Residues of human cytomegalovirus DNA polymerase catalytic subunit UL54 that are necessary and sufficient for interaction with the accessory protein UL44

Abstract

The human cytomegalovirus DNA polymerase contains a catalytic subunit, UL54, and an accessory protein, UL44. Recent studies suggested that UL54 might interact via its extreme C terminus with UL44 (A. Loregian, R. Rigatti, M. Murphy, E. Schievano, G. Palu', and H. S. Marsden, J. Virol. 77:8336-8344, 2003). To address this hypothesis, we quantitatively measured the binding of peptides corresponding to the extreme C terminus of UL54 to UL44 by using isothermal titration calorimetry. A peptide corresponding to the last 22 residues of UL54 was sufficient to bind specifically to UL44 in a 1:1 complex with a dissociation constant of ca. 0.7 microM. To define individual residues in this segment that are crucial for interacting with UL44, we engineered a series of mutations in the C-terminal region of UL54. The UL54 mutants were tested for their ability to interact with UL44 by glutathione S-transferase pulldown assays, for basal DNA polymerase activity, and for long-chain DNA synthesis in the presence of UL44. We observed that deletion of the C-terminal segment or substitution of alanine for Leu1227 or Phe1231 in UL54 greatly impaired both the UL54-UL44 interaction in pulldown assays and long-chain DNA synthesis without affecting basal polymerase activity, identifying these residues as important for subunit interaction. Thus, like the herpes simplex virus UL30-UL42 interaction, a few specific side chains in the C terminus of UL54 are crucial for UL54-UL44 interaction. However, the UL54 residues important for interaction with UL44 are hydrophobic and not basic. This information might aid in the rational design of new drugs for the treatment of human cytomegalovirus infection.

FIG. 1.

Purified GST, GST-UL44, GST-UL44ΔC290, and cleaved UL44ΔC290 preparations. Proteins were expressed in E. coli and purified by column chromatography as described in Materials and Methods. Samples after the final step of purification for each protein were analyzed by SDS-12% PAGE. The molecular masses based on protein markers analyzed on the same gel are indicated on the left.

FIG. 2.

Binding to UL44 of synthetic peptides corresponding to the C-terminal 22 residues (peptide 1) or 10 residues (peptide 9) of UL54 measured by ITC. Titrations were performed with 10-μl injections of peptide into a sample cell containing full-length GST-UL44, GST-UL44ΔC290, cleaved UL44ΔC290, or, as controls, GST or MBP-UL42Δ340. (A) Raw data for the titrations, in which the power output in microcalories per second is measured as a function of time in minutes. (B) The heats of dilution of both protein and ligand in A were subtracted, and the area under each injection curve was integrated to generate the points, which represent heat exchange in kilocalories per mole, which are plotted against the cumulative peptide-to-protein ratio for each injection.

FIG. 3.

Binding to GST-UL44 of a variant of UL54 peptide 1 lacking the two C-terminal cysteines (peptide 8) measured by ITC. Titrations and data analysis were performed as described in the legend to Fig. 2.

FIG. 4.

Detection of UL54-UL44 interaction by a GST-pulldown assay. Purified GST or GST-UL44 was incubated with in vitro-expressed and -radiolabeled wild-type or mutant UL54 proteins (as indicated at the top of the figure) or, where indicated, HSV-1 UL30 as a control and allowed to bind to glutathione columns. The columns were washed, and the proteins were eluted with 15 mM glutathione. The radiolabeled proteins were visualized by autoradiography after SDS-7.5% PAGE. The position of UL54 and UL30 are marked by arrows. Lanes: I, input; E, eluted by glutathione.

FIG. 5.

UL54 mutants. The sequence of the C-terminal 22 residues (amino acids 1221 to 1242) of wild-type UL54 protein, in single-letter code, is reported on the top. A series of mutations were engineered in this region of UL54 and at position 1217 as described in Materials and Methods. For each mutant, the sequence of the region containing the mutation is shown, with the mutated residue(s) indicated by underlined boldface letter(s).

FIG. 6.

Binding of mutant UL54 proteins to GST-UL44. The physical binding of wild-type or the mutant UL54 proteins, as indicated at the tops of the figure panels, to GST-UL44 was tested by GST-pulldown assays as described in legend to Fig. 4. Lanes: I, input; E, eluted by glutathione.

FIG. 7.

Long-chain DNA synthesis by UL54 in the presence of UL44. Wild-type UL54 expressed in reticulocyte lysates was assayed for the ability to be stimulated by purified baculovirus-expressed UL44 by measuring the incorporation of labeled TTP on a poly(dA)-oligo(dT) template. The reaction products were visualized by autoradiography after electrophoresis on a 4% alkaline agarose gel. Lane 1 contains UL54 alone; lanes 2, 3, and 4 contain UL54 plus 1,000, 500, or 200 fmol of UL44, respectively; lane 5 contains 1,000 fmol of UL44 alone.

FIG. 8.

Long-chain DNA synthesis by UL54 mutants in the presence of UL44. Wild-type or mutant UL54 proteins expressed in reticulocyte lysates were assayed for their ability to be stimulated by UL44 as described in legend to Fig. 7. Lanes 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, and 31 contain wild-type or mutant UL54 alone; lanes 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, and 32 and lanes 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, and 33 contain wild-type or mutant UL54 plus 400 or 800 fmol of UL44, respectively.

FIG. 9.

Basal DNA polymerase activity of UL54 mutants. (A) The DNA polymerase activity of wild-type or mutant UL54 proteins expressed in reticulocyte lysates was measured by determining the incorporation of [³²P]dTTP into a poly(dA)-oligo(dT) DNA template. Samples were taken after 0, 10, 20, and 30 min of incubation at 37°C and spotted onto DE81 filters. The filters were washed, and the radioactivity was counted. (B) The values were normalized to the amount of in vitro-translated proteins (lower panel), as estimated by SDS-PAGE analysis and phosphorimager quantification. The graph (upper panel) shows the average of data from three independent experiments, together with the standard deviations.