RESUMEN
The mechanism that herpesviruses use to enter cells is one of the most complex viral entry mechanisms studied so far. This complexity seems to mount as new participants, both cellular receptors and viral glycoproteins, are identified. Recent structural work on entry glycoproteins gD and gB from herpes simplex virus (HSV) 1 has illuminated the functional roles of these glycoproteins in the process of entry. In doing so, it provided information on the mechanism of two critical steps of HSV entry: receptor-mediated activation and membrane fusion. Remarkably, it is becoming clear that herpesviruses have a lot in common with other, simpler viruses.
Asunto(s)
Herpesviridae/fisiología , Proteínas del Envoltorio Viral , Internalización del Virus , Animales , Herpesviridae/clasificación , Humanos , Fusión de Membrana/fisiología , Modelos Moleculares , Estructura Terciaria de Proteína , Miembro 14 de Receptores del Factor de Necrosis Tumoral/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
The rapid spread of herpes simplex virus type 1 (HSV-1) in mucosal epithelia and neuronal tissue depends primarily on the ability of the virus to navigate within polarized cells and the tissues they constitute. To understand HSV entry and the spread of virus across cell junctions, we have previously characterized a human keratinocyte cell line, HaCaT. These cells appear to reflect cells infected in vivo more accurately than many of the cultured cells used to propagate HSV. HSV mutants lacking gE/gI are highly compromised in spread within epithelial and neuronal tissues and also show defects in cell-to-cell spread in HaCaT cells, but not in other, nonpolarized cells. HSV gD is normally considered absolutely essential for entry and cell-to-cell spread, both in cultured cells and in vivo. Here, an HSV-1 gD mutant virus, F-US6kan, was found to efficiently enter HaCaT cells and normal human keratinocytes and could spread from cell to cell without gD provided by complementing cells. By contrast, entry and spread into other cells, especially highly transformed cells commonly used to propagate HSV, were extremely inefficient. Further analyses of F-US6kan indicated that this mutant expressed extraordinarily low (1/500 wild-type) levels of gD. Neutralizing anti-gD monoclonal antibodies inhibited entry of F-US6kan, suggesting F-US6kan utilized this small amount of gD to enter cells. HaCaT cells expressed high levels of an HSV gD receptor, HveC, and entry of F-US6kan into HaCaT cells could also be inhibited with antibodies specific for HveC. Interestingly, anti-HveC antibodies were not fully able to inhibit entry of wild-type HSV-1 into HaCaT cells. These results help to uncover important properties of HSV and human keratinocytes. HSV, with exceedingly low levels of a crucial receptor-binding glycoprotein, can enter cells expressing high levels of receptor. In this case, surplus gD may be useful to avoid neutralization by anti-gD antibodies.
Asunto(s)
Queratinocitos/virología , Proteínas del Envoltorio Viral/fisiología , Línea Celular , Humanos , Receptores Virales/análisis , Receptores Virales/fisiología , Proteínas del Envoltorio Viral/análisisRESUMEN
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.
Asunto(s)
Iones/metabolismo , Receptores del Factor de Necrosis Tumoral/química , Receptores Virales/química , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Receptores del Factor de Necrosis Tumoral/metabolismo , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Receptores Virales/metabolismo , Alineación de Secuencia , Proteínas del Envoltorio Viral/metabolismoRESUMEN
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.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Inmunoglobulinas/metabolismo , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Bovinos , Moléculas de Adhesión Celular/genética , Membrana Celular/metabolismo , Cricetinae , Expresión Génica , Herpesvirus Bovino 1/metabolismo , Herpesvirus Bovino 1/fisiología , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/fisiología , Herpesvirus Suido 1/metabolismo , Herpesvirus Suido 1/fisiología , Herpesvirus Humano 2/metabolismo , Herpesvirus Humano 2/fisiología , Humanos , Inmunoglobulinas/genética , Datos de Secuencia Molecular , Nectinas , Plásmidos , Conformación Proteica , Receptores Virales/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
HSV-1 ICP34.5 mutants can slow progression of preformed tumors in rodent models. However, the current models available for study are limited due to the lack of a syngenic, low-immunogenic tumor model susceptible to HSV-1. Thus we have developed a new model to determine the role of the immune response in viral-mediated tumor destruction. The human herpesvirus entry (Hve) receptors (HveA, HveB, and HveC) and a control plasmid were transfected into B78H1 murine melanoma cells. Transfection of HveA and HveC conferred sensitivity to HSV-1 to these cells. A10 (HveA), C10 (HveC), and control cells were able to form tumors reproducibly in vivo. The transfection of the receptors into B78H1 cells did not induce a detectable in vivo immunogenicity to the tumors. Finally, A10 and C10 tumor-bearing mice treated with HSV-1 1716 had significant prolongation of survival compared to mock-treated mice. These data suggest that A10 and C10 will be useful as in vivo models for studying the role of the immune response in viral-mediated tumor destruction.
Asunto(s)
Herpesvirus Humano 1/genética , Melanoma Experimental/genética , Melanoma Experimental/terapia , Animales , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Plásmidos/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Receptores Virales/genética , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , Células VeroRESUMEN
Human herpesvirus entry mediator C (HveC) is an alphaherpesvirus receptor which binds to virion glycoprotein D (gD). We identified porcine HveC and studied its interaction with pseudorabies virus (PrV) and herpes simplex virus type 1 (HSV-1) gD. Porcine and human HveC have 96% amino acid identity and HveC from African green monkey, mouse, hamster, and cow are similarly conserved. Porcine HveC mediates entry of HSV-1, HSV-2, PrV, and bovine herpesvirus type 1. Truncated soluble forms of HSV-1 and PrV gD bind competitively to porcine HveC. Biosensor analysis shows that PrV gD binds with a 10-fold higher affinity than HSV-1 gD. Monoclonal antibodies against human HveC recognize the porcine homologue and can block gD binding and entry of HSV-1 and PrV. Porcine HveC is functionally indistinguishable from human HveC. Our results are consistent with the suggestion that HveC is a pan-alphaherpesvirus receptor that interacts with a conserved structural domain of gD.
Asunto(s)
Alphaherpesvirinae/fisiología , Moléculas de Adhesión Celular/metabolismo , Receptores Virales/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Unión Competitiva , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Herpesvirus Suido 1/fisiología , Humanos , Datos de Secuencia Molecular , Nectinas , Unión Proteica , Receptores Virales/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Porcinos , Células Vero , Proteínas del Envoltorio Viral/metabolismoRESUMEN
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.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Herpesvirus Bovino 1/metabolismo , Herpesvirus Humano 1/metabolismo , Herpesvirus Suido 1/metabolismo , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/metabolismo , Sitios de Unión , Unión Competitiva , Células CHO , Bovinos , Línea Celular , Cricetinae , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Nectinas , Solubilidad , Spodoptera/citología , Porcinos , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/genética , Proteínas Virales/biosíntesis , Proteínas Virales/genéticaRESUMEN
During virus entry, herpes simplex virus (HSV) glycoprotein D (gD) binds to one of several human cellular receptors. One of these, herpesvirus entry mediator A (HveA), is a member of the tumor necrosis factor receptor (TNFR) superfamily, and its ectodomain contains four characteristic cysteine-rich pseudorepeat (CRP) elements. We previously showed that gD binds the ectodomain of HveA expressed as a truncated, soluble protein [HveA(200t)]. To localize the gD-binding domain of HveA, we expressed three additional soluble forms of HveA consisting of the first CRP [HveA(76t)], the second CRP [HveA(77-120t)], or the first and second CRPs [HveA(120t)]. Biosensor and enzyme-linked immunosorbent assay studies showed that gD bound to HveA(120t) and HveA(200t) with the same affinity. However, gD did not bind to HveA(76t) or HveA(77-120t). Furthermore, HveA(200t) and HveA(120t), but not HveA(76t) or HveA(77-120t), blocked herpes simplex virus (HSV) entry into CHO cells expressing HveA. We also generated six monoclonal antibodies (MAbs) against HveA(200t). MAbs CW1, -2, and -4 bound linear epitopes within the second CRP, while CW7 and -8 bound linear epitopes within the third or fourth CRPs. None of these MAbs blocked the binding of gD to HveA. In contrast, MAb CW3 recognized a discontinuous epitope within the first CRP of HveA, blocked the binding of gD to HveA, and exhibited a limited ability to block virus entry into cells expressing HveA, suggesting that the first domain of HveA contains at least a portion of the gD binding site. The inability of gD to bind HveA(76t) suggests that additional amino acid residues of the gD binding site may reside within the second CRP.
Asunto(s)
Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Sitios de Unión , Técnicas Biosensibles , Células CHO , Chlorocebus aethiops , Cricetinae , Mapeo Epitopo , Glicosilación , Células HeLa , Humanos , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Proteínas Recombinantes/metabolismo , Células VeroRESUMEN
The human herpesvirus entry mediator C (HveC), also known as the poliovirus receptor-related protein 1 (PRR1) and as nectin-1, allows the entry of herpes simplex virus type 1 (HSV-1) and HSV-2 into mammalian cells. The interaction of virus envelope glycoprotein D (gD) with such a receptor is an essential step in the process leading to membrane fusion. HveC is a member of the immunoglobulin (Ig) superfamily and contains three Ig-like domains in its extracellular portion. The gD binding site is located within the first Ig-like domain (V domain) of HveC. We generated a panel of monoclonal antibodies (MAbs) against the ectodomain of HveC. Eleven of these, which detect linear or conformational epitopes within the V domain, were used to map a gD binding site. They allowed the detection of HveC by enzyme-linked immunosorbent assay, Western blotting, and biosensor analysis or directly on the surface of HeLa cells and human neuroblastoma cell lines, as well as simian Vero cells. The anti-HveC V-domain MAbs CK6, CK8, and CK41, as well as the previously described MAb R1.302, blocked HSV entry. Their binding to soluble HveC was blocked by the association of gD with the receptor, indicating that their epitopes overlap a gD binding site. Competition assays on an optical biosensor showed that CK6 and CK8 (linear epitopes) inhibited the binding of CK41 and R1.302 (conformational epitopes) to HveC and vice versa. Epitope mapping showed that CK6 and CK8 bound between residues 80 and 104 of HveC, suggesting that part of the gD binding site colocalizes in the same region.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Moléculas de Adhesión Celular/análisis , Receptores Virales/análisis , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Técnicas Biosensibles , Moléculas de Adhesión Celular/inmunología , Mapeo Epitopo , Humanos , Datos de Secuencia Molecular , Nectinas , Células Tumorales CultivadasRESUMEN
Herpes simplex virus (HSV) entry is dependent on the interaction of virion glycoprotein D (gD) with one of several cellular receptors. We previously showed that gD binds specifically to two structurally dissimilar receptors, HveA and HveC. We have continued our studies by using (i) a panel of baculovirus-produced gD molecules with various C-terminal truncations and (ii) a series of gD mutants with nonoverlapping 3-amino-acid deletions between residues 222 and 254. Binding of the potent neutralizing monoclonal antibody (MAb) DL11 (group Ib) was unaffected in forms of gD containing residues 1 to 250 but was greatly diminished in molecules truncated at residue 240 or 234. Both receptor binding and blocking of HSV infection were also affected by these C-terminal truncations. gD-1(234t) bound weakly to both HveA and HveC as determined by enzyme-linked immunosorbent assay (ELISA) and failed to block infection. Interestingly, gD-1(240t) bound well to both receptors but blocked infection poorly, indicating that receptor binding as measured by ELISA is not the only gD function required for blocking. Optical biosensor studies showed that while gD-1(240t) bound HveC with an affinity similar to that of gD-1(306t), the rates of complex formation and dissociation were significantly faster than for gD-1(306t). Complementation analysis showed that any 3-amino-acid deletion between residues 222 and 251 of gD resulted in a nonfunctional protein. Among this set of proteins, three had lost DL11 reactivity (those with deletions between residues 222 and 230). One of these proteins (deletion 222-224) was expressed as a soluble form in the baculovirus system. This protein did not react with DL11, bound to both HveA and HveC poorly as shown by ELISA, and failed to block HSV infection. Since this protein was bound by several other MAbs that recognize discontinuous epitopes, we conclude that residues 222 to 224 are critical for gD function. We propose that the potent virus-neutralizing activity of DL11 (and other group Ib MAbs) likely reflects an overlap between its epitope and a receptor-binding domain of gD.
Asunto(s)
Antígenos Virales/inmunología , Epítopos de Linfocito B/inmunología , Genes Sobrepuestos , Herpesvirus Humano 1/inmunología , Receptores del Factor de Necrosis Tumoral , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos Virales/genética , Baculoviridae , Sitios de Unión , Técnicas Biosensibles , Línea Celular , Chlorocebus aethiops , Epítopos de Linfocito B/genética , Expresión Génica , Prueba de Complementación Genética , Vectores Genéticos , Células HeLa , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/fisiología , Humanos , Datos de Secuencia Molecular , Mutagénesis , Pruebas de Neutralización , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Eliminación de Secuencia , Solubilidad , Spodoptera/citología , Células Vero , Proteínas del Envoltorio Viral/genéticaRESUMEN
The human herpesvirus entry mediator C (HveC/PRR1) is a member of the immunoglobulin family used as a cellular receptor by the alphaherpesviruses herpes simplex virus (HSV), pseudorabies virus, and bovine herpesvirus type 1. We previously demonstrated direct binding of the purified HveC ectodomain to purified HSV type 1 (HSV-1) and HSV-2 glycoprotein D (gD). Here, using a baculovirus expression system, we constructed and purified truncated forms of the receptor containing one [HveC(143t)], two [HveC(245t)], or all three immunoglobulin-like domains [HveC(346t)] of the extracellular region. All three constructs were equally able to compete with HveC(346t) for gD binding. The variable domain bound to virions and blocked HSV infection as well as HveC(346t). Thus, all of the binding to the receptor occurs within the first immunoglobulin-like domain, or V-domain, of HveC. These data confirm and extend those of Cocchi et al. (F. Cocchi, M. Lopez, L. Menotti, M. Aoubala, P. Dubreuil, and G. Campadelli-Fiume, Proc. Natl. Acad. Sci. USA 95:15700, 1998). Using biosensor analysis, we measured the affinity of binding of gD from HSV strains KOS and rid1 to two forms of HveC. Soluble gDs from the KOS strain of HSV-1 had the same affinity for HveC(346t) and HveC(143t). The mutant gD(rid1t) had an increased affinity for HveC(346t) and HveC(143t) due to a faster rate of complex formation. Interestingly, we found that HveC(346t) was a tetramer in solution, whereas HveC(143t) and HveC(245t) formed dimers, suggesting a role for the third immunoglobulin-like domain of HveC in oligomerization. In addition, the stoichiometry between gD and HveC appeared to be influenced by the level of HveC oligomerization.
Asunto(s)
Receptores del Factor de Necrosis Tumoral , Receptores Virales/metabolismo , Simplexvirus/fisiología , Proteínas del Envoltorio Viral/metabolismo , Animales , Bovinos , Línea Celular , Dimerización , Humanos , Inmunoglobulinas , Unión Proteica , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Receptores Virales/química , Replicación ViralRESUMEN
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.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Herpesvirus Humano 1/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Virales , Proteínas Recombinantes de Fusión/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae , Secuencia de Bases , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Línea Celular , ADN Viral , Vectores Genéticos , Glicosilación , Humanos , Datos de Secuencia Molecular , Mutagénesis , Nectinas , Conejos , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Solubilidad , Soluciones , Spodoptera , Virión/metabolismoRESUMEN
A human member of the immunoglobulin superfamily was shown to mediate entry of several alphaherpesviruses, including herpes simplex viruses (HSV) 1 and 2, porcine pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1). This membrane glycoprotein is poliovirus receptor-related protein 1 (Prr1), designated here as HveC. Incubation of HSV-1 with a secreted form of HveC inhibited subsequent infection of a variety of cell lines, suggesting that HveC interacts directly with the virus. Poliovirus receptor (Pvr) itself mediated entry of PRV and BHV-1 but not of the HSV strains tested. HveC was expressed in human cells of epithelial and neuronal origin; it is the prime candidate for the coreceptor that allows both HSV-1 and HSV-2 to infect epithelial cells on mucosal surfaces and spread to cells of the nervous system.
Asunto(s)
Alphaherpesvirinae/fisiología , Moléculas de Adhesión Celular/fisiología , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 2/fisiología , Proteínas de la Membrana , Receptores Virales , Animales , Secuencia de Bases , Células CHO , Moléculas de Adhesión Celular/genética , Células Cultivadas , Cricetinae , Células Epiteliales/virología , Expresión Génica , Herpesvirus Bovino 1/fisiología , Herpesvirus Suido 1/fisiología , Humanos , Datos de Secuencia Molecular , Nectinas , Neuronas/virología , Reacción en Cadena de la Polimerasa , Transfección , Células Tumorales Cultivadas , Proteínas del Envoltorio Viral/metabolismoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/metabolismo , Herpesvirus Humano 1/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Sitios de Unión , Células CHO , Línea Celular , Chlorocebus aethiops , Cricetinae , Humanos , Modelos Moleculares , Pruebas de Neutralización , Pruebas de Precipitina , Conejos , Spodoptera , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Herpes simplex virus type 1 latent infection in sensory neurons is characterized by the highly restricted transcription of viral genes. The latency-associated transcripts (LAT) family members are the only transcripts that can be identified in large amounts in latently infected cells. The most abundant LAT species is a 2-kb RNA that results from splicing of a rare primary transcript. Analysis of a LAT mutant virus (TB1) in cell culture revealed an aberrant splicing pattern and production of a stable small (0.95-kb) LAT intron. A panel of deletion constructs expressing truncated LAT in transiently transfected cells mapped the region influencing stability to the 3' end of the LAT intron. This region encompasses the branch point and a putative stable stem-loop hairpin structure immediately upstream of the splice acceptor consensus polypyrimidine tract. Mutagenic analysis of the sequence in this region confirmed our hypothesis that the stem-loop structure is important for efficient splicing by influencing the selection of a nonconsensus branch point. Changes in this structure correlate with changes in branch point selection and production of an unstable 2-kb LAT.
Asunto(s)
Herpesvirus Humano 2/genética , ARN Mensajero/química , ARN Viral/química , Empalme Alternativo , Bacteriófago lambda/genética , Secuencia de Bases , Intrones , Datos de Secuencia Molecular , MutagénesisRESUMEN
We have used a minigene construct of the herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene to analyze its transcripts in transient transfection assays. A 2.8-kb fragment of the approximately 8.5-kb LAT gene encompassing the 2.0-kb LAT was cloned into a eukaryotic expression vector downstream of the cytomegalovirus immediate-early gene promoter. Northern hybridization of RNA isolated from transfected COS-1 cells identified three LAT-specific transcripts, 3.4, 2.0, and 1.4 kb in size. Mapping of these transcripts by Northern hybridization indicated that the 1.4- and 2.0-kb RNAs are nonoverlapping, while the 3.4-kb RNA overlaps both smaller RNAs. Reverse transcription-PCR (RT-PCR) and partial sequencing of the 1.4-kb RNA revealed that this RNA is the spliced exons of the 3.4-kb primary transcript. The 2.0-kb LAT appears to be an intron accumulating after splicing of the minor LAT (mLAT) pre-mRNA. The splice donor and acceptor sites for the 2.0-kb LAT identified in transfected and HSV-1-infected cells are identical. Mapping of the branch point of this intron by RT-PCR in transfected and HSV-1-infected cells, as well as in latently infected murine trigemial ganglia, shows that it is a guanosine. This branch site does not bear homology to consensus mammalian branch site sequences. These data provide evidence that the 2.0-kb LAT is an intron of the mLAT pre-mRNA with a unique branch point.
Asunto(s)
Genes Virales , Intrones , Empalme del ARN , ARN Viral/genética , Simplexvirus/genética , Proteínas Estructurales Virales/genética , Animales , Secuencia de Bases , Células COS , Núcleo Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Citoplasma/metabolismo , Guanosina , Enlace de Hidrógeno , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas , Latencia del VirusRESUMEN
Early after infection, the mouse mammary tumor virus (MMTV) expresses a superantigen (SAg) at the surface of B lymphocytes. Interaction with the T-cell receptor Vbeta domain induces a polyclonal proliferative response of the SAg-reactive T cells. Stimulated T cells become anergic and are deleted from the T-cell repertoire. We have used a recombinant vaccinia virus encoding the MMTV(GR) SAg to dissect the effects of the retroviral SAg during an unrelated viral infection. Subcutaneous infection with this recombinant vaccinia virus induces a very rapid increase of Vbeta14 T cells in the draining lymph node. This stimulation does not require a large Plumber of infectious particles and is not strictly dependent on the expression of the major histocompatibility complex class II I-E molecule, as it is required after MMTV(GR) infection. In contrast to MMTV infection during which B cells are infected, we do not observe any clonal deletion of the reactive T cells following the initial stimulation phase. Our data show that contrary to the case with MMTV, macrophages but not B cells are the targets of infection by vaccinia virus in the lymph node, indicating the ability of these cells to present a retroviral SAg. The altered SAg expression in a different target cell observed during recombinant vaccinia virus infection therefore results in significant changes in the SAg response.
Asunto(s)
Antígenos Virales/inmunología , Virus del Tumor Mamario del Ratón/fisiología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Superantígenos/inmunología , Linfocitos T/inmunología , Virus Vaccinia/fisiología , Vaccinia/inmunología , Animales , Antígenos Virales/biosíntesis , Secuencia de Bases , Cartilla de ADN , Citometría de Flujo , Expresión Génica , Inmunidad Celular , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/virología , Virus del Tumor Mamario del Ratón/inmunología , Virus del Tumor Mamario del Ratón/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Recombinación Genética , Superantígenos/biosíntesis , Linfocitos T/virología , Factores de Tiempo , Virus Vaccinia/metabolismo , Ensayo de Placa Viral , Replicación ViralRESUMEN
The mouse mammary tumor virus proviral DNA contains an open reading frame in the 3' long terminal repeat which can code for a 36 kDa polypeptide with a putative transmembrane sequence and five N-linked glycosylation sites. This gene is known to code for a superantigen which deletes a specific subset of CD4+ T lymphocytes in vivo. The superantigen encoded by the exogenous mouse mammary tumor virus of the GR strain acts specifically on V beta 14 bearing T cells. We produced recombinant vaccinia viruses to express either the complete or a truncated ORF protein after infection of primate cells in culture. The complete ORF gene in mammalian cells leads to the production of a 47 kDa protein which is specifically detected with an anti-ORF-peptide antiserum. The 47 kDa protein can be labeled with D-[2-3H]mannose and its synthesis is inhibited by tunicamycin, an N-linked glycosylation inhibitor, indicating that it is a glycoprotein. The truncated ORF protein beginning at the second ATG of the open reading frame is also modified, but the C-terminal half of ORF, starting at the fifth ATG, has the expected size of the non modified polypeptide. Pulse-chase experiments indicate that the ORF protein has a short half-life of about 1.5-2 hr.