Herpes Simplex Virus DNA Polymerase Research Articles

Quantitative analysis of herpes simplex virus in cranial nerve ganglia.

A susceptible individual exposed to herpes simplex virus (HSV) will develop latent infection in multiple cranial nerve ganglia. There are a few quantitative studies of the viral load within the trigeminal ganglion, but none that investigate other cranial nerve ganglia. In this study, human trigeminal, geniculate, vestibular (Scarpa's) and cochlear (spiral) ganglia were obtained from willed body donors. Real time quantitative polymerase chain reaction (PCR) analysis of the HSV DNA polymerase gene was performed on ipsilateral ganglion sets from the same individual. Viral load, expressed as HSV genomes per 105 cells, was significantly greater in the vestibular ganglion (mean +/- SD, 176705 +/- 255916) than in the geniculate (9948 +/- 22066), cochlear (3527 +/- 9360), or trigeminal (2017 +/- 5578) ganglia. There was not a significant correlation among ganglia from the same individual. The results support the hypothesis that neuronal subpopulations have variable susceptibility to HSV infection.

The Herpes Simplex Virus Processivity Factor, UL42, Binds DNA as a Monomer

The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is a monomer in solution. However, UL42 is structurally similar to sliding clamp processivity factors, such as PCNA, which encircle DNA as a multimeric ring. We used chemical crosslinking and electrophoretic mobility-shift assays to investigate whether UL42 oligomerizes upon DNA binding. UL42 did not form intermolecular crosslinks upon treatment with glutaraldehyde in the presence of DNA, whereas proteins that are known to be multimers in solution were successfully crosslinked by this treatment. This result suggests that UL42 does not form multimers on DNA. We next analyzed the composition of UL42:DNA complexes using electrophoretic mobility-shift assays. UL42 was mixed with a maltose-binding protein–UL42 fusion protein before being added to DNA. The patterns of electrophoretic mobility of the resultant protein:DNA complexes were those predicted if each isoform of UL42 binds to DNA as a monomer. From this result and the failure of UL42 to form crosslinks, we infer that UL42 binds DNA as a monomer.

Herpes simplex virus and Meniere's disease.

This study was designed to investigate the hypothesis that Meniere's disease is associated with herpes simplex virus (HSV) reactivation in the vestibular ganglion. Case control study. Vestibular ganglia were obtained from archival surgical pathology specimens from patients undergoing vestibular neurectomy for vertigo caused by Meniere's disease. All patients met criteria for classification as definite Meniere's disease according to American Academy of Otolaryngology/Head and Neck Surgery (AAO-HNS) criteria. Control specimens were obtained from willed body donors. Sections from each ganglion were studied for prevalence of viral DNA using a nested polymerase chain reaction designed to amplify the HSV DNA polymerase gene. Quantitative analysis determined the number of viral copies per standard unit of ganglionic DNA. HSV DNA was more prevalent in paraffin embedded ganglia from patients with Meniere's disease (100%) than in fresh-frozen control ganglia (81%) (P =.02). Fixation and paraffin embedding substantially reduced recovery of HSV virus in selected control specimens. Quantitative analysis found no correlation between viral copy number in control ganglia processed frozen versus formalin fixed and paraffin embedded. HSV is more commonly isolated from vestibular ganglia of patients with Meniere's disease than the general population. The routine histologic preparation of formalin fixation and paraffin embedding significantly altered the quantity of virus detected though not in a predictable manner. The study provides supportive evidence for a viral etiology in Meniere's disease.

Inhibition of human cytomegalovirus DNA polymerase by C-terminal peptides from the UL54 subunit.

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.

Exonuclease-deficient polymerase mutant of herpes simplex virus type 1 induces altered spectra of mutations.

The effect of exonuclease activity of the herpes simplex virus DNA polymerase (Pol) on DNA replication fidelity was examined by using the supF mutagenesis assay. The recombinants with exonuclease-deficient Pol, containing an integrated supF gene in the thymidine kinase locus (tk), exhibited supF mutation frequencies ranging from 0.14 to 5.6%, consistent with the tk mutation frequencies reported previously (Y. T. Hwang, B.-Y. Liu, D. M. Coen, and C. B. C. Hwang, J. Virol. 71:7791-7798, 1997). The increased mutation frequencies were 10- to 500-fold higher than those observed for wild-type Pol recombinants. The increased mutation frequencies also were significantly higher than those of supF mutant replicated by exonuclease-deficient Pols in the plasmid-borne assay. Furthermore, characterization of supF mutants demonstrated that recombinants with a defective exonuclease induced types and distributions of supF mutations different from those induced by wild-type Pol recombinants. The types of supF mutations induced by exonuclease-deficient recombinants differed between the plasmid- and genome-based assays. The spectra of supF mutations also differed between the two assays. In addition, exonuclease-defective viruses also induced different spectra of supF and tk mutations. Therefore, both the assay methods and the target genes used for mutagenesis studies can affect the repication fidelity of herpes simplex virus type 1 Pol with defective exonuclease activity.

A double-blind, randomized, placebo-controlled trial of acyclovir in late pregnancy for the reduction of herpes simplex virus shedding and cesarean delivery

Objective: The purpose of this study was to assess the efficacy of acyclovir in the reduction of herpes simplex virus culture and polymerase chain reaction positivity and cesarean delivery. Study Design: Women with recurrent genital herpes simplex virus were randomized to acyclovir 400 mg three times daily or placebo from 36 weeks of gestation until delivery. A subset of daily specimens for herpes simplex virus culture and DNA polymerase chain reaction was self-collected. Analyses used χ2, Fisher exact, and Mann-Whitney U tests. Results: Lesions occurred at delivery among 11 of 78 women (14%) who received placebo and 4 of 84 women (5%) who received acyclovir (P =.08). Herpes simplex virus culture and polymerase chain reaction positivity near delivery occurred in 7% and 34% women in the placebo group and 0 and 2% in the acyclovir group (P =.03 and <.01, respectively). Cesarean delivery for herpes simplex virus occurred in 8 of the women (10%) in the placebo group and in 3 of the women (4%) in the acyclovir group (P =.17). Despite reductions in herpes simplex virus detection, 6% of the women who received acyclovir had herpes simplex virus detected by polymerase chain reaction on >20% of days. Neonatal outcomes were similar between groups. Conclusion: Acyclovir significantly reduced, but did not eliminate, herpes simplex virus lesions and detection in late pregnancy. (Am J Obstet Gynecol 2003;188:836-43.)

Evidence against a simple tethering model for enhancement of herpes simplex virus DNA polymerase processivity by accessory protein UL42.

The DNA polymerase holoenzyme of herpes simplex virus type 1 (HSV-1) is a stable heterodimer consisting of a catalytic subunit (Pol) and a processivity factor (UL42). HSV-1 UL42 differs from most DNA polymerase processivity factors in possessing an inherent ability to bind to double-stranded DNA. It has been proposed that UL42 increases the processivity of Pol by directly tethering it to the primer and template (P/T). To test this hypothesis, we took advantage of the different sensitivities of Pol and Pol/UL42 activities to ionic strength. Although the activity of Pol is inhibited by salt concentrations in excess of 50 mM KCl, the activity of the holoenzyme is relatively refractory to changes in ionic strength from 50 to 125 mM KCl. We used nitrocellulose filter-binding assays and real-time biosensor technology to measure binding affinities and dissociation rate constants of the individual subunits and holoenzyme for a short model P/T as a function of the ionic strength of the buffer. We found that as observed for activity, the binding affinity and dissociation rate constant of the Pol/UL42 holoenzyme for P/T were not altered substantially in high- versus low-ionic-strength buffer. In 50 mM KCl, the apparent affinity with which UL42 bound the P/T did not differ by more than twofold compared to that observed for Pol or Pol/UL42 in the same low-ionic-strength buffer. However, increasing the ionic strength dramatically decreased the affinity of UL42 for P/T, such that it was reduced more than 3 orders of magnitude from that of Pol/UL42 in 125 mM KCl. Real-time binding kinetics revealed that much of the reduced affinity could be attributable to an extremely rapid dissociation of UL42 from the P/T in high-ionic-strength buffer. The resistance of the activity, binding affinity, and stability of the holoenzyme for the model P/T to increases in ionic strength, despite the low apparent affinity and poor stability with which UL42 binds the model P/T in high concentrations of salt, suggests that UL42 does not simply tether the Pol to DNA. Instead, it is likely that conformational alterations induced by interaction of UL42 with Pol allow for high-affinity and high-stability binding of the holoenzyme to the P/T even under high-ionic-strength conditions.

Assessing the contribution of the herpes simplex virus DNA polymerase to spontaneous mutations.

BackgroundThe thymidine kinase (tk) mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV) replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1.MethodsA panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol) within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases.ResultsAlthough HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2–4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed signficant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA stucture.ConclusionsThis study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a) mutations may be modulated by other viral polypeptides cooperating with Pol, and (b) the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.

Resistance of herpesviruses to antiviral drugs: clinical impacts and molecular mechanisms.

Nucleoside analogues such as acyclovir and ganciclovir have been the mainstay of therapy for alphaherpesviruses (herpes simplex virus (HSV) and varicella-zoster virus (VZV)) and cytomegalovirus (CMV) infections, respectively. Drug-resistant herpesviruses are found relatively frequently in the clinic, almost exclusively among severely immunocompromised patients receiving prolonged antiviral therapy. For instance, close to 10% of patients with AIDS receiving intravenous ganciclovir for 3 months excrete a drug-resistant CMV isolate in their blood or urine and this percentage increases with cumulative drug exposure. Many studies have reported that at least some of the drug-resistant herpesviruses retain their pathogenicity and can be associated with progressive or relapsing disease. Viral mutations conferring resistance to nucleoside analogues have been found in either the drug activating/phosphorylating genes (HSV or VZV thymidine kinase, CMV UL97 kinase) and/or in conserved regions of the viral DNA polymerase. Currently available second line agents for the treatment of herpesvirus infections--the pyrophosphate analogue foscarnet and the acyclic nucleoside phosphonate derivative cidofovir--also inhibit the viral DNA polymerase but are not dependent on prior viral-specific activation. Hence, viral DNA polymerase mutations may lead to a variety of drug resistance patterns which are not totally predictable at the moment due to insufficient information on specific drug binding sites on the polymerase. Although some CMV and HSV DNA polymerase mutants have been found to replicate less efficiently in cell cultures, further research is needed to correlate viral fitness and clinical outcome.

Linear Diffusion on DNA Despite High-Affinity Binding by a DNA Polymerase Processivity Factor

The oligomeric “sliding clamp” processivity factors, such as PCNA, are thought to rely on a loose, topological association with DNA to slide freely along dsDNA. Unlike PCNA, the processivity subunit of the herpes simplex virus DNA polymerase, UL42, is a monomer and has an intrinsic affinity for dsDNA that is remarkably high for a sequence-independent DNA binding protein. Using a DNase footprinting assay, we demonstrate that UL42 translocates with the catalytic subunit of the polymerase during chain elongation. In addition, footprinting and electrophoretic mobility shift assays show that, despite its tight DNA binding, UL42 is capable of linear diffusion on DNA at a rate of between 17 and 47 bp/s. Our results thus suggest that, despite profound biochemical differences with the sliding clamps, UL42 can freely slide downstream with the catalytic subunit during DNA replication.

Utility of restriction fragment analysis for typing herpes simplex virus amplicons following PCR of targets in the DNA polymerase gene

We compared the utility of restriction endonuclease cleavage to type herpes simplex virus DNA polymerase gene amplicons from two well established PCR primer sets. Amplicons typed using Ava II had a 96% correlation to type determined by monoclonal antibody staining, while amplicons typed using Drd I had a 72% correlation to type determined by monoclonal antibody staining.

The Crystal Structure of an Unusual Processivity Factor, Herpes Simplex Virus UL42, Bound to the C Terminus of Its Cognate Polymerase

Herpes simplex virus DNA polymerase is a heterodimer composed of a catalytic subunit, Pol, and an unusual processivity subunit, UL42, which, unlike processivity factors such as PCNA, directly binds DNA. The crystal structure of a complex of the C-terminal 36 residues of Pol bound to residues 1–319 of UL42 reveals remarkable similarities between UL42 and PCNA despite contrasting biochemical properties and lack of sequence homology. Moreover, the Pol–UL42 interaction resembles the interaction between the cell cycle regulator p21 and PCNA. The structure and previous data suggest that the UL42 monomer interacts with DNA quite differently than does multimeric toroidal PCNA. The details of the structure lead to a model for the mechanism of UL42, provide the basis for drug design, and allow modeling of other proteins that lack sequence homology with UL42 or PCNA.

Secondary Structure and Structure-Activity Relationships of Peptides Corresponding to the Subunit Interface of Herpes Simplex Virus DNA Polymerase

The interaction of the catalytic subunit of herpes simplex virus DNA polymerase with the processivity subunit, UL42, is essential for viral replication and is thus a potential target for antiviral drug discovery. We have previously reported that a peptide analogous to the C-terminal 36 residues of the catalytic subunit, which are necessary and sufficient for its interaction with UL42, forms a monomeric structure with partial alpha-helical character. This peptide and one analogous to the C-terminal 18 residues specifically inhibit UL42-dependent long chain DNA synthesis. Using multidimensional (1)H nuclear magnetic resonance spectroscopy, we have found that the 36-residue peptide contains partially ordered N- and C-terminal alpha-helices separated by a less ordered region. A series of "alanine scan" peptides derived from the C-terminal 18 residues of the catalytic subunit were tested for their ability to inhibit long-chain DNA synthesis and by circular dichroism for secondary structure. The results identify structural aspects and specific side chains that appear to be crucial for interacting with UL42. These findings may aid in the rational design of new drugs for the treatment of herpesvirus infections.

Effects of exonuclease activity and nucleotide selectivity of the herpes simplex virus DNA polymerase on the fidelity of DNA replication in vivo.

A mutagenesis system was developed for the in vivo study of the fidelity of DNA replication mediated by wild-type herpes simplex virus type 1 (HSV-1) strain KOS and its polymerase (Pol) mutant derivatives PAAr5, Y7, and YD12. The pHOS1 shuttle plasmid, which contained the SupF mutagenesis marker gene and the HSV oris sequence, was used for analysis of the mutation frequency and the mutation spectrum. All three Pol mutants induced significant increases in the mutation frequencies of the target gene, despite the fact that PAAr5 was previously shown to have an antimutator phenotype by the thymidine kinase mutagenesis assay (J. D. Hall, D. M. Coen, B. L. Fisher, M. Weisslitz, S. Randall, R. E. Almy, P. Gelep, and P. A. Schaffer, Virology 132:26-37, 1984; C. B. C. Hwang and J.-H. Chen, Gene 152:191-193, 1995). Altered spectra of mutated target genes induced by these three mutants were also observed. The relative frequencies of both deletion and complex mutations found in mutants induced by exonuclease-proficient Pols were significantly higher than those induced by exonuclease-deficient Pols. On the other hand, the exonuclease-deficient Pols induced significant increases in the frequency of base substitutions, which comprised predominantly G. C-to-T. A transversions, as well as mutations at additional hot spots. These results suggest that the HSV-1 DNA Pol can incorporate purine-purine or pyrimidine-pyrimidine mispaired bases which may be preferentially proofread by its intrinsic exonuclease activity. Furthermore, the effects of the sequence context of the target gene and the assay method should also be considered carefully in any analysis of replication fidelity.

The Inherent Quantitative Capacity of the Reverse Transcription-Polymerase Chain Reaction

The quantitative capacity of the reverse transcription-polymerase chain reaction (RT-PCR) is generally underestimated. In this study, PCR and RT-PCR products were amplified from serially diluted DNA and RNA templates, respectively, using a 35-cycle PCR. In the approximate 30- to 100-fold range of template input above the lower limit of detection, herpes simplex virus ICP27 RT-PCR product yield was dependent on the logarithm of template mRNA input (r2= 0.99). Likewise, regression analysis indicated that yields of interleukin-12 p40, herpes simplex virus DNA polymerase, and interferon-γ PCR products were dependent on the logarithm of template DNA input over 40- (r2= 0.98), 60- (r2= 0.96), and 100-fold (r2= 0.99) ranges, respectively. This quantitative relationship appears to derive from the competition for reactants between specific PCR products and nonspecific primer–dimers that occurs at limiting concentrations of template. Although primer–dimers are not generally considered a common feature of PCR, 30 of 32 primer pairs tested in this study produced primer–dimer amplification in the absence of template. Because the coefficient of variation in replicate PCRs was typically 10–20% in the linear range, the precision of PCR was sufficient to measure 4-fold differences in template concentration. Thus, with statistically adequate sample numbers, an appropriate standard curve, and the inherent quantitative capacity of the method, differences in the abundance of a mRNA species are measurable by 35-cycle RT-PCR.

DNA and Protein Interactions of the Small Subunit of Herpes Simplex Virus Type 1 DNA Polymerase

Herpes simplex virus DNA polymerase (HSV pol) holoenzyme consists of a large catalytic (UL30 gene product) and a small auxiliary subunit (UL42 gene product). The DNA binding of HSV pol, its cofactor, and the assembled holoenzyme complex was studied by bandshift analysis using purified proteins expressed via recombinant baculovirus. The functional activity of the recombinant UL42, purified by phenyl-Sepharose chromatography, was confirmed by its ability (1) to convert the salt sensitivity of both, 3′-5′ exonuclease and polymerase, activities of HSV pol and (2) to enhance the processivity of polymerization. Bandshift analyses revealed that the HSV pol holoenzyme–DNA complex was stably formed up to a salt concentration of 50 mM ammonium sulfate, indicating that the restricted DNA and protein interactions of both HSV pol and UL42 are responsible for the observed salt preference of the HSV pol holoenzyme under standard assay conditionsin vitro. Studies of the assembly of the holoenzyme complex demonstrated that initial DNA binding of HSV pol was advantageous for the formation of the HSV pol holoenzyme–DNA complex.

Lobucavir is phosphorylated in human cytomegalovirus-infected and -uninfected cells and inhibits the viral DNA polymerase.

Lobucavir (LBV) is a deoxyguanine nucleoside analog with broad-spectrum antiviral activity. LBV was previously shown to inhibit herpes simplex virus (HSV) DNA polymerase after phosphorylation by the HSV thymidine kinase. Here we determined the mechanism of action of LBV against human cytomegalovirus (HCMV). LBV inhibited HCMV DNA synthesis to a degree comparable to that of ganciclovir (GCV), a drug known to target the viral DNA polymerase. The expression of late proteins and RNA, dependent on viral DNA synthesis, was also inhibited by LBV. Immediate-early and early HCMV gene expression was unaffected, suggesting that LBV acts temporally coincident with HCMV DNA synthesis and not through cytotoxicity. In vitro, the triphosphate of LBV was a potent inhibitor of HCMV DNA polymerase with a Ki of 5 nM. LBV was phosphorylated to its triphosphate form intracellularly in both infected and uninfected cells, with phosphorylated metabolite levels two- to threefold higher in infected cells. GCV-resistant HCMV isolates, with deficient GCV phosphorylation due to mutations in the UL97 protein kinase, remained sensitive to LBV. Overall, these results suggest that LBV-triphosphate halts HCMV DNA replication by inhibiting the viral DNA polymerase and that LBV phosphorylation can occur in the absence of viral factors including the UL97 protein kinase. Furthermore, LBV may be effective in the treatment of GCV-resistant HCMV.

Effects of mutations in the Exo III motif of the herpes simplex virus DNA polymerase gene on enzyme activities, viral replication, and replication fidelity.

The herpes simplex virus DNA polymerase catalytic subunit, which has intrinsic polymerase and 3'-5' exonuclease activities, contains sequence motifs that are homologous to those important for 3'-5' exonuclease activity in other polymerases. The role of one such motif, Exo III, was examined in this study. Mutated polymerases containing either a single tyrosine-to-histidine change at residue 577 or this change plus an aspartic acid-to-alanine at residue 581 in the Exo III motif exhibited defective or undetectable exonuclease activity, respectively, yet retained substantial polymerase activity. Despite the defects in exonuclease activity, the mutant polymerases were able to support viral replication in transient complementation assays, albeit inefficiently. Viruses replicated via the action of these mutant polymerases exhibited substantially increased frequencies of mutants resistant to ganciclovir. Furthermore, when the Exo III mutations were incorporated into the viral genome, the resulting mutant viruses displayed only modestly defect in replication in Vero cells and exhibited substantially increased mutation frequencies. The results suggest that herpes simplex virus can replicate despite severely impaired exonuclease activity and that the 3'-5' exonuclease contributes substantially to the fidelity of viral DNA replication.

Hepatitis B virus polymerase mutations during antiviral therapy in a patient following liver transplantation

Background/Aims: The purpose of this study was to investigate possible resistance mutations which arose in the polymerase gene of hepatitis B virus (HBV) in a patient with severe recurrent HBV infection following liver transplantation. The patient's management included antiviral chemotherapy for almost 4 years comprising ganciclovir, foscarnet and famciclovir. In the last 2.5 years of famciclovir treatment, an increase in serum HBV DNA levels and a reduced sensitivity of the virion-associated DNA polymerase to penciclovir triphosphate were observed. Methods: The viral polymerase gene and X gene were sequenced from serum samples collected at representative time intervals covering the entire treatment period. Results: No mutations were detected in the X gene. Three nucleotide mutations, each of which resulted in an altered amino acid sequence, were detected in the polymerase gene after 816 days of total antiviral therapy (370 days of famciclovir). Two of these mutations were detected by direct sequencing and the third was detected after cloning and was present in 10% of the clones. All three mutations occurred in “region B” of RNA-dependent DNA polymerases. The HBV polymerase has similarities to both RNA and DNA polymerases. These mutations in the HBV polymerase gene were located in a similar area to the penciclovir-induced mutations observed in the herpes simplex virus DNA polymerase gene. Conclusions: Three mutations within the HBV polymerase gene were detected which were associated with a reduced penciclovir sensitivity.

Sequence analysis of the bovine herpesvirus type 1 genes homologous to the DNA polymerase (UL30), the major DNA-binding protein (UL29) and ICP18.5 assembly protein (UL28) genes of herpes simplex virus.

The nucleotide sequence of a 10.5 kb region (map position 0.332 to 0.410) of bovine herpesvirus type 1 (BHV-1) was determined. This region contained three open reading frames (ORFs) homologous to herpes simplex virus DNA polymerase catalytic subunit (DNApol, UL30), major DNA-binding protein (MDBP, UL29) and ICP18.5 assembly protein (ICP18.5, UL28). The BHV-1 DNApol. MDBP and ICP18.5 ORFs were 1246, 1203 and 826 amino acids long with a calculated molecular mass of 134.2 kDa, 124.4 kDa and 86.9 kDa, respectively. They showed a high homology with alphaherpesvirus homologs despite large differences in the G + C content of the UL30-UL28 segment ranging from 44.4% for varicella zoster virus to 71.5% for BHV-1. Particularly well conserved among Alphaherpesvirinae are the putative functional domains of the DNApol and MDBP proteins which are discussed. Phylogenetic analysis revealed that BHV-1 clustered in the Varicellovirus genus with the animal D-type viruses. In this group, the BHV-1 position was shown to vary according to the investigated genes. Indeed, pseudorabies virus clustered with BHV-1 in the DNApol tree but with equine herpesvirus 1 in the ICP18.5 tree.