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.
Loregian
Arianna
A
Department of Histology, Microbiology and Medical Biotechnologies, University of Padua, via Gabelli 63, 35121 Padua, Italy. arianna.loregian@unipd.it
Case
Alessandro
A
Cancellotti
Enrico
E
Valente
Carlo
C
Marsden
Howard S
HS
Palù
Giorgio
G
D019943:Amino Acid Substitution; D016367:Baculoviridae; D020134:Catalytic Domain / Q000235:genetics; D002474:Cell-Free System; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D005090:Exodeoxyribonucleases / Q000235:genetics; D015870:Gene Expression; D004861:Herpesvirus 1, Equid / Q000201:enzymology / Q000235:genetics*; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*
D004279:DNA, Viral; D020035:Holoenzymes; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus