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Journal of Virology, July 2006, p. 6247-6258, Vol. 80, No. 13
0022-538X/06/$08.00+0     doi:10.1128/JVI.02551-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Arianna Loregian,^1* Alessandro Case,¹ Enrico Cancellotti,¹ Carlo Valente,² Howard S. Marsden,^3, and Giorgio Palù^1*

Department of Histology, Microbiology and Medical Biotechnologies, University of Padua, 35121 Padua, Italy,¹ Department of Diagnostic Pathology and Veterinary Clinic, University of Perugia, 06126 Perugia, Italy,² MRC Virology Unit, Institute of Virology, Glasgow G11 5JR, United Kingdom³

Received 6 December 2005/ Accepted 11 April 2006

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.

Present address: Briarwood, Helensburgh, United Kingdom.