Direct inactivation of viruses by human granulocyte defensins.

Human neutrophils contain a family of microbicidal peptides known as defensins. One of these defensins, human neutrophil peptide (HNP)-1, was purified, and its ability to directly inactivate several viruses was extensively tested. Herpes simplex virus (HSV) types 1 and 2, cytomegalovirus, vesicular stomatitis virus, and influenza virus A/WSN were inactivated by incubation with HNP-1. Two nonenveloped viruses, echovirus type 11 and reovirus type 3, were resistant to inactivation. Purified homologous peptides HNP-2 and HNP-3 were found to have HSV-1-neutralizing activities approximately equal to that of HNP-1. Inactivation of HSV-1 by HNP-1 depended on the time, temperature, and pH of incubation. Antiviral activity was abrogated by low temperature or prior reduction and alkylation of the defensins. Addition of serum or serum albumin to the incubation mixtures inhibited neutralization of HSV-1 by HNP-1. We used density gradient sedimentation techniques to demonstrate that HNP-1 bound to HSV-1 in a temperature-dependent manner. We speculate that binding of defensin peptides to certain viruses may impair their ability to infect cells.     

Theta defensins protect cells from infection by herpes simplex virus by inhibiting viral adhesion and entry

We tested the ability of 20 synthetic theta defensins to protect cells from infection by type 1 and type 2 herpes simplex viruses (HSV-1 and -2, respectively). The peptides included rhesus theta defensins (RTDs) 1 to 3, originally isolated from rhesus macaque leukocytes, and three peptides (retrocyclins 1 to 3) whose sequences were inferred from human theta-defensin (DEFT) pseudogenes. We also tested 14 retrocyclin analogues, including the retro, enantio, and retroenantio forms of retrocyclin 1. Retrocyclins 1 and 2 and RTD 3 protected cervical epithelial cells from infection by both HSV serotypes, but only retrocyclin 2 did so without causing cytotoxicity or requiring preincubation with the virus. Surface plasmon resonance studies revealed that retrocyclin 2 bound to immobilized HSV-2 glycoprotein B (gB2) with high affinity (K(d), 13.3 nM) and that it did not bind to enzymatically deglycosylated gB2. Temperature shift experiments indicated that retrocyclin 2 and human alpha defensins human neutrophil peptide 1 (HNP 1) to HNP 3 protected human cells from HSV-2 by different mechanisms. Retrocyclin 2 blocked viral attachment, and its addition during the binding or penetration phases of HSV-2 infection markedly diminished nuclear translocation of VP16 and expression of ICP4. In contrast, HNPs 1 to 3 had little effect on binding but reduced both VP16 transport and ICP4 expression if added during the postbinding (penetration) period. We recently reported that theta defensins are miniature lectins that bind gp120 of human immunodeficiency virus type 1 (HIV-1) with high affinity and inhibit the entry of R5 and X4 isolates of HIV-1. Given its small size (18 residues), minimal cytotoxicity, lack of activity against vaginal lactobacilli, and effectiveness against both HSV-2 and HIV-1, retrocyclin 2 provides an intriguing prototype for future topical microbicide development.     

Chemoattraction of macrophages, T lymphocytes, and mast cells is evolutionarily conserved within the human alpha-defensin family

Human defensins are natural peptide antibiotics. On the basis of the position and bonding of six conserved cysteine residues, they are divided into two families, designated alpha- and beta-defensins. Human alpha-defensins are expressed predominantly in neutrophils (human neutrophil peptides (HNP) 1-4) or intestinal Paneth cells (human defensins (HD) 5 and 6). Although alpha-defensins have been implicated in the pathogenesis of inflammatory bowel disease, their immunomodulatory functions are poorly understood. In the present study, HNP-1, HNP-3, and HD5 were found to be potent chemotaxins for macrophages but not dendritic cells using Galphai proteins and MAPK as signal transducers. Alpha-defensins were also chemoattractive for the human mast cell line HMC-1 but lacked, in contrast to beta-defensins, the ability to induce intracellular calcium fluxes. Furthermore, HNP-1, HNP-3, and HD5 comparably mobilized naive as well as memory T lymphocytes. Using the protein kinase C (PKC) inhibitors GF109 and G?6976, we observed a PKC-independent functional desensitization to occur between human alpha-defensins, which suggests a common receptor for HNP-1, HNP-3, and HD5 on immune cells. This alpha-defensin receptor was subject to heterologous desensitization by the PKC activator PMA and to PKC-dependent cross-desensitization by human beta-defensins. Conversely, alpha-defensins desensitized beta-defensin-mediated migration of immune cells in a PKC-dependent manner, suggesting unique receptors for both defensin families. Taken together, our observations indicate that chemoattraction of macrophages, T lymphocytes, and mast cells represents an immunomodulatory function which is evolutionarily conserved within the human alpha-defensin family and tightly regulated by beta-defensins.     

Herpes simplex virus glycoprotein B binds to cell surfaces independently of heparan sulfate and blocks virus entry

Virion glycoproteins gB, gD, and gH/gL play essential roles for herpes simplex virus (HSV) entry. The function of gD is to interact with a cognate receptor, and soluble forms of gD block HSV entry by tying up cell surface receptors. Both gB and the nonessential gC interact with cell surface heparan sulfate proteoglycan (HSPG), promoting viral attachment. However, cells deficient in proteoglycan synthesis can still be infected by HSV. This suggests another function for gB. We found that a soluble truncated form of gB bound saturably to the surface of Vero, A431, HeLa, and BSC-1 cells, L-cells, and a mouse melanoma cell line expressing the gD receptor nectin-1. The HSPG analog heparin completely blocked attachment of the gC ectodomain to Vero cells. In contrast, heparin only partially blocked attachment of soluble gB, leaving 20% of the input gB still bound even at high concentrations of inhibitor. Moreover, heparin treatment removed soluble gC but not gB from the cell surface. These data suggest that a portion of gB binds to cells independently of HSPG. In addition, gB bound to two HSPG-deficient cell lines derived from L-cells. Gro2C cells are deficient in HSPG, and Sog9 cells are deficient in HSPG, as well as chondroitin sulfate proteoglycan (CSPG). To identify particular gB epitopes responsible for HSPG-independent binding, we used a panel of monoclonal antibodies (MAbs) to gB to block gB binding. Only those gB MAbs that neutralized virus blocked binding of soluble gB to the cells. HSV entry into Gro2C and Sog9 cells was reduced but still detectable relative to the parental L-cells, as previously reported. Importantly, entry into Gro2C cells was blocked by purified forms of either the gD or gB ectodomain. On a molar basis, the extent of inhibition by gB was similar to that seen with gD. Together, these results suggest that soluble gB binds specifically to the surface of different cell types independently of HSPG and CSPG and that by doing so, the protein inhibits entry. The results provide evidence for the existence of a cellular entry receptor for gB.     

The importance of heparan sulfate in herpesvirus infection

Herpes simplex virus type-1 (HSV-1) is one of many pathogens that use the cell surface glycosaminoglycan heparan sulfate as a receptor.Heparan sulfate is highly expressed on the surface and extracellular matrix of virtually all cell types making it an ideal receptor.Heparan sulfate interacts with HSV-1 envelope glycoproteins gB and gC during the initial attachment step during HSV-1 entry.In addition,a modified form of heparan sulfate,known as 3-O-sulfated heparan sulfate,interacts with HSV-1 gD to induce fusion between the viral envelope and host cell membrane.The 3-O-sulfation of heparan sulfate is a rare modification which occurs during the biosynthesis of heparan sulfate that is carded out by a family of enzymes known as 3-O-sulfotransferases.Due to its involvement in multiple steps of the infection process,heparan sulfate has been a prime target for the development of agents to inhibit HSV entry.Understanding how heparan sulfate functions during HSV-1 infection may not only be critical for inhibiting infection by this virus,but it may also be crucial in the fight against many other pathogens as well.     

Human alpha-defensins inhibit BK virus infection by aggregating virions and blocking binding to host cells

BK virus (BKV) is a polyomavirus that establishes a lifelong persistence in most humans and is a major impediment to success of kidney grafts. The function of the innate immune system in BKV infection and pathology has not been investigated. Here we examine the role of antimicrobial defensins in BKV infection of Vero cells. Our data show that alpha-defensin human neutrophil protein 1 (HNP1) and human alpha-defensin 5 (HD5) inhibit BKV infection by targeting an early event in the viral lifecycle. HD5 treatment of BKV reduced viral attachment to cells, whereas cellular treatment with HD5 did not. Colocalization studies indicated that HD5 interacts directly with BKV. Ultrastructural analysis revealed HD5-induced aggregation of virions. HD5 also inhibited infection of cells by other related polyomaviruses. This is the first study to demonstrate polyomavirus sensitivity to defensins. We also show a novel mechanism whereby HD5 binds to BKV leading to aggregation of virion particles preventing normal virus binding to the cell surface and uptake into cells.     

Cyclic and Acyclic Defensins Inhibit Human Immunodeficiency Virus Type-1 Replication by Different Mechanisms

Defensins are antimicrobial peptides expressed by plants and animals. In mammals there are three subfamilies of defensins, distinguished by structural features: α, β and θ. Alpha and β-defensins are linear peptides with broad anti-microbial activity that are expressed by many mammals including humans. In contrast, θ-defensins are cyclic anti-microbial peptides made by several non-human primates but not humans. All three defensin types have anti-HIV-1 activity, but their mechanisms of action differ. We studied the anti-HIV-1 activity of one defensin from each group, HNP-1 (α), HBD-2 (β) and RTD-1 (θ). We examined how each defensin affected HIV-1 infection and demonstrated that the cyclic defensin RTD-1 inhibited HIV-1 entry, while acyclic HNP-1 and HBD-2 inhibited HIV-1 replication even when added 12 hours post-infection and blocked viral replication after HIV-1 cDNA formation. We further found that all three defensins downmodulated CXCR4. Moreover, RTD-1 inactivated X4 HIV-1, while HNP-1 and HBD-2 inactivated both X4 and R5 HIV-1. The data presented here show that acyclic and cyclic defensins block HIV-1 replication by shared and diverse mechanisms. Moreover, we found that HNP-1 and RTD-1 directly inhibited firefly luciferase enzymatic activity, which may affect the interpretation of previously published data.     

Specificity and affinity of binding of herpes simplex virus type 2 glycoprotein B to glycosaminoglycans.

Herpes simplex virus type 2 (HSV-2) interacts with cell surface glycosaminoglycans during virus attachment. Glycoprotein B of HSV-2 can potentially mediate the interaction between the virion and cell surface glycosaminoglycans. To determine the specificity, kinetics, and affinity of these interactions, we used plasmon resonance-based biosensor technology to measure HSV-2 glycoprotein binding to glycosaminoglycans in real time. The recombinant soluble ectodomain of HSV-2 gB (gB2) but not the soluble ectodomain of HSV-2 gD bound readily to biosensor surfaces coated with heparin. The affinity constants (Kds) were determined for gB2 (Kd = 7.7 x 10(-7) M) and for gB2 deltaTM (Kd = 9.9 x 10(-7) M), a recombinant soluble form of HSV-2 gB in which only its transmembrane domain has been deleted. gB2 binding to the heparin surface was competitively inhibited by low concentrations of heparin (50% effective dose [ED50] = 0.08 microg/ml). Heparan sulfate and dermatan sulfate glycosaminoglycans have each been suggested as cell surface receptors for HSV. Our biosensor analyses showed that both heparan sulfate and dermatan sulfate inhibited gB2 binding (ED50 = 1 to 5 microg/ml), indicating that gB2 interacts with both heparin-like and dermatan sulfate glycosaminoglycans. Chondroitin sulfate A, in contrast, inhibited gB2 binding to heparin only at high levels (ED50 = 65 microg/ml). The affinity and specificity of gB2 binding to glycosaminoglycans demonstrated in these studies support its role in the initial binding of HSV-2 to cells bearing heparan sulfate or dermatan sulfate glycosaminoglycans.     

Crystal structures of human alpha-defensins HNP4, HD5, and HD6

Six alpha-defensins have been found in humans. These small arginine-rich peptides play important roles in various processes related to host defense, being the effectors and regulators of innate immunity as well as enhancers of adoptive immune responses. Four defensins, called neutrophil peptides 1 through 4, are stored primarily in polymorphonuclear leukocytes. Major sites of expression of defensins 5 and 6 are Paneth cells of human small intestine. So far, only one structure of human alpha-defensin (HNP3) has been reported, and the properties of the intestine defensins 5 and 6 are particularly poorly understood. In this report, we present the high-resolution X-ray structures of three human defensins, 4 through 6, supplemented with studies of their antimicrobial and chemotactic properties. Despite only modest amino acid sequence identity, all three defensins share their tertiary structures with other known alpha- and beta-defensins. Like HNP3 but in contrast to murine or rabbit alpha-defensins, human defensins 4-6 form characteristic dimers. Whereas antimicrobial and chemotactic activity of HNP4 is somewhat comparable to that of other human neutrophil defensins, neither of the intestinal defensins appears to be chemotactic, and for HD6 also an antimicrobial activity has yet to be observed. The unusual biological inactivity of HD6 may be associated with its structural properties, somewhat standing out when compared with other human alpha-defensins. The strongest cationic properties and unique distribution of charged residues on the molecular surface of HD5 may be associated with its highest bactericidal activity among human alpha-defensins.     

A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry.

Herpes simplex virus type 1 (HSV-1) binds to cells through interactions of viral glycoproteins gB and gC with heparan sulfate chains on cell surface proteoglycans. This binding is not sufficient for viral entry, which requires fusion between the viral envelope and cell membrane. Here, we show that heparan sulfate modified by a subset of the multiple D-glucosaminyl 3-O-sulfotransferase isoforms provides sites for the binding of a third viral glycoprotein, gD, and for initiation of HSV-1 entry. We conclude that susceptibility of cells to HSV-1 entry depends on (1) presence of heparan sulfate chains to which virus can bind and (2) 3-O-sulfation of specific glucosamine residues in heparan sulfate to generate gD-binding sites or the expression of other previously identified gD-binding receptors.     

Neisseria gonorrhoeae-induced human defensins 5 and 6 increase HIV infectivity: role in enhanced transmission

Sexually transmitted infections (STIs) increase the likelihood of HIV transmission. Defensins are part of the innate mucosal immune response to STIs and therefore we investigated their role in HIV infection. We found that human defensins 5 and 6 (HD5 and HD6) promoted HIV infection, and this effect was primarily during viral entry. Enhancement was seen with primary viral isolates in primary CD4(+) T cells and the effect was more pronounced with R5 virus compared with X4 virus. HD5 and HD6 promoted HIV reporter viruses pseudotyped with vesicular stomatitis virus and murine leukemia virus envelopes, indicating that defensin-mediated enhancement was not dependent on CD4 and coreceptors. Enhancement of HIV by HD5 and HD6 was influenced by the structure of the peptides, as loss of the intramolecular cysteine bonds was associated with loss of the HIV-enhancing effect. Pro-HD5, the precursor and intracellular form of HD5, also exhibited HIV-enhancing effect. Using a cervicovaginal tissue culture system, we found that expression of HD5 and HD6 was induced in response to Neisseria gonorrhoeae (GC, for gonococcus) infection and that conditioned medium from GC-exposed cervicovaginal epithelial cells with elevated levels of HD5 also enhanced HIV infection. Introduction of small interfering RNAs for HD5 or HD6 abolished the HIV-enhancing effect mediated by GC. Thus, the induction of these defensins in the mucosa in the setting of GC infection could facilitate HIV infection. Furthermore, this study demonstrates the complexity of defensins as innate immune mediators in HIV transmission and warrants further investigation of the mechanism by which defensins modulate HIV infection.     

Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans

The role of cell surface heparan sulfate in herpes simplex virus (HSV) infection was investigated using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Binding of radiolabeled virus to the cells and infection were assessed in mutant and wild-type cells. Virus bound efficiently to wild-type cells and initiated an abortive infection in which immediate-early or alpha viral genes were expressed, despite limited production of late viral proteins and progeny virus. Binding of virus to heparan sulfate-deficient mutant cells was severely impaired and mutant cells were resistant to HSV infection. Intermediate levels of binding and infection were observed for a CHO cell mutant that produced undersulfated heparan sulfate. These results show that heparan sulfate moieties of cell surface proteoglycans serve as receptors for HSV.     

Bovine herpesvirus 1 glycoprotein B does not productively interact with cell surface heparan sulfate in a pseudorabies virion background.

Attachment to cell surface heparan sulfate proteoglycans is the first step in infection by several alphaherpesviruses. This interaction is primarily mediated by virion glycoprotein C (gC). In herpes simplex virus, in the absence of the nonessential gC, heparan sulfate binding is effected by glycoprotein B. In contrast, gC-negative pseudorabies virus (PrV) infects target cells via a heparan sulfate-independent mechanism, indicating that PrV virion gB does not productively interact with heparan sulfate. To assay whether a heterologous alphaherpesvirus gB protein will confer productive heparan sulfate binding on gC-negative PrV, gC was deleted from an infectious PrV recombinant, PrV-9112C2, which expresses bovine herpesvirus 1 (BHV-1) gB instead of PrV gB. Our data show that gC-negative PrV-BHV-1 gB recombinant 9112C2-delta gCbeta was not inhibited in infection by soluble heparin, in contrast to the gC-positive parental strain. Similar results were obtained when wild-type BHV-1 was compared with a gC-negative BHV-1 mutant. Moreover, infection of cells proficient or deficient in heparan sulfate biosynthesis occurred with equal efficiency by PrV-9112C2-delta gCbeta, whereas heparan sulfate-positive cells showed an approximately fivefold higher plating efficiency than heparan sulfate-negative cells with the parental gC-positive virus. In summary, our data show that in a PrV gC-negative virion background, BHV-1 gB is not able to mediate infection by productive interaction with heparan sulfate, and they indicate the same lack of heparin interaction for BHV-1 gB in gC-negative BHV-1.     

Toward understanding the cationicity of defensins. Arg and Lys versus their noncoded analogs

Human defensins are a family of small antimicrobial proteins found predominantly in leukocytes and epithelial cells that play important roles in the innate and adaptive immune defense against microbial infection. The most distinct molecular feature of defensins is cationicity, manifested by abundant Arg and/or Lys residues in their sequences. Sequence analysis indicates that Arg is strongly selected over Lys in alpha-defensins but not in beta-defensins. To understand this Arg/Lys disparity in defensins, we chemically synthesized human alpha-defensin 1 (HNP1) and several HNP1 analogs where three Arg residues were replaced by each of the following six alpha-amino acids: Lys, ornithine (Orn), diaminobutyric acid (Dab), diaminopropionic acid (Dap), N,N-dimethyl-Lys ((diMe)Lys), and homo-Arg ((homo)Arg). In addition, we prepared human beta-defensin 1 (hBD1) and (Lys-->Arg)hBD1 in which all four Lys residues were substituted for Arg. Bactericidal activity assays revealed the following. 1) Arg-containing HNP1 and (Lys-->Arg)hBD1 are functionally better than Lys-HNP1 and hBD1, respectively; the difference between Arg and Lys is more evident in the alpha-defensin than in the beta-defensin and is more evident at low salt concentrations than at high salt concentrations. 2) For HNP1, the Arg/Lys disparity is much more pronounced with Staphylococcus aureus than with Escherichia coli, and the Arg-rich HNP1 kills bacteria faster than its Lys-rich analog. 3) Arg and Lys appear to have optimal chain lengths for bacterial killing as shortening Lys or lengthening Arg in HNP1 invariably becomes functionally deleterious. Our findings provide insights into the Arg/Lys disparity in defensins, and shed light on the cationicity of defensins with respect to their antimicrobial activity and specificity.     

Delineation of Interfaces on Human Alpha-Defensins Critical for Human Adenovirus and Human Papillomavirus Inhibition

Human α-defensins are potent anti-microbial peptides with the ability to neutralize bacterial and viral targets. Single alanine mutagenesis has been used to identify determinants of anti-bacterial activity and binding to bacterial proteins such as anthrax lethal factor. Similar analyses of α-defensin interactions with non-enveloped viruses are limited. We used a comprehensive set of human α-defensin 5 (HD5) and human neutrophil peptide 1 (HNP1) alanine scan mutants in a combination of binding and neutralization assays with human adenovirus (AdV) and human papillomavirus (HPV). We have identified a core of critical hydrophobic residues that are common determinants for all of the virus-defensin interactions that were analyzed, while specificity in viral recognition is conferred by specific surface-exposed charged residues. The hydrophobic residues serve multiple roles in maintaining the tertiary and quaternary structure of the defensins as well as forming an interface for virus binding. Many of the important solvent-exposed residues of HD5 group together to form a critical surface. However, a single discrete binding face was not identified for HNP1. In lieu of whole AdV, we used a recombinant capsid subunit comprised of penton base and fiber in quantitative binding studies and determined that the anti-viral potency of HD5 was a function of stoichiometry rather than affinity. Our studies support a mechanism in which α-defensins depend on hydrophobic and charge-charge interactions to bind at high copy number to these non-enveloped viruses to neutralize infection and provide insight into properties that guide α-defensin anti-viral activity.     

Herpes simplex virus glycoprotein D bound to the human receptor HveA

Herpes simplex virus (HSV) infection requires binding of the viral envelope glycoprotein D (gD) to cell surface receptors. We report the X-ray structures of a soluble, truncated ectodomain of gD both alone and in complex with the ectodomain of its cellular receptor HveA. Two bound anions suggest possible binding sites for another gD receptor, a 3-O-sulfonated heparan sulfate. Unexpectedly, the structures reveal a V-like immunoglobulin (Ig) fold at the core of gD that is closely related to cellular adhesion molecules and flanked by large N- and C-terminal extensions. The receptor binding segment of gD, an N-terminal hairpin, appears conformationally flexible, suggesting that a conformational change accompanying binding might be part of the viral entry mechanism.     

Purification and characterization of human neutrophil peptide 4, a novel member of the defensin family

Primary (azurophil) granules of neutrophils contain proteins which play a major role in the killing and digestion of bacteria in the phagolysosome. We have isolated and characterized a novel antimicrobial peptide from the azurophil granule fraction of discontinuous Percoll gradients. We have named this peptide human neutrophil peptide 4 (HNP-4) based on its structural similarity to a group of antimicrobial polypeptides known as defensins (HNP 1-3). Using size exclusion and reverse-phase high performance liquid chromatography, HNP-4 was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. The amino acid sequence determined from isolated HNP-4 and from tryptic fragments of reduced and alkylated peptide is: NH2-Val-Cys-Ser-Cys-Arg-Leu-Val-Phe-Cys-Arg-Arg-Thr-Glu- Leu-Arg-Val-Gly-Asn-Cys-Leu-Ile-Gly-Gly-Val-Ser-Phe-Thr-Tyr-Cys-Cys-Thr- Arg-Val - COOH. Based on this sequence, HNP-4 has a calculated molecular weight of 3715 and a theoretical pI of 8.61. HNP-4 shows structural similarity to the family of three human defensins. HNP-4 and the defensins have identical cysteine backbones and, like the defensins, HNP-4 is rich in arginine (15.2 mol %). However, the amino acids at 22 of the 33 positions differ between HNP-4 and human defensins. Further, HNP-4 is significantly more hydrophobic than the defensins, as determined by its retention time on reverse-phase high performance liquid chromatography. In vitro, purified HNP-4 was shown to kill Escherichia coli, Streptococcus faecalis, and Candida albicans. Compared to a mixture of the other human defensins, HNP-4 was found to be approximately 100 times more potent against E. coli and four times more potent against both S. faecalis and C. albicans.     

Herpes simplex virus type 1 and pseudorabies virus bind to a common saturable receptor on Vero cells that is not heparan sulfate.

Herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) infect different natural hosts but are very similar in structure, replicative cycle, and entry into cultured cells. We determined whether HSV-1 and PRV use the same cellular components during entry into Vero cells, which are highly susceptible to each virus but are not from native hosts for either. UV-inactivated virions of either HSV-1 or PRV could saturate cell surfaces to block infection of challenge HSV-1 or PRV. In the presence of saturating levels for infection of either virus, radiolabeled virus bound well and in a heparin-sensitive manner. This result shows that heparan sulfate proteoglycans on Vero cells are not the limiting cellular component. To identify the virus component required for blocking, we used an HSV-1 null mutant virus lacking gB, gD, or gH as blocking virus. Virions lacking gB were able to block infection of challenge virus to the same level as did virus containing gB. In contrast, virions lacking gD lost all and most of the ability to block infection of HSV-1 and PRV, respectively. HSV-1 lacking gH and PRV lacking gp50 also were less competent in blocking infection of challenge virus. We conclude that HSV-1 and PRV bind to a common receptor for infection of Vero cells. Although both viruses bind a heparin-like cell component on many cells, including Vero cells, they also attach to a different and limited cell surface component that is bound at least by HSV-1 gD and possibly gH and to some degree by PRV gp50 but not gB. These results clearly demonstrate binding of both HSV-1 and PRV to a common cell receptor that is not heparan sulfate and demonstrate that several types of attachment occur for both viruses during infectious entry.