD could be infected with HSV-1 and HSV-2. If infectious HSV-1 was added prior to UV-inactivated virus particles containing gD, the infectious virus entered cells and replicated. Therefore, virus particles containing gD appear to block specific cell surface receptors which are very limited in number. Particles lacking gD are presumably unable to interact with these receptors, suggesting that gD is an essential receptor-binding polypeptide.     

Vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts

Vaccinia virus infects a wide variety of mammalian cells from different hosts, but the mechanism of virus entry is not clearly defined. The mature intracellular vaccinia virus contains several envelope proteins mediating virion adsorption to cell surface glycosaminoglycans; however, it is not known how the bound virions initiate virion penetration into cells. For this study, we investigated the importance of plasma membrane lipid rafts in the mature intracellular vaccinia virus infection process by using biochemical and fluorescence imaging techniques. A raft-disrupting drug, methyl-beta-cyclodextrin, inhibited vaccinia virus uncoating without affecting virion attachment, indicating that cholesterol-containing lipid rafts are essential for virion penetration into mammalian cells. To provide direct evidence of a virus and lipid raft association, we isolated detergent-insoluble glycolipid-enriched membranes from cells immediately after virus infection and demonstrated that several viral envelope proteins, A14, A17L, and D8L, were present in the cell membrane lipid raft fractions, whereas the envelope H3L protein was not. Such an association did not occur after virions attached to cells at 4 degrees C and was only observed when virion penetration occurred at 37 degrees C. Immunofluorescence microscopy also revealed that cell surface staining of viral envelope proteins was colocalized with GM1, a lipid raft marker on the plasma membrane, consistent with biochemical analyses. Finally, mutant viruses lacking the H3L, D8L, or A27L protein remained associated with lipid rafts, indicating that the initial attachment of vaccinia virions through glycosaminoglycans is not required for lipid raft formation.     

A novel role for phagocytosis-like uptake in herpes simplex virus entry

It is becoming increasingly clear that herpesviruses can exploit the endocytic pathway to infect cells, yet several important features of this process remain poorly defined. Using herpes simplex virus-1 (HSV-1) as a model, we demonstrate that endocytosis of the virions mimic many features of phagocytosis. During entry, HSV-1 virions associated with plasma membrane protrusions followed by a phagocytosis-like uptake involving rearrangement of actin cytoskeleton and trafficking of the virions in large phagosome-like vesicles. RhoA GTPase was activated during this process and the mode of entry was cell type-specific. Clathrin-coated vesicles had no detectable role in virion trafficking as Eps15 dominant-negative mutants failed to affect HSV-1 uptake. Binding and fusion of the virion envelope with the phagosomal membrane is likely facilitated by clustering of nectin-1 (or HVEM) in phagosomes, which was observed in infected cells. Collectively, our data suggests a novel mode of uptake by which the virus can infect both professional and nonprofessional phagocytes.     

Effects of herpes simplex virus on structure and function of nectin-1/HveC

Herpes simplex virus (HSV) entry requires the interaction between the envelope glycoprotein D (gD) and a cellular receptor such as nectin-1 (also named herpesvirus entry mediator C [HveC]) or HveA/HVEM. Nectin-1 is a cell adhesion molecule found at adherens junctions associated with the cytoplasmic actin-binding protein afadin. Nectin-1 can act as its own ligand in a homotypic interaction to bridge cells together. We used a cell aggregation assay to map an adhesive functional site on nectin-1 and identify the effects of gD binding and HSV early infection on nectin-1 function. Soluble forms of nectin-1 and anti-nectin-1 monoclonal antibodies were used to map a functional adhesive site within the first immunoglobulin-like domain (V domain) of nectin-1. This domain also contains the gD-binding site, which appeared to overlap the adhesive site. Thus, soluble forms of gD were able to prevent nectin-1-mediated cell aggregation and to disrupt cell clumps in an affinity-dependent manner. HSV also prevented nectin-1-mediated cell aggregation by occupying the receptor. Early in infection, nectin-1 was not downregulated from the cell surface. Rather, detection of nectin-1 changed gradually over a 30-min period of infection, as reflected by a decrease in the CK41 epitope and an increase in the CK35 epitope. The level of detection of virion gD on the cell surface increased within 5 min of infection in a receptor-dependent manner. These observations suggest that cell surface nectin-1 and gD may undergo conformational changes during HSV entry as part of an evolving interaction between the viral envelope and the cell plasma membrane.     

Glycoprotein D of herpes simplex virus (HSV) binds directly to HVEM, a member of the tumor necrosis factor receptor superfamily and a mediator of HSV entry.

Glycoprotein D (gD) is a structural component of the herpes simplex virus (HSV) envelope which is essential for virus entry into host cells. Chinese hamster ovary (CHO-K1) cells are one of the few cell types which are nonpermissive for the entry of many HSV strains. However, when these cells are transformed with the gene for the herpesvirus entry mediator (HVEM), the resulting cells, CHO-HVEM12, are permissive for many HSV strains, such as HSV-1(KOS). By virtue of its four cysteine-rich pseudorepeats, HVEM is a member of the tumor necrosis factor receptor superfamily of proteins. Recombinant forms of gD and HVEM, gD-1(306t) and HVEM(200t), respectively, were used to demonstrate a specific physical interaction between these two proteins. This interaction was dependent on native gD conformation but independent of its N-linked oligosaccharides, as expected from previous structure-function studies. Recombinant forms of gD derived from HSV-1(KOS)rid1 and HSV-1(ANG) did not bind to HVEM(200t), explaining the inability of these viruses to infect CHO-HVEM12 cells. A variant gD protein, gD-1(delta290-299t), showed enhanced binding to HVEM(200t) relative to the binding of gD-1(306t). Competition studies showed that gD-1(delta290-299t) and gD-1(306t) bound to the same region of HVEM(200t), suggesting that the differences in binding to HVEM are due to differences in affinity. These differences were also reflected in the ability of gD-1(delta290-299t) but not gD-1(306t) to block HSV type 1 infection of CHO-HVEM12 cells. By gel filtration chromatography, the complex between gD-1(delta290-299t) and HVEM(200t) had a molecular mass of 113 kDa and a molar ratio of 1:2. We conclude that HVEM interacts directly with gD, suggesting that HVEM is a receptor for virion gD and that the interaction between these proteins is a step in HSV entry into HVEM-expressing cells.     

Cross-linking of glycoprotein oligomers during herpes simplex virus type 1 entry.

Herpes simplex virus (HSV) has 10 glycoproteins in its envelope. Glycoprotein B (gB), gC, gD, gH, and gL have been implicated in virus entry. We previously used chemical cross-linking to show that these five glycoproteins were close enough to each other to be cross-linked into homodimeric and hetero-oligomeric forms; hetero-oligomers of gB-gC, gC-gD, gD-gB, gH-gL, gC-gL and gD-gL were found in purified virions. To better understand the roles of these glycoproteins in viral entry, we have modified a standard HSV penetration assay to include cross-linkers. This allowed us to examine changes in associations of viral glycoproteins during the entry process. HSV-1(KOS) was adsorbed at 4 degrees C to human neuroblastoma cells (SY5Y). The temperature was raised to 37 degrees C and cells were treated with cross-linker at various times after the temperature shift. Cytoplasmic extracts were examined by Western blotting (immunoblotting) for viral glycoproteins. We found that (i) as in virus alone, the length and concentration of the cross-linking agent affected the number of specific complexes isolated; (ii) the same glycoprotein patterns found in purified virions were also present after attachment of virions to cells; and (iii) the ability to cross-link HSV glycoproteins changed as virus penetration proceeded, e.g., gB and gD complexes which were present during attachment disappeared with increasing time, and their disappearance paralleled the kinetics of penetration. However, this phenomenon appeared to be selective since it was not observed with gC oligomers. In addition, we examined the cross-linking patterns of gB and gD in null viruses K082 and KOSgD beta. Neither of these mutants, which attach but cannot penetrate, showed changes in glycoprotein cross-linking over time. We speculate that these changes are due to conformational changes which preclude cross-linking or spatial alterations which dissociate the glycoprotein interactions during the penetration events.     

Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry

Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves upon receptor binding. Similarly, a point mutant that would destabilize the C-terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C-terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion.     

A herpes simplex virus mutant in which glycoprotein D sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells.

Herpes simplex virus (HSV) glycoprotein gD is a major component of the virion envelope and is thought to play an important role in the initial stages of viral infection and stimulates the production of high titers of neutralizing antibodies. We assumed that gD plays an essential role in virus replication, and so to complement viruses with mutations in the gD gene we constructed a cell line, denoted VD60, which is capable of expressing high levels of gD after infection with HSV. A recombinant virus, designated F-gD beta, in which sequences encoding gD and a nonessential glycoprotein, gI, were replaced by Escherichia coli beta-galactosidase sequences, was selected on the basis that it produced blue plaques on VD60 cell monolayers under agarose overlays containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). F-gD beta was able to replicate normally on complementing VD60 cells. However, F-gD beta was unable to form plaques on noncomplementing Vero cells. Virions lacking gD were produced in normal amounts by Vero cells infected with F-gD beta, and the virus particles were distributed throughout the cytoplasm and on the cell surface, suggesting that gD is not essential for HSV envelopment and egress. Virions lacking gD were able to bind to cells, but were unable to initiate synthesis of viral early polypeptides. Plaque production of F-gD beta particles lacking gD was enhanced by polyethylene glycol treatment, suggesting that gD is essential for penetration of HSV into cells. Other HSV glycoproteins have been implicated in the entry of virus into cells, and thus this process appears to involve multiple interactions at the cell surface.     

Herpes simplex virus: receptors and ligands for cell entry

Entry of herpes simplex virus (HSV) into cells depends upon multiple cell surface receptors and multiple proteins on the surface of the virion. The cell surface receptors include heparan sulphate chains on cell surface proteoglycans, a member of the tumor necrosis factor (TNF) receptor family and two members of the immunoglobulin superfamily related to the poliovirus receptor. The HSV ligands for these receptors are the envelope glycoproteins gB and gC for heparan sulphate and gD for the protein receptors and specific sites in heparan sulphate generated by certain 3-O-sulfotransferases. HSV gC also binds to the C3b component of complement and can block complement-mediated neutralization of virus. The purposes of this review are to summarize available information about these cell surface receptors and the viral ligands, gC and gD, and to discuss roles of these viral glycoproteins in immune evasion and cellular responses as well as in viral entry.     

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.     

Herpes Simplex Virus Glycoprotein D Can Bind to Poliovirus Receptor-Related Protein 1 or Herpesvirus Entry Mediator, Two Structurally Unrelated Mediators of Virus Entry

Several cell membrane proteins have been identified as herpes simplex virus (HSV) entry mediators (Hve). HveA (formerly HVEM) is a member of the tumor necrosis factor receptor family, whereas the poliovirus receptor-related proteins 1 and 2 (PRR1 and PRR2, renamed HveC and HveB) belong to the immunoglobulin superfamily. Here we show that a truncated form of HveC directly binds to HSV glycoprotein D (gD) in solution and at the surface of virions. This interaction is dependent on the native conformation of gD but independent of its N-linked glycosylation. Complex formation between soluble gD and HveC appears to involve one or two gD molecules for one HveC protein. Since HveA also mediates HSV entry by interacting with gD, we compared both structurally unrelated receptors for their binding to gD. Analyses of several gD variants indicated that structure and accessibility of the N-terminal domain of gD, essential for HveA binding, was not necessary for HveC interaction. Mutations in functional regions II, III, and IV of gD had similar effects on binding to either HveC or HveA. Competition assays with neutralizing anti-gD monoclonal antibodies (MAbs) showed that MAbs from group Ib prevented HveC and HveA binding to virions. However, group Ia MAbs blocked HveC but not HveA binding, and conversely, group VII MAbs blocked HveA but not HveC binding. Thus, we propose that HSV entry can be mediated by two structurally unrelated gD receptors through related but not identical binding with gD.     

Equine herpesvirus 1 entry via endocytosis is facilitated by alphaV integrins and an RSD motif in glycoprotein D

Equine herpesvirus 1 (EHV-1) is a member of the Alphaherpesvirinae, and its broad tissue tropism suggests that EHV-1 may use multiple receptors to initiate virus entry. EHV-1 entry was thought to occur exclusively through fusion at the plasma membrane, but recently entry via the endocytic/phagocytic pathway was reported for Chinese hamster ovary cells (CHO-K1 cells). Here we show that cellular integrins, and more specifically those recognizing RGD motifs such as αVβ5, are important during the early steps of EHV-1 entry via endocytosis in CHO-K1 cells. Moreover, mutational analysis revealed that an RSD motif in the EHV-1 envelope glycoprotein D (gD) is critical for entry via endocytosis. In addition, we show that EHV-1 enters peripheral blood mononuclear cells predominantly via the endocytic pathway, whereas in equine endothelial cells entry occurs mainly via fusion at the plasma membrane. Taken together, the data in this study provide evidence that EHV-1 entry via endocytosis is triggered by the interaction between cellular integrins and the RSD motif present in gD and, moreover, that EHV-1 uses different cellular entry pathways to infect important target cell populations of its natural host.     

Cell surface proteoglycans are not essential for infection by pseudorabies virus.

Cell surface proteoglycans, in particular those carrying heparan sulfate glycosaminoglycans, play a major role in primary attachment of herpesviruses to target cells. In pseudorabies virus (PrV), glycoprotein gC has been shown to represent the major heparan sulfate-binding virion envelope protein (T. C. Mettenleiter, L. Zsak, F. Zuckermann, N. Sugg, H. Kern, and T. Ben-Porat, J. Virol. 64:278-286, 1990). Since PrV gC is nonessential for viral infectivity in vitro and in vivo, either the interaction between virion envelope and cellular heparan sulfate is not necessary to mediate infection or other virion envelope proteins can substitute as heparan sulfate-binding components in the absence of gC. To answer these questions, we analyzed the infectivity of isogenic gC+ and gC- PrV on mouse L-cell derivatives with defects in glycosaminoglycan biosynthesis, using a rapid and sensitive fluorescence-based beta-galactosidase assay and single-cell counting in a fluorescence-activated cell sorter. Our data show that (i) in the virion, glycoprotein gC represents the only proteoglycan-binding envelope protein, and (ii) cellular proteoglycans are not essential for infectivity of PrV. Attachment studies using radiolabeled virions lacking either gC or the essential gD confirmed these results and demonstrated that PrV gD mainly contributes to binding of Pr virions to cell surface components other than proteoglycans. These data demonstrate the presence of a proteoglycan-independent mode of attachment for Pr virions leading to infectious entry into target cells.     

Site-specific proteolytic cleavage of the amino terminus of herpes simplex virus glycoprotein K on virion particles inhibits virus entry

Herpes simplex virus 1 (HSV-1) glycoprotein K (gK) is expressed on virions and functions in entry, inasmuch as HSV-1(KOS) virions devoid of gK enter cells substantially slower than is the case for the parental KOS virus (T. P. Foster, G. V. Rybachuk, and K. G. Kousoulas, J. Virol. 75:12431-12438, 2001). Deletion of the amino-terminal 68-amino-acid (aa) portion of gK caused a reduction in efficiency and kinetics of virus entry similar to that of the gK-null virus in comparison to the HSV-1(F) parental virus. The UL20 membrane protein and gK were readily detected on double-gradient-purified virion preparations. Immuno-electron microscopy confirmed the presence of gK and UL20 on purified virions. Coimmunoprecipitation experiments using purified virions revealed that gK interacted with UL20, as has been shown in virus-infected cells (T. P. Foster, V. N. Chouljenko, and K. G. Kousoulas, J. Virol. 82:6310-6323, 2008). Scanning of the HSV-1(F) viral genome revealed the presence of a single putative tobacco etch virus (TEV) protease site within gD, while additional TEV predicted sites were found within the UL5 (helicase-primase helicase subunit), UL23 (thymidine kinase), UL25 (DNA packaging tegument protein), and UL52 (helicase-primase primase subunit) proteins. The recombinant virus gDΔTEV was engineered to eliminate the single predicted gD TEV protease site without appreciably affecting its replication characteristics. The mutant virus gK-V5-TEV was subsequently constructed by insertion of a gene sequence encoding a V5 epitope tag in frame with the TEV protease site immediately after gK amino acid 68. The gK-V5-TEV, R-gK-V5-TEV (revertant virus), and gDΔTEV viruses exhibited similar plaque morphologies and replication characteristics. Treatment of the gK-V5-TEV virions with TEV protease caused approximately 32 to 34% reduction of virus entry, while treatment of gDΔTEV virions caused slightly increased virus entry. These results provide direct evidence that the gK and UL20 proteins, which are genetically and functionally linked to gB-mediated virus-induced cell fusion, are structural components of virions and function in virus entry. Site-specific cleavage of viral glycoproteins on mature and fully infectious virions utilizing unique protease sites may serve as a generalizable method of uncoupling the roles of viral glycoproteins in virus entry and virion assembly.