Construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein H coding sequences deleted.

A mutant of herpes simplex virus type 1 (HSV-1) in which glycoprotein H (gH) coding sequences were deleted and replaced by the Escherichia coli lacZ gene under the control of the human cytomegalovirus IE-1 gene promoter was constructed. The mutant was propagated in Vero cells which contained multiple copies of the HSV-1 gH gene under the control of the HSV-1 gD promoter and which therefore provide gH in trans following HSV-1 infection. Phenotypically gH-negative virions were obtained by a single growth cycle in Vero cells. These virions were noninfectious, as judged by plaque assay and by expression of beta-galactosidase following high-multiplicity infection, but partial recovery of infectivity was achieved by using the fusogenic agent polyethylene glycol. Adsorption of gH-negative virions to cells blocked the adsorption of superinfecting wild-type virus, a result in contrast to that obtained with gD-negative virions (D. C. Johnson and M. W. Ligas, J. Virol. 62:4605-4612, 1988). The simplest conclusion is that gH is required for membrane fusion but not for receptor binding, a conclusion consistent with the conservation of gH in all herpesviruses.     

Site-directed and linker insertion mutagenesis of herpes simplex virus type 1 glycoprotein H.

The gH-gL complex of herpes simplex virus type 1 (HSV-1) is essential for virion infectivity and virus-induced cell fusion, but functional domains of the gH molecule remain to be defined. We have addressed this question by mutagenesis. A set of linker insertion mutants in HSV-1 gH was generated and tested in transient assays for their ability to complement a gH-negative virus. Insertions at three sites in the C-terminal third of the external domain affected the ability of gH to function in cell-cell fusion and virus entry, while insertions at six sites in the N-terminal half of the external domain induced conformational changes in gH such that it was not recognized by monoclonal antibody LP11, although expression at the cell surface was unchanged. A recombinant virus in which a potential integrin-binding motif, RGD, in gH was changed to the triplet RGE entered cells as efficiently as the wild type, indicating that HSV-1 entry is not mediated by means of the gH-RGD motif binding to cell surface integrins. Furthermore, mutagenesis of the glycosylation site which is positionally conserved in all herpesvirus gH sequences in close proximity to the transmembrane domain generated a recombinant virus that grew in vitro with wild-type single-step kinetics.     

The transmembrane domain and cytoplasmic tail of herpes simplex virus type 1 glycoprotein H play a role in membrane fusion

Herpes simplex virus glycoprotein H (gH) is one of the four virion envelope proteins which are required for virus entry and for cell-cell fusion in a transient system. In this report, the role of the transmembrane and cytoplasmic tail domains of gH in membrane fusion was investigated by generating chimeric constructs in which these regions were replaced with analogous domains from other molecules and by introducing amino acid substitutions within the membrane-spanning sequence. gH molecules which lack the authentic transmembrane domain or cytoplasmic tail were unable to mediate cell-cell fusion when coexpressed with gB, gD, and gL and were unable to rescue the infectivity of a gH-null virus as efficiently as a wild-type gH molecule. Many amino acid substitutions of specific amino acid residues within the transmembrane domain also affected cell-cell fusion, in particular, those introduced at a conserved glycine residue. Some gH mutants that were impaired in cell-cell fusion were nevertheless able to rescue the infectivity of a gH-negative virus, but these pseudotyped virions entered cells more slowly than wild-type virions. These results indicate that the fusion event mediated by the coexpression of gHL, gB, and gD in cells shares common features with the fusion of the virus envelope with the plasma membrane, they point to a likely role for the membrane-spanning and cytoplasmic tail domains of gH in both processes, and they suggest that a conserved glycine residue in the membrane-spanning sequence is crucial for efficient fusion.     

Glycoprotein H of pseudorabies virus is essential for entry and cell-to-cell spread of the virus.

To study the function of the envelope glycoprotein gH of pseudorabies virus, a gH null mutant was constructed. A premature translation termination codon was introduced in the gH gene by linker insertion mutagenesis, and a mutant virus was rescued by using a cell line that expresses the wild-type protein. Mutant virus isolated from complementing cells was unable to form plaques on noncomplementing cells, indicating that gH is essential in the life cycle of the virus. Immunological staining and electron microscopy showed that the mutant virus produced noninfectious progeny and was unable to spread from infected to uninfected cells by cell-cell fusion. Thus, similar to gH of herpes simplex virus, gH of pseudorabies virus is required for entry and cell-to-cell spread.     

Characterization and sequence analyses of antibody-selected antigenic variants of herpes simplex virus show a conformationally complex epitope on glycoprotein H.

Thirteen antigenic variants of herpes simplex virus which were resistant to neutralization by monoclonal antibody 52S or LP11 were isolated and characterized. The antibodies in the absence of complement potently neutralize infectivity of wild-type virus as well as inhibit the transfer of virus from infected to uninfected cells ("plaque inhibition") and decrease virus-induced cell fusion by syncytial strains. The first variant isolated arose in vivo. Of 66 type 1 isolates analyzed from typing studies of 100 clinical isolates, one was identified as resistant to neutralization by LP11 antibody. The glycoprotein H (gH) sequence was derived and compared with those of wild-type and syncytial laboratory strains SC16, strain 17, and HFEM. The sequences were highly conserved in contrast to the diversity observed between gH sequences from herpesviruses of different subgroups. Only four coding changes were present in any of the comparisons, and only one unique coding change was observed between the laboratory strains and the clinical isolate (Asp-168 to Gly). These sequences were compared with those of antigenic variants selected by antibody in tissue culture. Twelve variants were independently selected with antibody LP11 or 52S from parent strain SC16 or HFEM. For each variant, the gH nucleotide sequence was derived and a point mutation was identified giving rise to a single amino acid substitution. The LP11-resistant viruses encoded gH sequences with amino acid substitutions at sites distributed over one-half of the gH external domain, Glu-86, Asp-168, or Arg-329, while the 52S-resistant mutant viruses had substitutions at adjacent positions Ser-536 and Ala-537. One LP11 mutant virus had a point mutation in the gH gene that was identical to that of the clinical isolate, giving rise to a substitution of Asp-168 with Gly. Both LP11 and 52S appeared to recognize distinct gH epitopes as mutant virus resistant to neutralization and immunoprecipitation with LP11 remained sensitive to 52S and the converse was shown for the 52S-resistant mutant virus. This is consistent with previous studies which showed that while the 52S epitope could be formed in the absence of other virus products, virus gene expression was required for stable presentation of the LP11 epitope, and for transport of gH to the cell surface (Gompels and Minson, J. Virol. 63:4744-4755, 1989). All mutant viruses produced numbers of infectious particles that were similar to those produced by the wild-type virus, with the exception of one variant which produced lower yields.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neutralizing antibodies specific for glycoprotein H of herpes simplex virus permit viral attachment to cells but prevent penetration.

Monoclonal antibodies specific for gH of herpes simplex virus were shown previously to neutralize viral infectivity. Results presented here demonstrate that these antibodies (at least three of them) block viral penetration without inhibiting adsorption of virus to cells. Penetration of herpes simplex virus is by fusion of the virion envelope with the plasma membrane of a susceptible cell. Electron microscopy of thin sections of cells exposed to virus revealed that neutralized virus bound to the cell surface but did not fuse with the plasma membrane. Quantitation of virus adsorption by measuring the binding of purified radiolabeled virus to cells revealed that the anti-gH antibodies had little or no effect on adsorption. Monitoring cell and viral protein synthesis after exposure of cells to infectious and neutralized virus gave results consistent with the electron microscopic finding that the anti-gH antibodies blocked viral penetration. On the basis of the results presented here and other information published elsewhere, it is suggested that gH is one of three glycoproteins essential for penetration of herpes simplex virus into cells.     

DNA sequence of the herpes simplex virus type 1 gene encoding glycoprotein gH, and identification of homologues in the genomes of varicella-zoster virus and Epstein-Barr virus.

We have determined the sequence of herpes simplex virus type 1 DNA around the previously mapped location of sequences encoding an epitope of glycoprotein gH, and have deduced the structure of the gH gene and the amino acid sequence of gH. The unprocessed polypeptide is predicted to contain 838 amino acids, and to possess an N-terminal signal sequence and a C-terminal transmembrane sequence. Temperature-sensitive mutant tsQ26 maps within the predicted gH coding sequence. Homologous genes were identified in the genomes of two other herpesviruses, namely varicella-zoster virus and Epstein-Barr virus.     

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.     

A heptad repeat in herpes simplex virus 1 gH, located downstream of the alpha-helix with attributes of a fusion peptide, is critical for virus entry and fusion

Entry of herpes simplex virus 1 (HSV-1) into cells occurs by fusion with cell membranes; it requires gD as the receptor binding glycoprotein and the trigger of fusion, and the trio of the conserved glycoproteins gB, gH, and gL to execute fusion. Recently, we reported that the ectodomain of HSV-1 gH carries a hydrophobic alpha-helix (residues 377 to 397) with attributes of an internal fusion peptide (T. Gianni, P. L. Martelli, R. Casadio, and G. Campadelli-Fiume, J. Virol. 79:2931-2940, 2005). Downstream of this alpha-helix, a heptad repeat (HR) with a high propensity to form a coiled coil was predicted between residues 443 and 471 and was designated HR-1. The simultaneous substitution of two amino acids in HR-1 (E450G and L453A), predicted to abolish the coiled coil, abolished the ability of gH to complement the infectivity of a gH-null HSV mutant. When coexpressed with gB, gD, and gL, the mutant gH was unable to promote cell-cell fusion. These defects were not attributed to a defect in heterodimer formation with gL, the gH chaperone, or in trafficking to the plasma membrane. A 25-amino-acid synthetic peptide with the sequence of HR-1 (pep-gH(wt25)) inhibited HSV replication if present at the time of virus entry into the cell. A scrambled peptide had no effect. The effect was specific, as pep-gH(wt25) did not reduce HSV-2 and pseudorabies virus infection. The presence of a functional HR in the HSV-1 gH ectodomain strengthens the view that gH has attributes typical of a viral fusion glycoprotein.     

Blocking immune evasion as a novel approach for prevention and treatment of herpes simplex virus infection

Many microorganisms encode immune evasion molecules to escape host defenses. Herpes simplex virus type 1 glycoprotein gC is an immunoevasin that inhibits complement activation by binding complement C3b. gC is expressed on the virus envelope and infected cell surface, which makes gC potentially accessible to blocking antibodies. Mice passively immunized with gC monoclonal antibodies prior to infection were protected against herpes simplex virus challenge only if the gC antibodies blocked C3b binding. Mice treated 1 or 2 days postinfection with gC monoclonal antibodies that block C3b binding had less severe disease than control mice treated with nonimmune immunoglobulin G (IgG). Mice immunized with gC protein produced antibodies that blocked C3b binding to gC. Immunized mice were significantly protected against challenge by wild-type virus, but not against a gC mutant virus lacking the C3b binding domain, suggesting that protection was mediated by antibodies that target the gC immune evasion domain. IgG and complement from subjects immunized with an experimental herpes simplex virus glycoprotein gD vaccine neutralized far more mutant virus defective in immune evasion than wild-type virus, supporting the importance of immune evasion molecules in reducing vaccine potency. These results suggest that it is possible to block immune evasion domains on herpes simplex virus and that this approach has therapeutic potential and may enhance vaccine efficacy.     

Pseudorabies virus glycoprotein L is necessary for virus infectivity but dispensable for virion localization of glycoprotein H.

Herpesviruses contain a number of envelope glycoproteins which play important roles in the interaction between virions and target cells. Although several glycoproteins are not present in all herpesviruses, others, including glycoproteins H and L (gH and gL), are conserved throughout the Herpesviridae. To elucidate common properties and differences in herpesvirus glycoprotein function, corresponding virus mutants must be constructed and analyzed in different herpesvirus backgrounds. Analysis of gH- mutants of herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PrV) showed that in both viruses gH is essential for penetration and cell-to-cell spread and that its presence is required for virion localization of gL. Since gH homologs are found complexed with gL, it was of interest to assess the phenotype of gL- mutant viruses. By using this approach, HSV-1 gL has been shown to be required for entry and for virion localization of gH (C. Roop, L. Hutchinson, and D. Johnson, J. Virol. 67:2285-2297, 1993). To examine whether a similar phenotype is associated with lack of gL in another alphaherpesvirus, PrV, we constructed two independent gL- PrV mutants by insertion and deletion-insertion mutagenesis. The salient findings are as follows: (i) PrV gL is required for penetration of virions and cell-to-cell spread; (ii) unlike HSV-1, PrV gH is incorporated into the virion in the absence of gL; (iii) virion localization of gH in the absence of gL is not sufficient for infectivity; (iv) in the absence of gL, N-glycans on PrV gH are processed to a greater extent than in the presence of gL, indicating masking of N-glycans by association with gL; and (v) an anti-gL polyclonal antiserum is able to neutralize virion infectivity but did not inhibit cell-to-cell spread. Thus, whereas PrV gL is essential for virus replication, as is HSV-1 gL, gL- PrV mutants exhibit properties strikingly different from those of HSV-1. In conclusion, our data show an important functional role for PrV gL in the viral entry process, which is not explained by a chaperone-type mechanism in gH maturation and processing.     

Characterization of a recombinant herpes simplex virus which expresses a glycoprotein D lacking asparagine-linked oligosaccharides.

Glycoprotein D (gD) is an envelope component of herpes simplex virus essential for virus penetration. gD contains three sites for addition of asparagine-linked carbohydrates (N-CHO), all of which are utilized. Previously, we characterized mutant forms of herpes simplex virus type 1 gD (gD-1) lacking one or all three N-CHO addition sites. All of the mutants complemented the infectivity of a gD-minus virus, F-gD beta, to the same extent as wild-type gD. Here, we show that recombinant viruses containing mutations in the gD-1 gene which eliminate the three N-CHO signals are viable. Two such viruses, called F-gD(QAA)-1 and F-gD(QAA)-2, were independently isolated, and the three mutations in the gD gene in one of these viruses were verified by DNA sequencing. We also verified that the gD produced in cells infected by these viruses is devoid of N-CHO. Plaques formed by both mutants developed more slowly than those of the wild-type control virus, F-gD(WT), and were approximately one-half the size of the wild-type. One mutant, F-gD(QAA)-2, was selected for further study. The QAA mutant and wild-type gD proteins extracted from infected cells differed in structure, as determined by the binding of monoclonal antibodies to discontinuous epitopes. However, flow cytometry analysis showed that the amount and structure of gD found on infected cell surfaces was unaffected by the presence or absence of N-CHO. Other properties of F-gD(QAA)-2 were quite similar to those of F-gD(WT). These included (i) the kinetics of virus production as well as the intracellular and extracellular virus titers; (ii) the rate of virus entry into uninfected cells; (iii) the levels of gB, gC, gE, gH, and gI expressed by infected cells; and (iv) the turnover time of gD. Thus, the absence of N-CHO from gD-1 has some effect on its structure but very little effect on its function in virus infection in cell culture.     

Physical mapping of the mutation in an antigenic variant of herpes simplex virus type 1 by use of an immunoreactive plaque assay.

Two mutations affecting herpes simplex virus type 1 glycoprotein B were mapped by marker rescue using cloned sequences of wild-type herpes simplex virus type 1 strain KOS DNA. One mutant, tsB5, is a temperature-sensitive mutant which does not express mature, functional glycoprotein B at the nonpermissive temperature. The other mutant, marB1.1, expresses an antigenic variant of glycoprotein B and was selected for resistance to neutralization by a monoclonal antibody. The mutation in tsB5 mapped to a 1.2-kilobase segment of the herpes simplex virus type 1 genome between coordinates 0.361 and 0.368, whereas the mutation in marB1.1 mapped to a 1.6-kilobase segment between coordinates 0.350 and 0.361. An in situ enzyme immunoassay was used to detect plaques of recombinant wild-type virus among the progeny of transfections with mutant marB1.1 DNA and wild-type DNA fragments.     

Mutant analysis of herpes simplex virus-induced cell surface antigens: resistance to complement-mediated immune cytolysis.

BHK-21 cells infected with temperature-sensitive mutants of herpes simplex virus type 1 strain KOS representing 16 complementation groups were tested for susceptibility to complement-mediated immune cytolysis at permissive (34 degrees C) and nonpermissive (39 degrees C) temperatures. Only cells infected by mutants in complementation group E were resistant to immune cytolysis in a temperature-sensitive manner compared with wild-type infections. The expression of group E mutant cell surface antigens during infections at 34 and 39 degrees C was characterized by a combination of cell surface radioiodination, specific immunoprecipitation, and gel electrophoretic analysis of immunoprecipitates. Resistance to immune lysis at 39 degrees C correlated with the absence of viral antigens exposed at the cell surface. Intrinsic radiolabeling of group E mutant infections with [14C]glucosamine revealed that normal glycoproteins were produced at 34 degrees C but none were synthesized at 39 degrees C. The effect of 2-deoxy-D-glucose on glycosylation of group E mutants at 39 degrees C suggested that the viral glycoprotein precursors were not synthesized. The complementation group E mutants failed to complement herpes simplex virus type 1 mutants isolated by other workers. These included the group B mutants of strain KOS, the temperature-sensitive group D mutants of strain 17, and the LB2 mutant of strain HFEM. These mutants should be considered members of herpes simplex virus type 1 complementation group 1.2, in keeping with the new herpes simplex virus type 1 nomenclature.     

Mutations in the cytoplasmic tail of herpes simplex virus glycoprotein H suppress cell fusion by a syncytial strain.

We have developed a complementation assay, using transiently transfected COS cells, to facilitate a molecular analysis of the herpes simplex virus type 1 glycoprotein gH. When infected by a gH-null syncytial virus, COS cells expressing wild-type gH generate infectious progeny virions and form a syncytium with neighboring cells. By deletion and point mutagenesis, we have found particular residues in the gH cytoplasmic tail to be essential for generation of a syncytium but apparently dispensable for production of infectious virions. This study emphasizes the different requirements for cell-cell and cell-envelope fusion and demonstrates that changes in the non-syn locus UL22-gH can reverse the syncytial phenotype.     

Fusogenic domains in herpes simplex virus type 1 glycoprotein H

Infection of eukaryotic cells by enveloped viruses requires fusion between the viral envelope and the cellular plasma or endosomal membrane. The actual merging of the two membranes is mediated by viral envelope glycoproteins, which generally contain a highly hydrophobic region termed the fusion peptide. The entry of herpesviruses is mediated by three conserved proteins: glycoproteins B, H (gH), and L. However, how fusion is executed remains unknown. Herpes simplex virus type 1 gH exhibits features typical of viral fusion glycoproteins, and its ectodomain seems to contain a putative internal fusion peptide. Here, we have identified additional internal segments able to interact with membranes and to induce membrane fusion of large unilamellar vesicles. We have applied the hydrophobicity-at-interface scale proposed by Wimley and White (Wimley, W. C., and White, S. H. (1996) Nat. Struct. Biol. 3, 842-848) to identify six hydrophobic stretches within gH with a tendency to partition into the membrane interface, and four of them were able to induce membrane fusion. Experiments in which equimolar mixtures of gH peptides were used indicated that different fusogenic regions may act in a synergistic way. The functional and structural characterization of these segments suggests that herpes simplex virus type 1 gH possesses several fusogenic internal peptides that could participate in the actual fusion event.     

Herpes simplex virus type 1 glycoprotein E is not indispensable for viral infectivity.

A mutant of the herpes simplex virus type 1 Angelotti was isolated in which 87% of the coding region of glycoprotein E (gE) was deleted and replaced by a functional neomycin resistance gene of the Tn5 transposon. The mutant was characterized by restriction enzyme analyses and Southern blotting. Western blotting of proteins and immunofluorescence assays revealed that gE was completely absent and that the Fc receptor was not expressed in cells infected with the mutant. The fact that this mutant was viable and that it replicated to a slightly lower titer than did the wild-type virus suggests that the presence of gE is not a prerequisite of viral infectivity in tissue culture.     

Serologic type conversion of a herpes simplex virus type 1 (HSV-1) to an HSV-2 epitope caused by a single amino acid substitution in glycoprotein C.

A monoclonal antibody to herpes simplex virus type 2 glycoprotein C (gC-2) did not recognize wild-type herpes simplex virus type 1 gC (gC-1) but did recognize a mutant gC-1 molecule. This conversion from a type 1 to a type 2 epitope was shown to be due to a single amino acid substitution in gC-1.     

The ectodomain of herpes simplex virus glycoprotein H contains a membrane alpha-helix with attributes of an internal fusion peptide, positionally conserved in the herpesviridae family

Human herpesviruses enter cells by fusion with target membranes, a process that requires three conserved glycoproteins: gB, gH, and gL. How these glycoproteins execute fusion is unknown. Neural network bioinformatics predicted a membrane alpha-helix contained within the ectodomain of herpes simplex virus (HSV) gH, positionally conserved in the gH of all examined herpesviruses. Evidence that it has attributes of an internal fusion peptide rests on the following lines of evidence. (i) The predicted membrane alpha-helix has the attribute of a membrane segment, since it transformed a soluble form of gD into a membrane-bound gD. (ii) It represents a critical domain of gH. Its partial or entire deletion, or substitution of critical residues inhibited HSV infectivity and fusion in the cell-cell fusion assay. (iii) Its replacement with the fusion peptide from human immunodeficiency virus gp41 or from vesicular stomatitis virus G partially rescued HSV infectivity and cell-cell fusion. The corresponding antisense sequences did not. (iv) The predicted alpha-helix located in the varicella-zoster virus gH ectodomain can functionally substitute the native HSV gH membrane alpha-helix, suggesting a conserved function in the human herpesviruses. We conclude that HSV gH exhibits features typical of viral fusion glycoproteins and that this property is likely conserved in the Herpesviridae family.