Herpesvirus DNA Polymerase Research Articles

A paradigm linking herpesvirus immediate-early gene expression apoptosis and myalgic encephalomyelitis chronic fatigue syndrome

A paradigm linking herpesvirus immediate-early gene expression apoptosis and myalgic encephalomyelitis chronic fatigue syndrome A Martin Lerner1, Safedin Beqaj21Department of Medicine, William Beaumont Hospital, Royal Oak, MI, USA; 2DCL Medical Laboratories, Indianapolis, IN, USAAbstract: There is no accepted science to relate herpesviruses (Epstein–Barr virus [EBV], human cytomegalovirus [HCMV], and human herpesvirus 6 [HHV6]) as causes of myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS). ME/CFS patients have elevated serum immunoglobulin (Ig)G serum antibody titers to EBV, HCMV, and HHV6, but there is no herpesvirus DNA-emia, herpesvirus antigenemia, or uniformly elevated IgM serum antibody titers to the complete virions. We propose that herpesvirus EBV, HCMV, and HHV6 immediate-early gene expression in ME/CFS patients leads to host cell dysregulation and host cell apoptosis without lytic herpesvirus replication. Specific antiviral nucleosides, which alleviate ME/CFS, namely valacyclovir for EBV ME/CFS and valganciclovir for HCMV/HHV6 ME/CFS, inhibit herpesvirus DNA polymerases and/or thymidine kinase functions, thus inhibiting lytic virus replication. New host cell recruitment thus ceases. In the absence of new herpesvirus, nonpermissive herpesvirus replication stops, and ME/CFS recovery ensues.Keywords: ME/CFS, Epstein–Barr virus (EBV), human cytomegalovirus (HCMV), HHV6, abortive replication

Discovery of Selective Inhibitors Against EBNA1 via High Throughput In Silico Virtual Screening

BackgroundEpstein-Barr Virus (EBV) latent infection is associated with several human malignancies and is a causal agent of lymphoproliferative diseases during immunosuppression. While inhibitors of herpesvirus DNA polymerases, like gancyclovir, reduce EBV lytic cycle infection, these treatments have limited efficacy for treating latent infection. EBNA1 is an EBV-encoded DNA-binding protein required for viral genome maintenance during latent infection.MethodologyHere, we report the identification of a new class of small molecules that inhibit EBNA1 DNA binding activity. These compounds were identified by virtual screening of 90,000 low molecular mass compounds using computational docking programs with the solved crystal structure of EBNA1. Four structurally related compounds were found to inhibit EBNA1-DNA binding in biochemical assays with purified EBNA1 protein. Compounds had a range of 20–100 µM inhibition of EBNA1 in fluorescence polarization assays and were further validated for inhibition using electrophoresis mobility shift assays. These compounds exhibited no significant inhibition of an unrelated DNA binding protein. Three of these compounds inhibited EBNA1 transcription activation function in cell-based assays and reduced EBV genome copy number when incubated with a Burkitt lymphoma cell line.ConclusionsThese experiments provide a proof-of-principle that virtual screening can be used to identify specific inhibitors of EBNA1 that may have potential for treatment of EBV latent infection.

Compounds that target host cell proteins prevent varicella-zoster virus replication in culture, ex vivo, and in SCID-Hu mice

Varicella-zoster virus (VZV) replicates in quiescent T cells, neurons, and skin cells. In cultured fibroblasts (HFFs), VZV induces host cyclin expression and cyclin-dependent kinase (CDK) activity without causing cell cycle progression. CDK1/cyclin B1 phosphorylates the major viral transactivator, and the CDK inhibitor roscovitine prevents VZV mRNA transcription. We investigated the antiviral effects of additional compounds that target CDKs or other cell cycle enzymes in culture, ex vivo, and in vivo. Cytotoxicity and cell growth arrest doses were determined by Neutral Red assay. Antiviral effects were evaluated in HFFs by plaque assay, genome copy number, and bioluminescence. Positive controls were acyclovir (400 μM) and phosphonoacetic acid (PAA, 1 mM). Test compounds were roscovitine, aloisine A, and purvalanol A (CDK inhibitors), aphidicolin (inhibits human and herpesvirus DNA polymerase), l-mimosine (indirectly inhibits human DNA polymerase), and DRB (inhibits casein kinase 2). All had antiviral effects below the concentrations required for cell growth arrest. Compounds were tested in skin organ culture at EC 99 doses; all prevented VZV replication in skin, except for aloisine A and purvalanol A. In SCID mice with skin xenografts, roscovitine (0.7 mg/kg/day) was as effective as PAA (36 mg/kg/day). The screening systems described here are useful models for evaluating novel antiviral drugs for VZV.

Hepatitis and Enteritis Caused by a Novel Herpesvirus in Two Monitor Lizards (Varanus Spp.)

Reported cases of herpesvirus-induced disease are uncommon in most species of reptiles, with the majority of reports in chelonians. Two monitor lizards (Varanus spp.) presented for postmortem examination at the Veterinary Medical Diagnostic Laboratory at the University of Missouri. Tan, 1-2-mm foci were grossly visible on the mucosal surface of the intestine and in the liver. Microscopically, there was multifocal necrosis in the lamina propria of the small intestine and in the liver. Many of the degenerate cells contained large, eosinophilic intranuclear inclusions. Enveloped icosahedral virions consistent with herpesvirus were detected by electron microscopy. A 180-bp DNA fragment was amplified by polymerase chain reaction from samples of small intestine and liver using primers that targeted a portion of the herpesvirus DNA polymerase gene. The sequence of the fragment was determined to be most closely related to Varanid herpesvirus 2 (80% nucleotide identity, 82% amino acid identity). Based on histological and molecular findings, a novel pathogenic herpesvirus of lizards in the family Varanidae is proposed.

A novel herpesvirus in the sanctuary chimpanzees on Ngamba Island in Uganda

Recent studies in non-human primates have led to the discovery of novel primate herpesviruses. In order to get more information on herpesvirus infections in apes, we studied wild born captive chimpanzees. Chimpanzees of the Ngamba island sanctuary, Uganda, were analyzed with pan-herpes polymerase chain reaction (PCR) targeting the herpesvirus DNA polymerase gene and the glycoprotein B gene. The obtained sequences were connected by long-distance PCR, and analyzed phylogenetically. Twenty-one of 40 individuals were infected with members of the Gammaherpesvirinae, two of them with a novel member of this subfamily. Phylogenetically, the novel virus fell into a clade of primate rhadinoviruses and the Kaposi sarcoma herpesvirus (human herpesvirus 8), representing a third distinct rhadinovirus in chimpanzees. Non-human primates harbor several herpesviruses many of which are still unknown. This has implications to management of primates in sanctuaries requiring continuous updates on the management protocols to deal with potential occupational pathogens.

The Crystal Structure of PF-8, the DNA Polymerase Accessory Subunit from Kaposi's Sarcoma-Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi's sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.8 A. This structure reveals that each monomer of PF-8 shares a fold common to processivity factors. Like human cytomegalovirus UL44, PF-8 forms a head-to-head dimer in the form of a C clamp, with its concave face containing a number of basic residues that are predicted to be important for DNA binding. However, there are several differences with related proteins, especially in loops that extend from each monomer into the center of the C clamp and in the loops that connect the two subdomains of each protein, which may be important for determining PF-8's mode of binding to DNA and to Pol-8. Using the crystal structures of PF-8, the herpes simplex virus catalytic subunit, and RB69 bacteriophage DNA polymerase in complex with DNA and initial experiments testing the effects of inhibition of PF-8-stimulated DNA synthesis by peptides derived from Pol-8, we suggest a model for how PF-8 might form a ternary complex with Pol-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins.

Detection of a new bat gammaherpesvirus in the Philippines

A new bat herpesvirus was detected in the spleen of an insectivorous bat (Hipposideros diadema, family Hipposideridae) collected on Panay Island, the Philippines. PCR analyses were performed using COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOPs) targeting the herpesvirus DNA polymerase (DPOL) gene. Although we obtained PCR products with CODEHOPs, direct sequencing using the primers was not possible because of high degree of degeneracy. Direct sequencing technology developed in our rapid determination system of viral RNA sequences (RDV) was applied in this study, and a partial DPOL nucleotide sequence was determined. In addition, a partial gB gene nucleotide sequence was also determined using the same strategy. We connected the partial gB and DPOL sequences with long-distance PCR, and a 3741-bp nucleotide fragment, including the 3′ part of the gB gene and the 5′ part of the DPOL gene, was finally determined. Phylogenetic analysis showed that the sequence was novel and most similar to those of the subfamily Gammaherpesvirinae.

Sheep-associated Malignant Catarrhal Fever in Free-ranging Moose (Alces alces) in Saskatchewan, Canada

Malignant catarrhal fever (MCF) is a sporadic disease of artiodactyls caused by several viruses in the Gammaherpesvirinae. We report two cases of MCF in free-living moose (Alces alces) from Saskatchewan. One was a thin, dehydrated, adult male found recumbent in 2006. At necropsy, ulcers were found in the intestine, bladder, and corneas. Microscopically, there was lymphocytic vasculitis and perivasculitis in many organs with infrequent fibrinoid necrosis. Ovine herpes virus-2 (OHV-2) was identified by polymerase chain reaction. A segment of the herpesviral DNA polymerase gene was 99% identical to published OHV-2 sequences. During a retrospective search of earlier cases, a female moose with lymphoplasmacytic meningoencephalitis examined in 2003 was identified and OHV-2 was amplified from paraffin-embedded tissues from this animal. We believe this to be the first description of MCF in free-ranging moose in North America. Infection requires contact with infected sheep or goats, and MCF in moose may become more prevalent as moose distribution continues to expand into agricultural prairie.

Herpesvirus-Associated Neurological Disease in a Donkey

A 4-year-old donkey was evaluated for progressive neurological abnormalities consisting of depression, stupor, weakness, and recumbency. Diagnostic evaluation for viral involvement identified an asinine herpesvirus in DNA extracted from deep pharyngeal swabs. Specific primers were designed based on comparison with equine herpesviral DNA polymerase sequences and yielded an 875-base pair product from the donkey. This sequence had complete identity with short sequences of asinine herpesvirus previously identified in donkeys with interstitial pneumonia. Amino acid analysis of the entire sequence indicated high similarity with Equid herpesvirus 7 (91%), Zebra herpesvirus 1 (90%), and Equid herpesvirus 2 (89%). With supportive treatment and physical therapy, the donkey gradually recovered over 5 days of hospitalization and returned to normal function. The current case illustrates the potential of a novel asinine herpesvirus to induce neurological disease in donkeys and provides a large viral sequence allowing confident assignment of this virus to the subfamily Gammaherpesvirinae.

Acyclovir Is Activated into a HIV-1 Reverse Transcriptase Inhibitor in Herpesvirus-Infected Human Tissues

For most viruses, there is a need for antimicrobials that target unique viral molecular properties. Acyclovir (ACV) is one such drug. It is activated into a human herpesvirus (HHV) DNA polymerase inhibitor exclusively by HHV kinases and, thus, does not suppress other viruses. Here, we show that ACV suppresses HIV-1 in HHV-coinfected human tissues, but not in HHV-free tissue or cell cultures. However, addition of HHV-6-infected cells renders these cultures sensitive to anti-HIV ACV activity. We hypothesized that such HIV suppression requires ACV phosphorylation by HHV kinases. Indeed, an ACV monophosphorylated prodrug bypasses the HHV requirement for HIV suppression. Furthermore, phosphorylated ACV directly inhibits HIV-1 reverse transcriptase (RT), terminating DNA chain elongation, and can trap RT at the termination site. These data suggest that ACV anti-HIV-1 activity may contribute to the response of HIV/HHV-coinfected patients to ACV treatment and could guide strategies for the development of new HIV-1 RT inhibitors.

Herpesviral Inclusion Body Disease in Owls and Falcons is Caused by the Pigeon Herpesvirus (Columbid herpesvirus 1).

A herpesviral disease of Rock Pigeons (Columba livia), called "inclusion body disease" or "inclusion body hepatitis," was first described in the 1940s. The disease involves hepatic and splenic necrosis with associated intranuclear inclusion bodies and occurs primarily in young squabs. A similar herpesviral disease occurs in falcons and owls. Serologic and restriction endonuclease digestion studies indicate that herpesviruses from pigeons, falcons, and owls are very closely related and that most reported cases of disease in falcons and owls involve prior documented or possible ingestion of pigeons. These findings led to the hypothesis that an endemic herpesvirus of pigeons may be causing disease in falcons and owls. In order to test this hypothesis, we sequenced a fragment of the herpesviral DNA polymerase gene from naturally infected owls, falcons, and pigeons with inclusion body disease collected between 1991 and 2006. Sequences from all three sources were almost identical, and we therefore propose that the usual agent of inclusion body hepatitis in owls and falcons is columbid herpesvirus 1.

Effect of phosphorylation on the transactivation activity of Epstein–Barr virus BMRF1, a major target of the viral BGLF4 kinase

Modification of human herpesvirus DNA polymerase processivity factors (PFs) by phosphorylation occurs frequently during viral lytic replication. However, functional regulation of the herpesvirus PFs through phosphorylation is not well understood. In addition to processivity, the PF BMRF1 of Epstein-Barr virus can function as a transactivator to activate the BHLF1 promoter within the lytic origin of replication (oriLyt), which is assumed to facilitate DNA replication through remodelling viral chromatin structure. BMRF1 is known to be phosphorylated by the viral BGLF4 kinase, but its impact on BMRF1 function is unclear. Seven candidate BGLF4 target sites were predicted within a proline-rich region between the DNA-processivity and nuclear-localization domains of BMRF1. We show that four of these residues, Ser-337, Thr-344, Ser-349 and Thr-355, are responsible for the BGLF4-induced hyperphosphorylation of BMRF1. In functional analyses, a phosphorylation-mimicking mutant of BMRF1 shows similar nuclear localization, as well as DNA-binding ability, to the wild type; however, it displays stronger synergistic activation of the BHLF1 promoter with Zta. Notably, BGLF4 downregulates BMRF1 transactivation and enhances the transactivation activity of Zta and the synergistic activation of BMRF1 and Zta on the BHLF1 promoter. Our findings suggest that BGLF4 may modulate the activation of the oriLyt BHLF1 promoter coordinately through multiple mechanisms to facilitate optimal oriLyt-dependent viral DNA replication.

Isolation of a gammaherpesvirus similar to asinine herpesvirus-2 (AHV-2) from a mule and a survey of mules and donkeys for AHV-2 infection by real-time PCR

Equids are commonly infected by herpesviruses, but isolation of herpesviruses from mules has apparently not been previously reported. Furthermore, the genomic relationships among the various equid herpesviruses are poorly characterized. We describe the isolation and preliminary characterization of a mule gammaherpesvirus tentatively identified as asinine herpesvirus-2 (AHV-2; also designated equid herpesvirus-7 (EHV-7)) from the nasal secretions (NS) of a healthy mule in northern California. The virus was initially identified by transmission electron microscopic examination of lysates of cell culture inoculated with NS collected from the mule. A 913 nucleotide sequence of the DNA polymerase gene was amplified using degenerate primers, and comparison of this sequence with those of various other herpesviruses showed that the mule herpesvirus was most closely related to EHV-2 (AHV-2 sequences were not available for comparison). The sequence of a shorter portion (166 nucleotides) of the mule herpesvirus DNA polymerase gene was identical to that of the published sequence of an asinine gammaherpesvirus, previously designated as AHV-4-3 ( AY054992). AHV-2 was detected by real-time polymerase chain reaction assay in the NS of approximately 8% of a cohort of 114 healthy mules and 13 donkeys.

Equine Multinodular Pulmonary Fibrosis: A Newly Recognized Herpesvirus-Associated Fibrotic Lung Disease

Pulmonary fibrosis and interstitial lung disease are poorly understood in horses; the causes of such conditions are rarely identified. Equine herpesvirus 5 (EHV-5) is a gamma-herpesvirus of horses that has not been associated with disease in horses. Pathologic and virologic findings from 24 horses with progressive nodular fibrotic lung disease associated with EHV-5 infection are described and compared with 23 age-matched control animals. Gross lesions consisted of multiple nodules of fibrosis throughout the lungs. Histologically, there was marked interstitial fibrosis, often with preservation of an "alveolar-like" architecture, lined by cuboidal epithelial cells. The airways contained primarily neutrophils and macrophages. Rare macrophages contained large eosinophilic intranuclear viral inclusion bodies; similar inclusion bodies were also found cytologically. The inclusions were identified as herpesviral-like particles by transmission electron microscopy in a single horse. In situ hybridization was used to detect EHV-5 nucleic acids within occasional macrophage nuclei. With polymerase chain reaction (PCR), the herpesviral DNA polymerase gene was detected in 19/24 (79.2%) of affected horses and 2/23 (8.7%) of the control horses. Virus genera-specific PCR was used to detect EHV-5 in all of the affected horses and none of the control horses. EHV-2 was detected in 8/24 (33.3%) of affected horses and 1/9 (11.1%) of the control horses. This disease has not been reported before, and the authors propose that based upon the characteristic gross and histologic findings, the disease be known as equine multinodular pulmonary fibrosis. Further, we propose that this newly described disease develops in association with infection by the equine gamma-herpesvirus, EHV-5.

Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach

Targeting the highly conserved herpes DNA polymerase (DPOL) gene with PCR using panherpes degenerate primers is a powerful tool to universally detect unknown herpesviruses. However, vertebrate hosts are often infected with more than one herpesvirus in the same tissue, and pan-herpes DPOL PCR often favors the amplification of one viral sequence at the expense of the others. Here we present two different technical approaches that overcome this obstacle: (i) Pan-herpes DPOL PCR is carried out in the presence of an oligonucleotide substituted with locked nucleic acids (LNA).This suppresses the amplification of a specific herpesvirus DPOL sequence by a factor of approximately 1000, thereby enabling the amplification of a second, different DPOL sequence. (ii) The less conserved glycoprotein B (gB) gene is targeted with several sets of degenerate primers that are restricted to gB genes of different herpesvirus subfamilies or genera. These techniques enable the amplification of gB and DPOL sequences of multiple viruses from a single specimen. The partial gB and DPOL sequences can be connected by long-distance PCR, producing final contiguous sequences of approximately 3.5 kbp. Such sequences include parts of two genes and therefore allow for a robust phylogenetic analysis. To illustrate this principle, six novel herpesviruses of the genera Rhadinovirus, Lymphocryptovirus and Cytomegalovirus were discovered in multi-infected samples of non-human primates and phylogenetically characterized.

IBEX-ASSOCIATED MALIGNANT CATARRHAL FEVER IN A BONGO ANTELOPE (TRAGELAPHUS EURYCEROS)

A 4-yr-old male bongo antelope (Tragelaphus euryceros) died after an acute clinical course involving a febrile illness, anorexia, lethargy, minor oculonasal discharge, and diarrhea. Histologic lesions were compatible with malignant catarrhal fever (MCF). Polymerase chain reaction (PCR) revealed an amplified region of a herpesviral DNA polymerase gene sequence nearly identical to that of a MCF virus previously identified in Nubian ibex (Capra nubiana). The bongo had been housed across from an exhibit containing Nubian ibex that tested positive for MCF viral antibodies by competitive inhibition enzyme-linked immunosorbent assay. Further testing of the zoo's ibex via PCR also revealed viral DNA sequences nearly identical to those found in the bongo's tissues.

Binding parameters and thermodynamics of the interaction of the human cytomegalovirus DNA polymerase accessory protein, UL44, with DNA: implications for the processivity mechanism

The mechanisms of processivity factors of herpesvirus DNA polymerases remain poorly understood. The proposed processivity factor for human cytomegalovirus DNA polymerase is a DNA-binding protein, UL44. Previous findings, including the crystal structure of UL44, have led to the hypothesis that UL44 binds DNA as a dimer via lysine residues. To understand how UL44 interacts with DNA, we used filter-binding and electrophoretic mobility shift assays and isothermal titration calorimetry (ITC) analysis of binding to oligonucleotides. UL44 bound directly to double-stranded DNA as short as 12 bp, with apparent dissociation constants in the nanomolar range for DNAs >18 bp, suggesting a minimum DNA length for UL44 interaction. UL44 also bound single-stranded DNA, albeit with lower affinity, and for either single- or double-stranded DNA, there was no apparent sequence specificity. ITC analysis revealed that UL44 binds to duplex DNA as a dimer. Binding was endothermic, indicating an entropically driven process, likely due to release of bound ions. Consistent with this hypothesis, analysis of the relationship between binding and ionic strength indicated that, on average, 4 ± 1 monovalent ions are released in the interaction of each monomer of UL44 with DNA. The results taken together reveal interesting implications for how UL44 may mediate processivity.

7-Oxo-4,7-dihydrothieno[3,2- b]pyridine-6-carboxamides: Synthesis and biological activity of a new class of highly potent inhibitors of human cytomegalovirus DNA polymerase

We report a new class of non-nucleoside antivirals, the 7-oxo-4,7-dihydrothieno[3,2- b]pyridine-6-carboxamides, some of which possess remarkable potency versus a broad spectrum of herpesvirus DNA polymerases and excellent selectivity compared to human DNA polymerases. A critical factor in the level of activity is hypothesized to be conformational restriction of the key 2-aryl-2-hydroxyethylamine sidechain by an adjacent methyl group.

Greyhound meningoencephalitis: PCR-based detection methods highlight an absence of the most likely primary inducing agents

Greyhound meningoencephalitis is currently classified as a breed-associated idiopathic central nervous system inflammatory disorder. The non-suppurative inflammatory response can be distinguished from the other breed-associated disorders based on histopathology and lesion topography, however the nature of the response primarily suggests a viral infection. In the present study PCR and RT-PCR technologies were employed on frozen cerebral tissue from confirmed cases of meningoencephalitis to target specific viruses and protozoa likely to be implicated and to exclude the presence of bacterial 16SrRNA. Secondly, degenerate primers were used to detect viruses of the herpesvirus and flavivirus families. In addition cerebral tissues were probed for West Nile Virus. Viral nucleic acid sequences to Borna disease virus, to louping ill, tick borne encephalitis, West Nile and other flaviviruses were not detected. Canine distemper virus was detected in one animal with 97% homology to strain A75/15. Degenerate PCR for herpesviruses detected viral amplification products in one animal with 90% homology to canine herpesvirus DNA polymerase gene. Protozoal amplification products were only detected in a single dog with pathological confirmation of a combination of lesions of greyhound meningoencephalitis and a protozoal encephalomyelitis. Neospora was confirmed with sequence homology to Austrian strain 1. Bacterial 16SrRNA was not detected. The present study supports previous observations that many of the known microbial causes of canine meningoencephalitis are not involved. Findings could reflect that the causal agent was not specifically targeted for detection, or that the agent is at undetectable levels or has been eliminated from brain tissue. The potential roles of genetics and of molecular mimicry also cannot be discounted.

Cloning, Expression, and Functional Characterization of the Equine Herpesvirus 1 DNA Polymerase and Its Accessory Subunit

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.