The herpes simplex virus type 1 Fc receptor discriminates between IgG1 allotypes.

Herpes simplex virus type 1 (HSV-1) expresses a complex of two virally encoded glycoproteins, gE and gl, which is capable of binding nonimmune human IgG. The gE-gl complex has thus become known as an Fc receptor (FcR), which reportedly binds human IgG subclasses in the order IgG4 > IgG1 > or = IgG2 and does not bind IgG3 from many individuals. There is, however, allelic variation in the genes encoding the human IgG1 heavy chain constant region and this gives rise to allotypes of IgG1. Using recombinant monoclonal IgG molecules of known isotype and mutants thereof we have unexpectedly discovered that the HSV-1 FcR discriminates between IgG1 allotypes. This is evidence of functional differences between IgG1 allotypes that may account for their distribution in populations. Furthermore, these findings suggest HSV-1 FcR binding sites on the IgG molecule some distance from the proposed binding site in the CH2-CH3 domain interface.

A broad spectrum secreted chemokine binding protein encoded by a herpesvirus.

Chemokines are a family of small proteins that interact with seven-transmembrane domain receptors and modulate the migration of immune cells into sites of inflammation and infection. The murine gammaherpesvirus 68 M3 gene encodes a secreted 44-kD protein with no sequence similarity to known chemokine receptors. We show that M3 binds a broad range of chemokines, including CC, CXC, C, and CX(3)C chemokines, but does not bind human B cell-specific nor mouse neutrophil-specific CXC chemokines. This herpesvirus chemokine binding protein (hvCKBP) blocks the interaction of chemokines with high-affinity cellular receptors and inhibits chemokine-induced elevation of intracellular calcium levels. hvCKBP is the first soluble chemokine receptor identified in herpesviruses; it represents a novel protein structure with the ability to bind all subfamilies of chemokines in solution and has potential therapeutic applications.

A study of primary neuronal infection by mutants of herpes simplex virus type 1 lacking dispensable and non-dispensable glycoproteins.

Cultures of primary rat dorsal root ganglia neurones were inoculated with various doses of herpes simplex virus mutants deficient in glycoproteins B, D, H, C, G, E, I or J, and the proportion of infected neurones was determined. The behaviour of these mutants on primary neurones was broadly similar to their behaviour on fibroblasts or epithelial cells. Thus, virions lacking the 'nondispensable' glycoproteins B, D or H were incapable of infecting primary neurones, whereas mutants lacking glycoproteins G, E, I or J infected primary neurones with the same efficiency as wild-type virions. Two independently derived mutants lacking gC displayed a marginal phenotype, infecting neurones with a five- to tenfold reduced efficiency relative to wild-type virus and relative to non-neuronal cells in the same cultures. We conclude that the virion glycoprotein requirements for infection of mammalian neurones are similar to those required for infection of fibroblasts and epithelial cells but that glycoprotein C may enhance infection of neurones.

In vivo complementation studies of a glycoprotein H-deleted herpes simplex virus-based vector.

The utilization of herpes simplex virus (HSV) as a vector for gene delivery to the nervous system or as a live vaccine delivery system is dependent on the construction and characterization of disabled virus mutants which are unable to cause disease. Under certain circumstances, however, replication-defective vectors may carry a potential risk if they can be efficiently complemented by a co-infecting wild-type virus. Stocks of defective vectors should, therefore, be free from replication-competent virus, and helper cell lines should be incapable of generating replication-competent virus by recombination between the vector and the complementary gene. We describe a glycoprotein H-negative (gH-) virus/helper cell line combination which generates helper-free defective virus stocks containing replication-competent virus at a frequency no higher than 1 in 10(9) p.f.u. This virus/helper cell system provides a suitable background for the construction of safe replication-defective gene delivery vectors. In vivo studies demonstrate that gH- virus is unable to initiate disease in mice and establishes latency at low efficiency compared to wild-type HSV. To determine whether gH- virus can be complemented by wild-type virus in vivo, mice were infected with a variety of mixtures of these viruses. Complementation was observed in a minority of animals infected with more than 10(6) p.f.u. of both wild-type and defective virus but the most common observation was that the presence of defective virus suppressed entry of wild-type virus into the nervous system.

Characterization of herpes simplex virus type 1 recombinants with mutations in the cytoplasmic tail of glycoprotein H.

Herpes simplex virus (HSV) type 1 glycoprotein H is essential for fusion of virus envelopes with cellular membranes and for the fusion of an infected cell membrane with an uninfected neighbour. Previous studies have pointed to a requirement for certain amino acid residues of the cytoplasmic tail of gH in these processes. Results from transient transfection experiments suggested that the serine-valine-proline (SVP) motif in the cytoplasmic tail may be important for gH-mediated fusion. HSV recombinants expressing gH molecules with mutations in the cytoplasmic tail were constructed and analysed in terms of their abilities to fuse cellular membranes and to function in virus entry. Viruses containing a deletion of the SVP motif, or in which the valine residue of this triplet was replaced by alanine, entered cells less efficiently than wild-type virus and were unable to induce syncytium formation on Vero cells.

Herpes virus-based vectors.

Herpesviruses are a diverse family of large DNA viruses, all of which have the capacity to establish lifelong latent infections. Many different herpesviruses may have potential as gene delivery vehicles, but exploitation of this potential has, to date, been explored only using Herpes simplex virus (HSV), a virus which naturally establishes a silent, latent infection of neurones in man and in a number of experimental animal models. Delivery of reporter genes in vitro and in vivo has been demonstrated using a variety of replication competent and replication defective vectors, and significant physiological modification in the CNS has been achieved by HSV-mediated gene delivery. Much remains to be done using animal models and, in particular, the requirements for long-term gene expression from latent virus genomes needs to be defined in different cell types in vivo.

Protective vaccination against primary and recurrent disease caused by herpes simplex virus (HSV) type 2 using a genetically disabled HSV-1.

The vaccine potential of a mutant herpes simplex virus (HSV) type 1, with a deletion in the glycoprotein H (gH) gene, was evaluated. The virus requires a gH-expressing cell line for multi-cycle growth but can complete a single cycle of infection in noncomplementing cells. Such viruses, termed DISC (disabled infectious single cycle) viruses, should be safe, yet still able to stimulate humoral and cell-mediated responses against a broad range of virus antigens in vaccinated hosts. Prophylactic vaccination of guinea pigs with DISC HSV-1, by ear scarification or direct infection of the vaginal mucosa, afforded a high degree of protection against HSV-2-induced primary genital disease and reduced significantly the frequency of subsequent disease recurrence. There was also a trend toward reduced recurrence following therapeutic vaccination of animals already infected with HSV-2. DISC HSV vaccination, therefore, offers an effective route for control of HSV disease.

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.

Vaccine potential of a herpes simplex virus type 1 mutant with an essential glycoprotein deleted.

Several approaches to the production of vaccines to human herpesviruses have been proposed. Subunit vaccines, subunits delivered by live vectors, and rationally attenuated vaccines have all been shown to be efficacious in animal models but suffer from uncertainties as to the roles of individual genes involved in pathogenesis and the most relevant components of the immune response required for protection in humans and the target antigens involved. With these problems in mind, we examined the vaccine potential of a fully disabled herpes simplex virus type 1 mutant that is capable of only a single round of replication, since a virus of this type should induce the full spectrum of immune responses but has no pathogenic potential. A virus has been described which lacks essential glycoprotein H (gH) and can be propagated in a cell line which supplies gH in trans (A. Forrester, H. Farrell, G. Wilkinson, J. Kaye, N. Davis-Poynter, and T. Minson, J. Virol. 66:341-348, 1992). Infection of normal cells with this mutant is indistinguishable from a wild-type infection, except that the resulting progeny are gH negative and noninfectious: the virus is self-limiting. Infection of mice by the ear pinna route was similarly self-limiting in that input infectivity decreased rapidly at the inoculation site and no infectivity was detected in sensory ganglia. Animals given a wide range of doses of the gH-negative mutant produced both humoral and T-cell responses to herpes simplex virus type 1 and proved solidly resistant to challenge with a high dose of wild-type virus. The gH-negative mutant is presumably capable of establishing a latent infection, but since no infectious virus was detected in numerous attempts to reactivate the mutant, the risk of a pathogenic outcome is minimal.

Glycoprotein H of human cytomegalovirus (HCMV) forms a stable complex with the HCMV UL115 gene product.

The human cytomegalovirus (HCMV) UL75 gene product is the homologue of herpes simplex virus type 1 (HSV-1) glycoprotein H (gH), a virion glycoprotein that is essential for infectivity and which is conserved among members of the alpha-, beta- and gamma-herpesviruses. It has previously been shown that HSV-1 gH forms a stable complex with HSV-1 gL, the product of the UL1 gene, and the formation of this complex facilitates the cell surface expression of gH. None of the open reading frames within the HCMV genome encode a product with discernible sequence homology with HSV-1 gL, but an examination of the arrangement of conserved genes in HCMV suggested that the UL115 gene is a 'positional homologue' of HSV-1 UL1 which, like UL1, encodes a small secreted glycoprotein. Co-expression of HCMV gH (the UL75 gene product) and the UL115 gene product revealed that these proteins form a disulphide-linked complex and that the formation of this complex results in cell surface expression of gH. This complex is analogous to the gH:gL complex of HSV-1 and the HCMV UL115 gene product is therefore the functional homologue of HSV-1 gL.

Conservation of HPV-16 E6/E7 ORF sequences in a cervical carcinoma.

A cervical carcinoma that contained human papillomavirus (HPV)-16 homologous DNA was analyzed. Each tumor cell genome contained a single, incomplete copy of HPV-16 DNA. The E6 and E7 open reading frames (ORFs) were completely conserved relative to other published HPV-16 sequences. Much of the non-coding region (NCR) was free of base changes, including complete conservation of several regulatory elements. Multiple mutations were identified in the remaining integrated HPV-16 DNA, which was composed of parts of the L1 and E1 ORFs. The extraordinary conservation of the E6/E7 DNA sequence, as compared with other regions of the integrated HPV-16 DNA, supports the role of E6/E7 in tumorigenesis.

A novel herpes simplex virus glycoprotein, gL, forms a complex with glycoprotein H (gH) and affects normal folding and surface expression of gH.

A glycoprotein encoded by the UL1 gene of herpes simplex virus type 1 (HSV-1) was detected in infected cells with antipeptide sera. The UL1 gene has previously been implicated in virus-induced cell fusion (S. Little and P. A. Schaffer, Virology 112:686-697, 1981). Two protein species, a 30-kDa precursor form and a 40-kDa mature form of the glycoprotein, both of which were modified with N-linked oligosaccharides, were observed. This novel glycoprotein is the 10th HSV-1 glycoprotein to be described and was named glycoprotein L (gL). A complex was formed between gL and gH, a glycoprotein known to be essential for entry of HSV-1 into cells and for virus-induced cell fusion. Previously, it had been reported that gH expressed in the absence of other viral proteins was antigenically abnormal, not processed, and not expressed at the cell surface (U.A. Gompels and A. C. Minson, J. Gen. Virol. 63:4744-4755, 1989; A. J. Forrester, V. Sullivan, A. Simmons, B. A. Blacklaws, G. L. Smith, A. A. Nash, and A. C. Minson, J. Gen. Virol. 72:369-375, 1991). However, gH coexpressed with gL by using vaccinia virus recombinants was antigenically normal, processed normally, and transported to the cell surface. Similarly, gL was dependent on gH for proper posttranslational processing and cell surface expression. These results suggest that it is a hetero-oligomer of gH and gL which is incorporated into virions and transported to the cell surface and which acts during entry of virus into cells.

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)

Induction of protective immunity with antibody to herpes simplex virus type 1 glycoprotein H (gH) and analysis of the immune response to gH expressed in recombinant vaccinia virus.

Passive administration of neutralizing monoclonal antibody (MAb) to glycoprotein H (gH) of herpes simplex virus type 1 (HSV-1) was found to protect mice from an HSV-1 strain SC16 challenge infection. To investigate further the protective potential of gH, recombinant vaccinia viruses were constructed which expressed the HSV-1 gH open reading frame under the control of the vaccinia virus 7.5K early/late promoter or the 4b late promoter. Immunization with recombinant viruses, however, did not induce the production of neutralizing antisera and the mice were not protected from zosteriform spread or the establishment of latent infection following viral challenge. The gH produced by the recombinant vaccinia viruses differed in electrophoretic mobility and antigenicity from authentic HSV-1 gH. Only one of three neutralizing MAbs specific for conformational epitopes on gH was able to immunoprecipitate gH synthesized in recombinant vaccinia virus-infected cells. In addition cell surface expression of gH was not detected in cells infected with the recombinant vaccinia viruses.

Immunogenicity of herpes simplex virus type 1 glycoproteins expressed in vaccinia virus recombinants.

Vaccinia virus recombinants expressing glycoproteins B (vgB11), D (VgD52), E (gE/7.5 and gE/4B), G (gG-vac), H (gH-vac), and I (gI-vac) of HSV-1 were used to compare the protective response to these individual glycoproteins in the mouse. Glycoprotein D induced the best neutralizing antibody titers and the most increased rates of HSV clearance from the ear as well as good protection from the establishment of latent HSV infections in the sensory ganglia. Glycoprotein B also induced good neutralizing antibody titers and as great a protection from the establishment of latency as gD although the rate of virus clearance from the ear was not as great as after immunization with gD. Glycoprotein E induced weak neutralizing antibody but gG, gH, and gI did not show a neutralizing antibody response. At higher challenge doses of virus (10(6) PFU HSV-1 in the ear), gE induced a protective response by increasing the rate of virus clearance and reducing the acute infection of ganglia as compared to negative control immunized mice. However there was no protection from the establishment of latent infections after immunization with gE. No protective response was seen to gG, gH, or gl.

Cloning and molecular characterization of the murine herpesvirus 68 genome.

Murine herpesvirus 68 (MHV-68) is a naturally occurring herpesvirus of small free-living rodents. In order to facilitate the molecular characterization of the virus genome, a library of cloned restriction fragments has been produced and restriction enzyme cleavage maps deduced for the enzymes BamHI, EcoRI and HindIII. The MHV-68 genome comprises a region of unique DNA of approximately 118 kbp which is flanked by variable numbers of a 1.23 kb repeat unit. The organization of the MHV-68 genome is, therefore, most similar to that of the lymphotropic gamma 2 group of herpesviruses which include herpesvirus saimiri and herpesvirus ateles.

Production and characterisation of a monoclonal antibody to human papillomavirus type 16 using recombinant vaccinia virus.

A monoclonal antibody was raised against the major capsid protein L1 of human papillomavirus type 16, using a recombinant vaccinia virus that expresses the L1 protein, as a target for screening. This antibody, designated CAMVIR-1, reacted with a 56 kilodalton protein in cells infected with L1-vaccinia virus, and the protein was present in a predominantly nuclear location. The antibody also detects the HPV-16 L1 antigen in formalin fixed, paraffin wax embedded biopsy specimens and on routine cervical smears. The antibody reacts strongly and consistently with biopsy specimens containing HPV-16 or HPV-33, but very weak reactions were occasionally observed with biopsy specimens or smears containing HPV-6 or HPV-11. The potential advantages of using a vaccinia recombinant are (i) the target protein is synthesised in a eukoryotic cell so that its "processing" and location are normal; (ii) cells infected with vaccinia recombinants can be subjected to various fixing procedures similar to those used for routine clinical material. This greatly increases the probability that an identified antibody will be useful in a clinical setting.

Antigenic properties and cellular localization of herpes simplex virus glycoprotein H synthesized in a mammalian cell expression system.

Herpes simplex virus type 1 glycoprotein H (HSV-1 gH) was synthesized in an inducible mammalian cell expression system, and its properties were examined. The gH coding sequence, together with the stable 5' untranslated leader sequence from xenopus beta-globin, was placed under control of the strong promoter from the human cytomegalovirus major immediate-early gene in an amplifiable plasmid which contains the simian virus 40 (SV40) virus origin for replication (ori). This expression vector was transfected into ts COS cells constitutively expressing a temperature-sensitive SV40 T antigen which allows utilization of the SV40 ori at permissive temperatures. The results of transient expression assays at the permissive temperature showed that HSV-1 gH could be synthesized in greater amounts than those produced by a high-multiplicity virus infection. The proteins produced were detected in Western blots (immunoblots) with a HSV-1 gH-specific polyclonal serum raised against a TrpE-gH fusion protein. The transfected gH had an apparent molecular weight of approximately 105,000, intermediate in size to those of the precursor (100,000) and fully processed forms (110,000) of HSV-1 gH from infections. Antigenicity was investigated by reactions with three virus-neutralizing monoclonal antibodies specific for conformational epitopes on gH. Only one of these monoclonal antibodies could immunoprecipitate the synthesized gH. However, equal recognition of the transfected gH was achieved by superinfection with virus. In addition, detectable amounts of gH were not expressed on the cell surface unless the cells were superinfected with virus. Studies with a temperature-sensitive mutant, ts1201, defective in encapsidation showed that the changes in antigenic structure and cell surface expression caused by superinfection with virus were not due simply to incorporation of gH into virions. These results suggest that gH requires additional virus gene products for cell surface localization and formation of an antigenic structure important for its function in mediating infectivity.