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Herpes simplex virus type 1 (HSV-1) is transmitted by close contact, both sexual and nonsexual, and infections are acquired during childhood and adolescence. Herpes simplex virus type 2 (HSV-2), however, is thought to be transmitted mainly by sexual contact. Most HSV-2 infections are consequently expected to occur after the onset of sexual activity. Recent reports indicate an increasing prevalence of HSV-2 on the African continent, but most studies have been performed on adult cohorts. In the present study, we collected sera from Tanzanian children and young persons from 1 to 20 years old, with at least 100 individuals in each age group. Antibodies against HSV-1 and HSV-2 were detected by an in-house Western blot method which was shown to perform well in comparison with a commercial Western blot assay. Type-specific antibodies were also analyzed by two noncommercial enzyme-linked immunosorbent assay methods based upon the antigenicities of branched synthetic oligopeptides corresponding to epitopes in glycoprotein G of HSV-1 or HSV-2. The prevalence of HSV-1 antibodies increased gradually from 73% for the age group of 1 to 4 years to 92% for the age group of 17 to 20 years. The prevalence of HSV-2 antibodies was unexpectedly high, as 15% of the children were infected by the age of 8 years, with the incidence increasing gradually to 40% in the age group of 17 to 20 years. The reason for this unexpectedly high frequency is not clear but could suggest that nonsexual transmission of HSV-2 is more common than previously thought. There was no statistically significant association between seropositivities for HSV-2 and human immunodeficiency virus.

We report the expression and characterization of the putative catalytic subunit (pORF30) and accessory protein (pORF18) of equine herpesvirus 1 DNA polymerase, which are encoded by open reading frames 30 and 18 and are homologous to herpes simplex virus type 1 UL30 and UL42, respectively. In vitro transcription-translation of open reading frames 30 and 18 generated proteins of 136 and 45 kDa, respectively. In vitro-expressed pORF30 possessed basal DNA polymerase activity that was stimulated by pORF18, as measured by DNA polymerase assays in vitro. Purified baculovirus-expressed pORF30 exhibited DNA polymerase activity similar to that of the in vitro-expressed protein, and baculovirus-expressed pORF18 could stimulate both nucleotide incorporation and long-chain DNA synthesis by pORF30 in a dose- and time-dependent manner. The salt optima for activity of both pORF30 and the holoenzyme were substantially different from those for other herpesvirus DNA polymerases. As demonstrated by yeast two-hybrid assays, pORF30 and pORF18 could physically interact, most likely with a 1:1 stoichiometry. Finally, by mutational analysis of the 1,220-residue pORF30, we demonstrated that the extreme C terminus of pORF30 is important for physical and functional interaction with the accessory protein, as reported for UL30 and other herpesvirus DNA polymerases. In addition, a C-proximal region of pORF30, corresponding to residues 1114 to 1172, is involved in binding to, and stimulation by, pORF18. Taken together, the results indicate that pORF30 and pORF18 are the equine herpesvirus 1 counterparts of herpes simplex virus type 1 UL30 and UL42 and share many, but not all, of their characteristics.

Herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2), which are common worldwide, are so similar that antibodies directed against one serotype may crossreact with antigens from the other one. Methods for specific detection of antibodies against HSV-1 or HSV-2 are based upon the antigenicities of glycoproteins G. However, due to the cost, the available commercial methods may not readily be used in developing countries. A different enzyme-linked immunosorbent assay (ELISA) method, based upon a synthetic oligopeptide corresponding to an immunogenic region in glycoprotein G of HSV-2, has been used recently and successfully for detection of HSV-2 antibodies. In the present study, the sequences of a newly identified immunogenic and type-specific region in glycoprotein G of HSV-1 was used to synthesize three different, branched oligopeptides. The performances of these peptides in an ELISA were investigated by testing Scandinavian and African sera which were characterized by commercial ELISA and Western blotting methods and divided into four groups either lacking HSV antibodies, containing antibodies against one or the other virus, or against both types. The peptide which corresponded in sequence to the immunodominant region was as specific and sensitive by an ELISA as were the commercial methods. The method is inexpensive and reliable.

In common with other herpesviruses, the human cytomegalovirus (HCMV) DNA polymerase contains a catalytic subunit (Pol or UL54) and an accessory protein (UL44) that is thought to increase the processivity of the enzyme. The observation that antisense inhibition of UL44 synthesis in HCMV-infected cells strongly inhibits viral DNA replication, together with the structural similarity predicted for the herpesvirus processivity subunits, highlights the importance of the accessory protein for virus growth and raises the possibility that the UL54/UL44 interaction might be a valid target for antiviral drugs. To investigate this possibility, overlapping peptides spanning residues 1161 to 1242 of UL54 were synthesized and tested for inhibition of the interaction between purified UL54 and UL44 proteins. A peptide, LPRRLHLEPAFLPYSVKAHECC, corresponding to residues 1221 to 1242 at the very C terminus of UL54, disrupted both the physical interaction between the two proteins and specifically inhibited the stimulation of UL54 by UL44. A mutant peptide lacking the two carboxy-terminal cysteines was markedly less inhibitory, suggesting a role for these residues in the UL54/UL44 interaction. Circular dichroism spectroscopy indicated that the UL54 C-terminal peptide can adopt a partially alpha-helical structure. Taken together, these results indicate that the two subunits of HCMV DNA polymerase most likely interact in a way which is analogous to that of the two subunits of herpes simplex virus DNA polymerase, even though there is no sequence homology in the binding site, and suggest that the UL54 peptide, or derivatives thereof, could form the basis for developing a new class of anti-HCMV inhibitors that act by disrupting the UL54/UL44 interaction.

Most cellular and viral processes depend on the coordinated formation of protein-protein interactions. With a better understanding of the molecular biology and biochemistry of human viruses it has become possible to screen for and detect inhibitors with activity against specific viral functions and to develop new approaches for the treatment of viral infections. A novel strategy to inhibit viral replication is based on the disruption of viral protein-protein complexes by peptides that mimic either face of the interaction between subunits. Peptides and peptide mimetics capable of dissociating protein-protein interactions have such exquisite specificity that they hold great promise as the next generation of therapeutic agents. This review is focused on recent developments using peptides and small molecules to inhibit protein-protein interactions between cellular and/or viral proteins with comments on the practicalities of transforming chemical leads into derivatives with the characteristics desired of medicinal compounds., (Copyright 2002 John Wiley & Sons, Ltd.)

The herpes simplex virus type 1 DNA polymerase consists of a catalytic subunit (POL or UL30) and a processivity factor (UL42). The POL/UL42 interaction, which occurs through the extreme C-terminus of POL, is essential for HSV-1 replication and thus represents a valid target for drug inhibition. We recently showed (A. Loregian et al. (1999) Proc. Natl. Acad. Sci. USA 96, 5221-5226) that an oligopeptide corresponding to the 27 C-terminal amino acids of POL, when delivered into herpes simplex virus type 1-infected cells by a protein carrier, was able to localize into the nucleus and to inhibit viral replication by disruption of the POL/UL42 interaction. In this report, to further characterize the 27 mer (Pol peptide), we investigated whether its nuclear localization was due to the presence of a nuclear localization signal. By testing the ability of the Pol peptide to localize the beta-galactosidase, a normally cytoplasmic protein, to the nucleus, we confirmed that the Pol peptide contained a functional nuclear localization signal, corresponding to the RRMLHR motif. This sequence proved not only necessary but also sufficient for nuclear localization, because its substitution with a six-alanine stretch prevented nuclear translocation of the beta-galactosidase-Pol peptide fusion. Site-directed mutagenesis experiments on this revealed that both the three basic arginines and the two hydrophobic residues Met and Leu were crucial for nuclear targeting. Finally, functionally equivalent sequences were also found in the C-terminus of the catalytic subunits of human cytomegalovirus (RRLHL) and of equine herpesvirus-1 DNA polymerase (RRILH)., (Copyright 2000 Academic Press.)

Glycoprotein G of HSV-2 (gG2) and a peptide, corresponding to a previously recognised immunodominant epitope spanning residues 561-578 of the protein, were compared directly for type-specific serodiagnosis of HSV-2. The protein was affinity purified and obtained in a commercially available EIA kit while the peptide, previously designated as peptide 55, was made as a multiple antigenic peptide. A panel of 100 characterised serum samples (60 HSV-2 positive, 20 HSV-1 positive and 20 HSV negative) was screened using the two antigens. The intact protein and peptide 55 showed the same sensitivity for antibodies in the serum of HSV-2 infected individuals, reacting with 96.7% (58/60) of the samples. The peptide did not react with any of the HSV-1 positive or HSV negative sera. In contrast, gG2 gave a number of false positive results, reacting with 20% (4/20) of the HSV-1 positive sera and 10% (2/20) of the HSV negative sera. The superior performance of peptide 55, together with the very much lower costs of its production, compared with gG2 suggest that the peptide will become the antigen of choice in enzyme immunoassays for type-specific serodiagnosis of HSV-2.

We report an intracellular peptide delivery system capable of targeting specific cellular compartments. In the model system we constructed a chimeric protein consisting of the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) fused to a 27-mer peptide derived from the DNA polymerase of herpes simplex virus 1. Viral DNA synthesis takes places in the nucleus and requires the interaction with an accessory factor, UL42, encoded by the virus. The peptide, designated Pol, is able to dissociate this interaction. The chimeric protein, EtxB-Pol, retained the functional properties of both EtxB and peptide components and was shown to inhibit viral DNA polymerase activity in vitro via disruption of the polymerase-UL42 complex. When added to virally infected cells, EtxB-Pol had no effect on adenovirus replication but specifically interfered with herpes simplex virus 1 replication. Further studies showed that the antiviral peptide localized in the nucleus, whereas the EtxB component remained associated with vesicular compartments. The results indicate that the chimeric protein entered through endosomal acidic compartments and that the Pol peptide was cleaved from the chimeric protein before being translocated into the nucleus. The system we describe is suitable for delivery of peptides that specifically disrupt protein-protein interactions and may be developed to target specific cellular compartments.

Herpes simplex virus type 1 gene UL4 is predicted to encode a 199-amino-acid protein with a molecular mass of 21 5 kDa. We report here identification of this protein and its localization in the nuclei of infected cells. Antisera raised against oligopeptides corresponding to the C terminus of the predicted UL4 protein were used for identification of a 25 kDa protein as the product of the UL4 gene. This protein was not detected in cells infected with a UL4 defective mutant virus, but was synthesized by coupled in vitro transcription-translation of the UL4 gene. Synthesis of the 25 kDa protein was blocked by phosphonoacetic acid, an inhibitor of DNA synthesis, indicating that the UL4 gene is expressed with gamma kinetics. Subcellular fractionation showed the protein to be localized in the nucleus. It was not detected in virions or light particles.

A series of 67 oligopeptides that spanned the open reading frame of herpes simplex virus type 2 (HSV-2) glycoprotein G (gG2) were synthesized and tested for reactivity with 173 serum specimens collected from 117 individuals. The oligopeptides were made as multiple antigenic peptides consisting of four copies of a unique sequence attached to a branched lysine core and separated from the core by four glycine residues. The sera included HSV antibody-negative samples as well as sera from individuals from whom HSV had been isolated. Isolated viruses were typed by indirect fluorescence using a panel of type-specific monoclonal antibodies. One peptide, corresponding to residues 561 to 578 of gG2, did not react with any sera lacking HSV-specific antibodies of with sera from HSV-1-infected individuals, but did react with sera from HSV-2-infected individuals. For sera taken seven or more days after initialclinical lesions, the detection rate of the peptide was 92% (47/51), comparable with the 98% (50/51) of truncated glycoprotein D, a sensitive type-common reagent. We conclude that this peptide, of structure (PEEFEGAGDGEPPEDDDSG4)K3A, is an immunodominant type-specific epitope for human antibodies and should be useful for type-specific serodiagnosis of HSV-2. Surprisingly, the epitope lies within one of the most conserved regions of gG1 and gG2. The test can distinguish an initial HSV-2 infection in the presence of a preexisting HSV-1 infection.

The UL4 gene of herpes simplex virus type 1 is predicted to encode a 21.5 kDa protein of 199 amino acids. Although UL4 is dispensable for growth in cell culture, its function is not known. In the present study, the promoter of UL4 was examined and found to contain a cAMP-response element which bound the transcription factor CREB, and was strongly activated by cAMP. A recombinant virus, termed UL4HS, was constructed with a nonsense linker inserted into the UL4 open reading frame, to make a truncated UL4 protein of 60 amino acids. In addition, a marker-rescued virus, UL4HSMR, was constructed. Western immunoblot analysis revealed a 23 kDa band in extracts of wild-type and marker-rescued virus infected cells which was missing for UL4HS. Only modest differences were observed in the abilities of wild-type and UL4-mutant viruses to grow in Vero cells or in contact-inhibited mouse C3H/10T1/2 cells. In addition, there were only modest differences between the ability of UL4HS to replicate in murine corneas and trigeminal ganglia relative to wild-type viruses, and reactivation of UL4HS from latency was unaffected. Taken together, these data demonstrate that UL4 is dispensable for latency and pathogenesis in mice.

The herpes simplex virus type 1 (HSV-1) UL8 DNA replication protein is a component of a trimeric helicase-primase complex. Sixteen UL8-specific monoclonal antibodies (MAbs) were isolated and characterized. In initial immunoprecipitation experiments, one of these, MAb 804, was shown to coprecipitate POL, the catalytic subunit of the HSV-1 DNA polymerase, from extracts of insect cells infected with recombinant baculoviruses expressing the POL and UL8 proteins. Coprecipitation of POL was dependent on the presence of UL8 protein. Rapid enzyme-linked immunosorbent assays (ELISAs), in which one protein was bound to microtiter wells and binding of the other protein was detected with a UL8- or POL-specific MAb, were developed to investigate further the interaction between the two proteins. When tested in the ELISAs, five of the UL8-specific MAbs consistently inhibited the interaction, raising the possibility that these antibodies act by binding to epitopes at or near a site(s) on UL8 involved in its interaction with POL. The epitopes recognized by four of the inhibitory MAbs were approximately located by using a series of truncated UL8 proteins expressed in mammalian cells. Three of these MAbs recognized an epitope near the C terminus of UL8, which was subjected to fine mapping with a series of overlapping peptides. The C-terminal peptides were then tested in the ELISA for their ability to inhibit the POL-UL8 interaction: the most potent exhibited a 50% inhibitory concentration of approximately 5 microM. Our findings suggest that the UL8 protein may be involved in recruiting HSV-1 DNA polymerase into the viral DNA replication complex and also identify a potential new target for antiviral therapy.

The nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a convenient carrier molecule for the attachment and delivery of heterologous peptides into eukaryotic cells. To evaluate the properties of such EtxB-based fusion proteins an efficient method for their production and purification is required. High-level production and purification of native EtxB has been achieved using heterologous expression and secretion in a marine Vibrio (Amin, T., and Hirst, T. R., 1994, Protein Expression Purif. 5, 198-204). However, the use of this method to isolate EtxB fusion proteins has been precluded because of their susceptibility to degradation by extracellular proteases secreted by members of the Vibrionaceae. In this paper a method is described for production of EtxB-pol, comprising the enterotoxin B subunit linked to a 27-residue C-terminal fragment of Pol, the catalytic subunit of DNA polymerase of herpes simplex virus type 1 (HSV-1). Following assessment of the relative efficacy of different Vibrio strains as hosts for EtxB-pol expression, the chimera was produced at the highest level of 3.5 mg/liter by cultures of Vibrio sp.60. Addition of 0.3 mM EDTA to the growth medium blocked proteolysis of the secreted EtxB-pol fusion protein, which was then purified to homogeneity using ammonium sulfate fractionation and hydrophobic interaction chromatography, with a yield of 57%. Purified EtxB-pol reacted with both anti-EtxB and anti-Pol peptide antibodies, and was able to specifically bind UL42, a processivity factor which normally binds to the C-terminal region of HSV-1 Pol. This modified method for expression and purification of EtxB-pol should be of general utility for the preparation of other EtxB-based fusion proteins.

We have developed a panel of 14 monoclonal antibodies (MAbs) to POL, the catalytic subunit of herpes simplex virus type 1 (HSV-1) DNA polymerase encoded by gene UL30, and one MAb to the UL52 protein, another of the seven proteins essential for replication of HSV DNA. The approximate locations of the epitopes of the polymerase-specific MAbs were identified using truncated polymerase molecules, and the antibodies were characterized in a number of immunological assays allowing eight different specificities to be recognized. These MAbs, together with a polyclonal antibody raised in rabbits against a third DNA replication protein, ICP8, were used to localize the respective proteins by immunofluorescence in cells infected with wild-type HSV-1 or the DNA replication-defective mutants ambUL8 or 2-2. In BHK cells infected with ambUL8, a mutant with an amber termination codon within the coding region of gene UL8, the UL52 protein did not enter the nucleus, although ICP8 and POL entered the nucleus in a normal fashion. The failure of the UL52 protein to be correctly transported to the nucleus was also observed in both HFL and Vero cells infected with ambUL8. In contrast, UL52 protein was transported to the nucleus in BHK cells infected with wild-type HSV-1 or with 2-2, a mutant lacking a functional UL9 protein.

We have investigated the ability of monkey kidney cell lines (SupD3 and SupD12) inducibly expressing an amber suppressor tRNA(ser) to suppress amber nonsense mutations in three genes of herpes simplex virus type 1 (HSV-1). HSV-1 mutant TK4, which contains a nonsense mutation in the non-essential viral thymidine kinase (TK) gene, synthesized a full-length TK polypeptide at about 30% of the wild-type (wt) level in induced SupD3 cells but not in the parental non-suppressor (Sup0) cells. Using complementing cells, we constructed HSV-1 mutants carrying nonsense mutations in an essential gene, UL8, encoding a protein essential for viral DNA replication (ambUL8) or in a partially dispensable gene, UL12, encoding alkaline nuclease (ambUL12). The growth of the mutants in Vero or Sup0 cells was either totally (ambUL8) or severely (ambUL12) impaired, whereas in cells expressing suppressor tRNA the mutants produced infectious virus. However, the yields were much lower than obtained with wt HSV-1. In Vero or Sup0 cells the mutants ambUL8 and ambUL12 failed to synthesize full-length UL8 and UL12 protein products, respectively. Western immunoblotting showed that the virus ambUL12 produced full-length UL12 protein in SupD12 cells which yielded a level of 25.9% of the alkaline nuclease activity of the wt HSV-1 control. Our results show that the levels of suppression of the nonsense mutations in ambUL8 and ambUL12 are insufficient to allow their continuing propagation in the available Sup+ cells. Possible reasons are discussed.

The amplification and analysis of a 468bp fragment from the gB gene of the serologically related ruminant alphaherpesviruses bovine herpesvirus-1.1 and 1.2 (BHV-1.1 and BHV-1.2), caprine herpesvirus-1 (CapHV-1), cervine herpesvirus-1 (CerHV-1) and rangiferine herpesvirus-1 (RanHV-1) by PCR and restriction endonuclease analysis is described. As primers, 22bp oligomers selected from the BHV-1 gB gene sequences were used for the amplification of the DNA from the five viruses. The amplification product from each virus was analysed by the restriction endonuclease enzymes BglI, HinfI, SmaI and AvaI. The specific amplification obtained demonstrate the existence of the gB gene sequences for each of the five alphaherpesviruses. However, sequences from some of the fragments were found to be different from those predicted from the gB gene following restriction endonuclease analysis. All five amplification products generated the same number of fragments after digestion with HinfI except for two additional bands evident in CapHV-1. The CerHV-1 and RanHV-1 fragments contained slightly different BglI restriction sites from those of the other three. While BHV-1.1, BHV-1.2, CapHV-1 and CerHV-1 contained SmaI and AvaI restriction sites, the RanHV-1 amplification product lacked both SmaI and AvaI restriction sites.

We have analysed the intracellular localisation of herpes simplex virus type 1 ribonucleotide reductase during infection of cultured cells by indirect immunofluorescence using polyclonal and monoclonal antibodies specific for the R1 and R2 subunits. Three different viruses were used to infect cells, wild-type strain 17+ and two temperature-sensitive mutants, ts 1222, which produces R1 only, and ts 1207, which expresses a normal R2 and an altered R1 that fails to interact with R2 at the nonpermissive temperature because of an amino acid substitution in R1. R1 was detected 2 hr postinfection with all three viruses and remained evenly distributed throughout the cytoplasm. R2 was not observed until 4 hr postinfection and, in contrast to the even distribution of R1, was localised in discrete cytoplasmic foci close to the nucleus. In double-labelling experiments both R1 and R2 were found in these foci where they presumably associate to form the active enzyme. As expected R2 was not detectable in cells infected with ts 1222. In ts 1207-infected cells it formed wild-type-like foci, indicating that interaction with R1 is not required for R2 focus formation. R1 was present in a twofold excess over R2 in wild-type-infected cells. We suggest that the uncomplexed R1 could perform a role associated with the protein kinase present in the N-terminal domain.

Several recent reports implicate sequences at or near the C terminus of the catalytic subunit (POL) of herpes simplex virus type 1 (HSV-1) DNA polymerase in its interaction with the accessory protein UL42. We have investigated further the involvement of this region by three different approaches: anti-idiotype antibodies, a competition ELISA and inhibition of the interaction by peptides. Antibodies raised in rabbits to peptides corresponding to regions of POL all reacted in Western blots with POL. Surprisingly, the sera raised against C-terminal peptides (amino acids 1221 to 1235 and 1224 to 1235) also reacted with UL42. The UL42 reactivity was shown to be due to the presence of anti-idiotype antibodies, providing direct evidence for complementarity of the structure of the extreme C terminus of POL to a region of UL42. To measure the contribution of the C terminus of POL to UL42 binding we developed a competition ELISA using POL, a truncated polymerase lacking the carboxyl-terminal 27 amino acids (POLd1) and UL42. UL42 binding to immobilized POL was inhibited approximately four times more effectively by competition, in solution, with POL than with POLd1, indicating that the C-terminal 27 amino acids of POL are responsible for at least 75% of the binding energy. A peptide corresponding to these 27 amino acids (residues 1209 to 1235) inhibited both the POL-UL42 interaction and the stimulation of POL by UL42 and did so more effectively than peptides corresponding to amino acids just away from the C terminus (residues 1195 to 1223 and 1177 to 1195).

The products of herpes simplex virus type 1 (HSV-1) genes UL5, UL8 and UL52 form a complex in virus-infected cells that exhibits both DNA helicase and DNA primase activities. UL8 protein was purified from insect cells infected with a recombinant baculovirus and used to generate monoclonal antibodies (MAbs). MAb 0811 was shown to recognize the UL8 protein in both Western blots and immunoprecipitation assays and to co-precipitate the other two proteins in the complex from insect cells triply infected with recombinants expressing the UL5, UL8 and UL52 polypeptides. Experiments performed using extracts from doubly infected cells indicated that UL8 could interact separately with both the UL5 and UL52 proteins. Similar experiments using a recombinant virus that expressed the HSV-1 origin-binding protein (OBP), UL9, demonstrated a direct physical interaction between the helicase-primase complex and OBP which involved the UL8 subunit. The C-terminal DNA-binding domain of OBP is dispensable for this interaction, as evidenced by the ability of MAb 0811 to co-precipitate a truncated UL9 protein, containing only the N-terminal 535 amino acids, with UL8.

The region between the 'a' sequence and the 5' end of the IE1 gene within the long repeat sequence of the herpes simplex virus (HSV) genome plays an important role in the neurovirulence of both HSV-1 strain F and HSV-1 strain 17. However, there has been controversy over the protein-coding potential of this region. Although an open reading frame (ORF) was predicted in HSV-1(F) and shown to encode a polypeptide called ICP34.5, only recently has a corresponding ORF, designated RL1, been recognized in HSV-1(17). To determine whether the HSV-1(17) ORF is expressed, we raised antipeptide sera against predicted amino acid sequences from RL1; one serum specifically recognized a 37K protein in HSV-1(17)-infected cell extracts. Compared with the corresponding HSV-1(F) polypeptide the HSV-1(17) protein has a lower apparent M(r), shows similar kinetics of accumulation and intracellular localization but may accumulate to lower levels than the HSV-1(F) protein. The non-neurovirulent HSV-1(17) deletion variant 1716 fails to synthesize detectable levels of ICP34.5. Thus we have established that HSV-1(17), like HSV-1(F), expresses ICP34.5, a protein important for HSV neurovirulence.

The large subunits of herpes simplex virus types 1 and 2 ribonucleotide reductases contain unique amino-terminal regions comprising 311 and 318 residues respectively, which are not found in ribonucleotide reductases from other sources. We report the mapping of the epitope recognized by monoclonal antibody 1026, which is specific for the large subunit (R1) of HSV-1, and then deduce the structural relationship of the amino-terminal region of R1 with the rest of the protein. A panel of 10 fusion proteins containing sequences spanning the entire R1 subunit were constructed. They were used together with proteolytic fragments of R1 and several synthetic peptides to show that the epitope is discontinuous and appears to be a loop structure centered on a previously located trypsin-sensitive site at residue 305. The existence of the loop was suggested by the observation that reactivity of the antibody with R1 could be blocked by peptides corresponding to residues 289 to 303 and 308 to 313 which flank the trypsin-sensitive site. Our results suggest that the unique amino-terminal region of R1 consists of a structurally distinct domain which is linked to the conserved carboxy region by an exposed loop.

A rapid and simple two-step scheme for the purification of herpes simplex virus type 1 UL8 protein from insect cells infected with a recombinant baculovirus has been developed. The scheme involves DEAE-Sepharose and phenyl-Sepharose chromatography and yields approximately 1.5 mg of protein from 2.4 x 10(8) infected cells. The protein remains intact during purification as judged by its reactivity with amino and carboxy termini-specific antisera. Gel filtration chromatography showed that the protein exists as a monomer in solution. No binding of the protein to ssDNA or dsDNA or to a DNA/RNA hybrid could be demonstrated using a gel mobility shift assay.

To identify regions in the UL42 protein of herpes simplex virus type 1 which affect viral DNA polymerase activity, a series of 96 overlapping pentadecapeptides spanning the entire 488 amino acids of the UL42 protein were synthesized and tested for their ability to inhibit polymerase activity on a primed single-stranded M13 DNA template. Two assays were used: formation of full-length double-stranded M13 molecules and rate of incorporation of deoxyribonucleoside triphosphates. Peptides from five noncontiguous regions of the UL42 protein were found to inhibit herpes simplex virus type 1 polymerase activity in both the presence and absence of UL42 protein. The most active peptides from each region correspond to amino acids 23 to 38 (peptide 6), 64 to 78 (peptide 14), 89 to 102 (peptide 19), 229 to 243 (peptide 47), and 279 to 293 (peptide 57). By two different methods (DNA mobility shift and DNA precipitation), peptides 14, 19, 47, and 57 were found to bind DNA; they most probably inhibit enzyme activity by this mechanism. Peptide 6 did not bind DNA and must act by some mechanism other than competing for DNA. The inhibitory peptides were also tested for activity against mammalian polymerase alpha and the Klenow fragment of Escherichia coli polymerase. Although some limited specificity was demonstrated (up to 10-fold for peptide 6), all the peptides showed significant activity against both polymerase alpha and E. coli polymerase.

We describe a rapid method of fragment condensation to couple a helper T cell epitope, active in BALB/c mice, to the amino terminus of branched peptides (or 'multiple antigenic peptides', MAPs). The helper T cell epitope-MAP conjugate considerably enhanced the immunogenicity in BALB/c mice of branched peptides. The method of fragment condensation, whereby the helper T cell epitope portion of the immunogen is added as a 'cassette' in a one step addition process, is both faster and more convenient than continuous step-by-step addition of individual amino acids and is likely to be generally applicable. The method should be advantageous in the development of peptide based vaccines.

Immunodominant antibody-binding sites were mapped using overlapping synthetic peptides of the structural proteins p17 and p24 of human immunodeficiency virus type 1 (HIV-1). Using sera from HIV-1-infected individuals at a variety of disease states, three major epitopes were identified within p17 and one within p24. Antibodies which recognized these epitopes were present in all risk groups throughout all stages of HIV infection, regardless of the presence of high levels of serum p24 antigen.

Fourteen hybridoma cell lines secreting monoclonal antibodies (Mab) to cervine herpesvirus-1 (CerHV-1) produced following the fusion of NSO myeloma cells with splenocytes of BALB/c mice previously immunized with gradient purified CerHV-1 were selected using an indirect enzyme-linked immunosorbent assay (ELISA) employing CerHV-1 antigen and tested by the ELISA against four other ruminant alphaherpesviruses from cattle (bovine herpesvirus type 1.1 and 1.2) goat (caprine herpesvirus-2) and reindeer (rangiferine herpesvirus-1). Comparison of all five ruminant alphaherpesviruses with these Mabs confirmed their close antigenic relationships, with two Mabs reacting against all viruses. Ten Mabs which were able to differentiate between the viruses reacted with a 64 kDa polypeptide in a western blot. Four Mabs including two specific only for CerHV-1 with neutralizing activity against the virus used for immunization were directed against a 74 kDa viral protein.

A retrospective study of sera from mothers infected with human immunodeficiency virus (HIV-1) was undertaken to investigate whether the titers or affinities of antibodies against the third hypervariable region (V3 loop) of gp120 correlated with transmission of the virus from mother to child. The cohort comprised 7 mothers who transmitted HIV-1 to their children and 20 who did not. Sera were screened for reactivity against two synthetic peptides, one encompassing the entire V3 loop of gp120 (amino acids 297-330) and the other containing an immunodominant epitope from gp41 (amino acids 596-614). Doubling dilutions of sera were tested to obtain antibody titers against both peptides: Anti-gp41 titers were used to normalize the anti-V3 titers. Maternal sera were also screened for the presence of high-affinity antibodies against the V3 peptide. No differences were observed in either titers or affinities of maternal antibodies to the V3 sequence from transmitters and nontransmitters.

We have raised antisera and monoclonal antibodies (MAbs) to the C4 region of HIV-1 gp120, using an antigen chimaera of poliovirus as immunogen. These MAbs and sera, together with MAbs to the same region raised by other methods, fall into three groups defined by their abilities to bind to recombinant gp120 and/or the immunogenic peptide. In some cases, the amino acids recognized by the MAbs have been identified by pep-scan and by solution phase peptide inhibition of binding to recombinant gp120. Our results indicate that the amino acids WQEVGKAMYA are exposed on the surface of recombinant gp120. Antibodies to these amino acids on recombinant gp120 compete for soluble CD4 binding in vitro, but only weakly neutralize HIV.

The reactivities of antibodies with branched and monomeric peptides were compared in ELISA assays. We found that lower amounts of antibodies could be detected with branched peptides than with monomeric peptides. This was observed with a monoclonal antibody and with antibodies in the sera of various HIV-positive individuals. To investigate the physical aspects of branched peptides important for the observed increase in sensitivity, glycine spacers of different lengths were introduced between the branched lysine core and the epitope reacting with the monoclonal antibody. The effect of the number of glycine residues, both on the sensitivity of antibody detection and on the amount of branched peptide needed to produce a given signal, was studied and the optimum was found at 4-5 residues. We discuss the basis for these findings and conclude that the routine use of branched peptides for serodiagnosis will give both greater sensitivity and appreciable cost savings.

The herpes simplex virus type 1 (HSV-1) gene encoding the ribonucleotide reductase (RR) small subunit (R2) was cloned as an unfused and intact open reading frame into a T7 RNA polymerase expression system in Escherichia coli. The expressed product was recovered from bacteria in soluble form and constituted 7% of the soluble protein. Protein purification yielded 3.5 mg of 95% pure R2 per litre of bacterial culture. The correct composition of the purified protein was verified by amino acid analysis and N-terminal sequencing. The isoelectric point of the protein was 5.3. Atomic emission spectroscopy indicated that the iron content of the E. coli-expressed R2 was 0.2 to 0.5 atoms of iron per R2 protomer as compared with a theoretical maximum value of 2. The E. coli-expressed HSV-1 R2 existed as a combination of a stable dimer and monomer. Combination of the E. coli-expressed R2 with the E. coli-expressed large subunit (R1) gave an active holoenzyme. Thus, the T7 expression system provides a rich source of enzymically active HSV-1 RR.

The open reading frame of the large subunit (R1) of herpes simplex virus type 1 (HSV-1) ribonucleotide reductase has been positioned downstream of the phage T7 gene 10 promoter in the expression vector, pET. Transformation of this recombinant plasmid into Escherichia coli BL21 DE3 cells containing the T7 RNA polymerase, under the control of the lac UV5 promoter, allows expression of the subunit on induction of the T7 RNA polymerase by isopropyl thiodigalactoside. The expressed protein is soluble and can be purified with yields up to 0.5 mg of R1 per litre of bacterial culture. The subunit can complement R2 produced in BHK cells or E. coli to give specific activities comparable to that produced in BHK cells infected with HSV-1. Enzyme activity reconstituted from E. coli-expressed R1 and R2 is inhibited by the nonapeptide YAGAVVNDL with an IC50 comparable to that obtained with enzyme extracted from BHK cells infected with HSV-1. Results suggest that the E. coli produced enzyme is a good source of protein for further structural and functional studies.

The technique of Fmoc chemistry has been applied successfully to the synthesis of branched peptides. The immunogenicity of branched peptides has been compared quantitatively with those of protein-conjugated and resin-linked peptides. Six different peptide sequences were used to immunise rabbits and both antipeptide and anti-protein titres were determined for each serum. The data show that the titres of sera from rabbits immunised with branched peptides were higher than those of rabbits immunised with protein-conjugated peptides which in turn were higher than those immunised with resin-linked peptides. The effect was demonstrated with two strains of rabbits.

The herpes simplex virus type 1 temperature-sensitive (ts) mutant ts1207 does not induce detectable levels of ribonucleotide reductase activity at the non-permissive temperature (NPT, 39.5 degrees C). The ts lesion prevents the association of the enzyme's large (RR1) and small (RR2) subunits to give an active holoenzyme and maps within the gene specifying RR1. Here, it is shown that the ts mutant phenotype is due to the substitution of an asparagine for the wild-type (wt) serine at RR1 position 961, which is located within a region highly conserved between herpesviral and cellular RR1 subunit polypeptides. This ts1207 asparagine is predicted to alter a wt alpha-helix to a beta-strand. We have used synthetic oligopeptides, corresponding to the wt amino acid sequence of the mutation site, and antisera raised against them to determine whether this region is involved in subunit association. Neither the oligopeptides nor the antisera inhibit the enzyme activity, or the reconstituted activity formed by mixing intact RR2 and RR1 subunits present in partially purified extracts of cells infected at the NPT with ts1207 or ts1222 (an HSV-1 mutant with a lesion in the RR2 subunit), respectively. We infer from these results that the site of the mutation is unlikely to be positioned at the surface of RR1 and hence is probably not directly involved in subunit association. We suggest that the mutation site identifies an important RR1 region whose alteration in ts1207 changes the structure of a contact region(s) positioned at the RR1/RR2 interface.

We have previously shown that aminoglycosides such as neomycin and the polyamino acids polylysine and polyarginine selectively inhibit the binding of herpes simplex virus type 1 (HSV-1) to the cellular receptor, whereas HSV-2 infection is unaffected. In the present study we took advantage of this difference between HSV-1 and HSV-2 by using HSV(-1)-HSV(-2) intertypic recombinants to locate a region on the HSV-1 genome encoding proteins affecting the binding of the virion to the cellular receptor. The results were consistent with those obtained by marker rescue experiments. The identified region, which mapped between coordinates 0.580 and 0.687, contains two partial and eight complete genes, including the glycoprotein C (gC) gene and two others with potential transmembrane sequences. Various gC monoclonal antibody-resistant mutants of HSV-1 as well as a mutant completely lacking gC were found to be fully sensitive to neomycin, suggesting that gC is not the site of drug sensitivity and is not essential for adsorption of virus to the cellular receptor. However, the rate of adsorption was reduced in the absence of gC, indicating a facilitating function of the glycoprotein. The universal nature of this HSV-1 receptor binding was revealed by the similarity in drug sensitivity of infectivity in four different cell lines from various tissues and species.

Using the drug tunicamycin we have investigated the nature of the oligosaccharides on herpes simplex virus type 1 (HSV-1)-induced glycoproteins E and Y (gY is a newly identified glycoprotein which has the same apparent mol. wt. as gC but a more basic isoelectric point). Synthesis of both glycoproteins was inhibited by the drug, suggesting they contain N-linked oligosaccharides. Our finding, combined with the previous results of other workers, suggests that all the major HSV-induced glycoproteins have this type of carbohydrate modification. All of the major HSV-1-induced glycoproteins are modified by addition of inorganic sulphate. This modification occurs late in their maturation. Most inorganic sulphate appears to be attached to N-linked oligosaccharides but some is attached to other parts of glycoprotein E. Using HSV-1/HSV-2 intertypic recombinants, the mapping limits of that part of the glycoprotein E gene coding for differences in mobility between the two serotypes have been further narrowed and are located between coordinates 0.886 and 0.935.

Two hundred and thirty virus-induced polypeptides have been detected in BHK cells infected with herpes simplex virus type 1 (HSV-1; strain 17) by means of two-dimensional gel electrophoresis. The polypeptides have been characterized by both relative mobility following isoelectric focusing and apparent mol. wt. in SDS-polyacrylamide gels. Some polypeptides, visualized as a single band on a one-dimensional SDS-polyacrylamide gel, were resolved into several spots. Three were identified in Vmw43, the band thought to contain thymidine kinase activity. Not all the observed polypeptides are unique species: some appear to be related and have altered mobilities as a consequence of post-translational modification events. Pulse-chase experiments and treatment of infected cells with neuraminidase suggested that glycoproteins gB, gC and gD contain sialic acid and that synthesis of gB and gD occurs by at least 15 and 10 discrete steps respectively.

Evidence is presented showing that the 92,000-dalton glycoprotein (g92K) induced by herpes simplex virus (HSV) type 2 has properties distinct from those assigned to any other HSV glycoprotein. First, the carbohydrate composition and extent of sulfation differ from those of glycoproteins D and E. Second, two clonally unrelated monoclonal antibodies, AP1 and LP5, shown in this paper to specifically immunoprecipitate g92K, do not react with any of the known processed forms of glycoproteins B, C, D, and E. Third, by using HSV type 1/HSV type 2 intertypic recombinants and a simple radioimmunoassay, the target antigen of the two monoclonal antibodies was shown to map in the same region as g92K (0.846 to 0.924). Fourth, the intertypic recombinant R12-3 was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of infected cells to induce the HSV type 2 g92K and HSV type 1 gD and GE, whereas R12-1, which did not induce g92K, induced HSV-2 gE and an altered gD, providing genetic evidence that g92K is encoded, at least in part, by a different region of the genome from that encoding gD and gE.

Gene US9 of herpes simplex virus type 1 has been predicted, from DNA sequence analysis, to encode a protein of mol wt 10,026, designated 10K (D.J. McGeoch, A. Dolan, S. Donald, and F.J. Rixon (1985). J. Mol. Biol. 181, 1-13). We have investigated this protein by using a synthetic peptide corresponding to the 11 amino acids adjacent to the amino-terminal methionine and rasing antisera in rabbits. One antiserum was able to precipitate at least 12 electrophoretically distinct polypeptide species from extracts of BHK cells infected with HSV-1. The estimated molecular weights of these polypeptides ranged from 12K to 20K and immunoblotting showed them to be related proteins. The primary translation product has an apparent mol wt of 13K. The various forms of 10K differ in their relative abundance in the infected cell and also in their degree of phosphorylation. Lower molecular weight forms of the 10K protein can be precipitated from NP-40 extracts of HSV-1 virions, suggesting that these forms of 10K are contained in the virion tegument or envelope. An association between this protein and nucleocapsids has also been observed in the nuclei of infected cells by immunoelectron microscopy. These observations imply that the product of US9 is a tegument protein which becomes associated with nucleocapsids at, or soon after, their formation in the nuclei of infected cells.

The genes of herpes simplex virus type 1 (HSV-1) can be divided into at least three temporally regulated groups termed immediate early (IE), early and late. We have studied in detail the expression of a member of the late class of genes, US11, which encodes a polypeptide of apparent molecular weight 21K. Highly specific and sensitive probes were used to monitor US11 RNA and protein synthesis during HSV-1 infection of tissue culture cells in the presence and absence of phosphonoacetic acid, an inhibitor of viral DNA replication. The results were compared with a similar study of the products of a delayed early gene, US6, encoding glycoprotein D (gD). It was found that the patterns of RNA and protein synthesis from US11 were significantly different to those of gD. US11 products appeared later and accumulated until late in infection, while gD RNA was significantly reduced at late times. In the presence of the inhibitor of DNA synthesis, US11 gene expression was reduced 50- to 100-fold while gD expression was reduced five- to tenfold. We conclude that US11 behaves as a true late gene during HSV-1 infection. However, the use of sensitive assays, which allowed the detection of very low levels of US11 gene products under conditions designed to eliminate DNA replication, brings into question the absolute requirement for DNA replication for the expression of a true late HSV-1 gene. These results are discussed in terms of current models for the regulation of late gene expression.

Gene US4 of herpes simplex virus type 1 (HSV-1) has been predicted, from DNA sequence analysis, to encode a protein of molecular weight 25237 and its properties suggest it to be a membrane-associated protein. We have investigated this protein by raising antiserum to a synthetic oligopeptide corresponding to a stretch of amino acids from an internal hydrophilic region of the predicted sequence. This antiserum immunoprecipitates three glycoprotein species of apparent mol. wt. 37 000, 48 000 and 56 000 from extracts of cells infected with HSV-1. These species are also specifically immunoprecipitated from purified virions. The in vitro translation product of gene US4 has an apparent mol. wt. of 35 000. Sequence comparisons of the short unique regions of the HSV-1 and HSV-2 genomes, in combination with published mapping data for glycoprotein G (gG) of HSV-2, has led to the conclusion that the product of gene US4 of HSV-1 is the equivalent of gG.

A hybridoma line was isolated which produced antibody reacting with polypeptides of apparent molecular weights 32,000, 34,000 and 35,000 (32K/34K/35K) and 55,000 and 57,000 (55K/57K). These were sulphated glycoproteins that have previously been found in the medium of herpes simplex virus type 1 (HSV-1)-infected cells. By tryptic peptide mapping and serological cross-reactions the polypeptides were shown to be related to HSV-1 glycoprotein E (gE-1) but they lacked the Fc binding function. The 32K/34K/35K and 55K/57K polypeptides were not found in the medium of HSV-1-infected cells incubated in serum-free medium. They could be generated in vitro from purified gE-1 in the presence of serum. It is likely that 32K/34K/35K and 55K/57K are derived from gE-1 by the action of serum proteases.

Previously we have described the isolation of seven monoclonal antibodies (MAbs) and two polyvalent rabbit sera directed against the product of herpes simplex virus type 1 (HSV-1) gene UL42, a 65K DNA-binding protein (65KDBP) which is essential for HSV DNA replication and virus growth. We now report the synthesis of all 483 overlapping hexapeptides of this 488 amino acid protein and describe their use for the identification of epitopes recognized by these MAbs and polyvalent sera. MAb 6898, derived from one fusion, recognizes the peptides EDLDGA and DLDGAA which correspond to amino acids 363 to 369 of 65KDBP. MAbs Z4D4, Z6F3, Z1A8, Z10Cl, Z3H12 and Z1F11, derived from a second fusion, all recognize the peptides GDPEDL and DPEDLD which correspond to amino acids 360 to 366. As expected both polyvalent sera recognize several different epitopes.

Analysis of the polypeptides induced by 29 herpes simplex virus type 1/type 2 intertypic recombinants and correlation of the data with the crossover points in the recombinant DNAs have enabled the map positions of many polypeptides to be deduced. These include 25 polypeptides which label with [35S]methionine, 11 which label with [32P]orthophosphate, and 4 which label with [14C]glucosamine. Together with the data of Preston et al. (J. Virol., in press) on the mapping of five immediate-early polypeptides, the results show that representatives of four groups of proteins--immediate-early, late, phosphorylated, and glycosylated--map in both long and short regions. The functional organization of the herpes simplex virus genome does not therefore restrict any of these four groups to either the long or the short region.

Herpes simplex virus type 1 (HSV-1) encodes a polypeptide of apparent mol. wt. 136 000 (Vmw136) known to be a component of the virus-specified ribonucleotide reductase. Monoclonal antibodies that precipitate this polypeptide also precipitate a polypeptide of mol. wt. 38 000 (Vmw38) from extracts of HSV-1-infected cells. The basis for this co-precipitation has been investigated using a monoclonal antibody directed against Vmw136 and an oligopeptide-induced antiserum directed against the carboxy terminus of Vmw38. We have also made use of a temperature-sensitive (ts) mutant of HSV-1 which maps within the sequences encoding Vmw136 and which induces a thermolabile ribonucleotide reductase. Our experiments show (i) Vmw136 and Vmw38 form a complex in infected cells and (ii) the mutation in the ts mutant results in the two polypeptides being unable to form the complex at the non-permissive temperature. We speculate that association of the two polypeptides is necessary for ribonucleotide reductase activity. No evidence was found for involvement of host proteins in the proposed virus-induced ribonucleotide reductase complex. The terms RR1 and RR2 are suggested for the large and small subunits of the HSV-induced enzyme.

A protein factor has been found in serum which converts the M form of placental alkaline phosphatase (PLAP) to the A and B forms. The identity of the conversion products has been confirmed by analysis of their dimers and polypeptides. Proteolysis is not implicated in this phenomenon. This report establishes microvillous M-PLAP as the precursor of the A and B forms.

Previous studies (H.S. Marsden, L. Haarr, and C.M. Preston, J. Virol. 46:434-445, 1983) have shown that at least three polypeptides, with molecular weights of 43,000, 39,000, and 38,000, are encoded by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene. It has been suggested that the 39,000- and 38,000-molecular-weight polypeptides arise from preinitiation complexes bypassing the first and second AUG codons before commencement of translation since, according to previous work (M. Kozak, Nucleic Acids Res. 9:5233-5252, 1981), these codons are not of the most efficient structure for initiation. This possibility was investigated by using specific herpes simplex virus mutants with alterations in the TK gene. Mutant TK4 has an amber mutation between the first and second AUG codons, whereas mutant delta 1 has a deletion which removes the first AUG codon but leaves other AUG codons, as well as transcriptional promoter sequences, intact. Both mutants synthesized only the 39,000- and 38,000-molecular-weight polypeptides, and the amounts produced were normal in TK4-infected cells but increased in delta 1-infected cells. Furthermore, the levels of TK produced after infection with the mutant viruses correlated with the amounts of the 39,000- and 38,000-molecular-weight polypeptides synthesized. The 43,000-, 39,000-, and 38,000-molecular-weight polypeptides were shown to be related by their positive reaction with anti-TK serum in both immunoprecipitation and immunoblotting experiments. The production of the 39,000- and 38,000-molecular-weight polypeptides through bypassing of the first AUG codon was examined by hybrid arrest experiments with a DNA fragment complementary to only 50 bases at the 5' terminus of TK mRNA. This fragment arrested the synthesis of the 30,000- and 38,000-molecular-weight polypeptides when annealed to mRNA from wild-type HSV-1- or TK4-infected cells, showing that those polypeptides arise from an mRNA initiated upstream from the first AUG codon. mRNA from cells infected with mutant delta 1, which lacks DNA sequences upstream from the first AUG, was not affected by the 50-base-pair fragment. The data therefore confirm that three polypeptides encoded by the HSV-1 TK gene arise by differential use of in-phase AUG codons for the initiation of protein synthesis. This mechanism for the production of related but distinct polypeptides has not previously been demonstrated in a eucaryotic system, and the implications for the regulation of TK enzyme activities are discussed.

The HSV-1/HSV-2 intertypic recombinant Bx1 (28-1) is heterotypic for the repeat sequences flanking the long unique region of the genome (IRL and TRL) and expresses both the immediate-early polypeptide IE 110 of HSV-1 and its functionally equivalent polypeptide IE 118 of HSV-2. The genome structures of five subclones of this recombinant and the immediate-early polypeptides they induce have been analysed. Subclone 14 lacked most of the IRL sequence, including the region from which part of the mRNA for IE 110 is transcribed, and expressed only the HSV-2 IE 118. Subclone 22 lacked almost all of TTL including the gene for IE 118 and induced only the HSV-1 IE 110. Since both subclones produced viable progeny in HFL cells it follows that expression of only one copy of the equivalent genes in TRL and IRL, here coding for the distinguishable polypeptides IE 110 or IE 118, is sufficient for successive complete cycles of virus replication.