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

Abstract

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 transcriptiontranslation of open reading frames 30 and 18 generated proteins of 136 and 45 kDa, respectively. In vitroexpressed 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.Equine herpesvirus 1 (EHV-1) is a pathogen of major importance in horses that can induce a wide spectrum of diseases (1, 45). Like other herpesviruses, EHV-1 establishes a lifelong infection, via a quiescent state known as latency. The outcomes of infection, either following primary infection or reactivation from latency, vary from mild/unapparent respiratory disease to the induction of abortion in pregnant mares and, in extreme cases, neurological disease resulting in paralysis and ultimately death.EHV-1 is classified on biological grounds as a member of the Alphaherpesvirinae, a subfamily of the herpesviruses which is typified by herpes simplex virus type 1 (HSV-1) and also includes varicella-zoster virus (VZV) and pseudorabies virus (PRV) (49). The complete DNA sequence of a pathogenic British isolate (strain Ab4) of EHV-1 was recently determined (56). The genome is approximately 150 kbp in size, is similar in arrangement to the HSV-1, VZV, and PRV genomes, and contains 80 open reading frames (ORFs), corresponding to 76 distinct genes, likely to encode proteins. Comparison of predicted amino acid sequences to those of HSV-1, VZV, and PRV homologs allowed the functions of many EHV-1 proteins to be putatively assigned. However, only a few of these proteins have been studied so far, although the number of characterized proteins is rapidly increasing....

A Point Mutation in a Herpesvirus Polymerase Determines Neuropathogenicity

“…As is the case in all herpesviruses studied to date, an accessory protein, EHV-1 pORF18, which stimulates nucleotide incorporation by Pol, forms the viral DNA polymerase holoenzyme together with the catalytic subunit [20]. Thus, we compared DNA polymerase activities of Pol D752 and Pol N752 in the absence or presence of pORF18 under different assay conditions (Figure 7C).…”

Regulated Transport into the Nucleus of Herpesviridae DNA Replication Core Proteins

“…The pol subunit is known to perform the synthesis of leading and lagging strands, while the PAP tethers the holoenzyme to the DNA, thus conferring processivity [65,66,67]. Since the replication of all known herpesviruses takes place in the host cell nucleus [68], these high molecular weight proteins need to be actively transported through the NPC to reach the site of viral DNA replication.…”

Molecular characterisation of the ORF68 region of equine herpesvirus-1 strains isolated from aborted fetuses in Hungary between 1977 and 2008

“…These differences occurred mostly as single nucleotide polymorphisms (SNPs), causing coding changes in 31 ORFs, although the significance of these substitutions has been proved only in the case of a few ORFs, of which the two most important are ORF30 and ORF68. The SNPs in ORF30 could be associated with the neuropathogenic attributes of virus strains (Loregian et al, 2006;Nugent et al, 2006;Goodman et al, 2007), although in some cases the non-neuropathogenic genotype was found to be the causative agent in a disease with neurological signs (Heerkens, 2009). In a previous study a primer-probe energy transfer method was developed for the differentiation of neuropathogenic and non-neuropathogenic genotypes of EHV-1 strains.…”