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 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.

FIG. 1.

In vitro transcription-translation products of the EHV-1 ORF30 and ORF18 genes. The ORF30 and ORF18 coding sequences were introduced into the pTM1 expression vector under the control of a T7 promoter and the EMV 5′ UTR and were in vitro expressed with the Promega T7-TNT coupled transcription-translation system. The [³⁵S]methionine-labeled ORF30 and ORF18 translation products were fractionated by SDS-7.5% PAGE and examined by autoradiography. Mock, translation from the pTM1 vector lacking a coding insert. The molecular masses of protein markers, in kilodaltons, analyzed on the same gel are indicated on the left; the apparent masses of ORF30 and ORF18 translation products are shown on the right.

FIG. 2.

pORF30 possesses DNA polymerase activity which is stimulated by pORF18. The DNA synthesis activity of in vitro-transcribed-translated pORF30, pORF18, or pORF30 plus pORF18 was analyzed by measuring the incorporation of [³H]dTTP into a poly(dA)-oligo(dT) template. Mock represents the DNA polymerase activity of the translation reaction from the pTM1 vector lacking a coding insert. □, mock; ○, pORF18; ⋄, pORF30; ▵, pORF30 plus pORF18.

FIG. 3.

Expression and purification of recombinant baculovirus-expressed pORF30 and pORF18. Proteins were expressed in insect cells infected with recombinant baculoviruses and purified by column chromatography as described in Materials and Methods. Samples after the final step of pORF30 (A) and pORF18 (B) purification were analyzed by 5% and 10% SDS-PAGE, respectively. The positions of the molecular mass markers are indicated on the left.

FIG. 4.

Stimulation of DNA polymerase activity of purified baculovirus-expressed pORF30 by pORF18. The effects of different amounts of purified pORF18 on the activity of 200 fmol of pORF30 were examined by measuring the incorporation of [³H]dTTP into a poly(dA)-oligo(dT) template in a 60-min reaction.

FIG. 5.

The functional interaction between the EHV-1 DNA polymerase subunits, pORF30 and pORF18, is specific. Stimulation of DNA polymerase activity of pORF30 by UL42, the HSV-1 DNA polymerase accessory protein, and stimulation of UL30, the HSV-1 DNA polymerase catalytic subunit, by pORF18 were examined by measuring the incorporation of [³H]dTTP into a poly(dA)-oligo(dT) template by individual or combined proteins. As a control, stimulation of UL30 by UL42 was also assayed. Equimolar amounts (200 fmol) of purified baculovirus-expressed proteins were used in these assays. •, pORF30 alone; ○, pORF30 plus UL42; ▪, UL30 alone; □, UL30 plus pORF18; ♦, UL30 plus UL42.

FIG. 6.

pORF18 enables pORF30 to synthesize long-chain DNA products. Long-chain DNA synthesis by purified baculovirus-expressed pORF30 in the absence and in the presence of purified baculovirus-expressed pORF18 was assayed by measuring the incorporation of [³²P]TTP on a poly(dA)-oligo(dT) template. The reaction products were visualized by autoradiography following electrophoresis on a 4% alkaline agarose gel. Positions of DNA size markers (bases) and the length of short DNA products are indicated at left. Lane 1 contains 200 fmol of pORF30 alone; lane 2 contains 200 fmol of pORF18 alone; and lanes 3 and 4 contain 200 fmol of pORF30 plus 100 or 200 fmol of pORF18, respectively.

FIG. 7.

Effects of salt concentration on DNA polymerase activities of pORF30 and the pORF30-pORF18 complex. The DNA polymerase activities of pORF30 alone (○) and of pORF30 plus pORF18 at an equimolar ratio (□), determined by filter-based DNA polymerase assays employing poly(dA)-oligo(dT) as the template, are plotted against the concentration of KCl present in the reaction mixtures. NaCl (1 mM or less) was carried over into assays from protein solutions.

FIG. 8.

Schematic representation and summary of the properties of full-length pORF30 and pORF30 deletion mutants. Several pORF30 mutants were created by deleting different portions of the EHV-1 ORF30 coding sequence (pORF30 residues that have been deleted are indicated within parentheses). Deleted portions of pORF30 are represented by black lines, while regions still expressed are depicted as gray bars. The representations of the mutants are not drawn to scale. The ability of full-length and mutant pORF30 proteins to physically interact with pORF18, as determined by yeast two-hybrid assays (Table 2), and their catalytic activity both in the absence and in the presence of pORF18, as measured by DNA polymerase assays (Fig. 9), are scored as follows: +, wild-type levels of activity; ±, partially impaired activity; ±/−, almost completely impaired activity; −, no detectable activity.

FIG. 9.

Stimulation of DNA polymerase activity of pORF30 C-proximal and C-terminal mutants by pORF18. The DNA synthesis activities of in vitro-transcribed-translated pORF30 deletion mutants in the absence and in the presence of in vitro-expressed pORF18 were analyzed in a DNA polymerase assay by measuring the incorporation of [³H]dTTP into a poly(dA)-oligo(dT) template. ▵, pORF30 alone; ▴, pORF30 plus pORF18; □, pORF30_{(Δ1114-1172)} alone; ▪, pORF30_{(Δ1114-1172)} plus pORF18; ○, pORF30_{(Δ1188-1220)} alone; •, pORF30_{(Δ1188-1220)} plus pORF18; ⋄, pORF30_{(Δ1114-1220)} alone; ♦, pORF30_{(Δ1114-1220)} plus pORF18.