Specific residues in the connector loop of the human cytomegalovirus DNA polymerase accessory protein UL44 are crucial for interaction with the UL54 catalytic subunit

Abstract

The human cytomegalovirus DNA polymerase includes an accessory protein, UL44, which has been proposed to act as a processivity factor for the catalytic subunit, UL54. How UL44 interacts with UL54 has not yet been elucidated. The crystal structure of UL44 revealed the presence of a connector loop analogous to that of the processivity subunit of herpes simplex virus DNA polymerase, UL42, which is crucial for interaction with its cognate catalytic subunit, UL30. To investigate the role of the UL44 connector loop, we replaced each of its amino acids (amino acids 129 to 140) with alanine. We then tested the effect of each substitution on the UL44-UL54 interaction by glutathione S-transferase pulldown and isothermal titration calorimetry assays, on the stimulation of UL54-mediated long-chain DNA synthesis by UL44, and on the binding of UL44 to DNA-cellulose columns. Substitutions that affected residues 133 to 136 of the connector loop measurably impaired the UL44-UL54 interaction without altering the ability of UL44 to bind DNA. One substitution, I135A, completely disrupted the binding of UL44 to UL54 and inhibited the ability of UL44 to stimulate long-chain DNA synthesis by UL54. Thus, similar to the herpes simplex virus UL30-UL42 interaction, a residue of the connector loop of the accessory subunit is crucial for UL54-UL44 interaction. However, while alteration of a polar residue of the UL42 connector loop only partially reduced binding to UL30, substitution of a hydrophobic residue of UL44 completely disrupted the UL54-UL44 interaction. This information may aid the discovery of small-molecule inhibitors of the UL44-UL54 interaction.

FIG. 1.

UL44 C-terminal truncations. UL44 contains five glycine-rich strings in the C terminus, which begin at residue 291. The truncation mutants studied here are depicted as white bars, with the location of each glycine string shown as a black bar.

FIG. 2.

DNA-binding activity of C-terminal truncation mutants of UL44. Radiolabeled full-length and truncated GST-UL44 fusion proteins (as indicated to the left of the panels) were expressed in vitro and applied to a dsDNA-cellulose column equilibrated in buffer containing 50 mM NaCl. Elution was carried out by washing with buffer containing the concentrations of NaCl indicated above the panels. Fractions were analyzed by SDS-PAGE and autoradiography. Input, unfractionated input protein.

FIG. 3.

UL44ΔC290 binds to UL54. The physical binding of GST-full-length UL44 and of GST-UL44ΔC290 proteins to UL54 was tested by GST pulldown experiments. Purified GST, GST-full-length UL44 (FL), and GST-UL44ΔC290 (ΔC290) proteins, as indicated at the tops of the figure panels, were incubated with in vitro-expressed and radiolabeled HCMV UL54 (leftmost six lanes) or HSV-1 UL30 (rightmost two lanes) 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 following electrophoresis on an SDS-7.5% polyacrylamide gel. The positions of UL54 and UL30 are marked by arrows. I, input; E, eluted by glutathione.

FIG. 4.

UL44ΔC290 stimulates DNA synthesis by UL54. (A) The DNA polymerase activity of purified baculovirus-expressed UL54 alone (•) and in the presence of 200 (▾), 500 (*), or 1,000 (▴) fmol of GST-UL44 (full length) or of 200 (×), 500 (⧫), or 1,000 (+) fmol of GST-UL44ΔC290 was measured by incorporation of [³H]dTTP into a poly(dA)-oligo(dT) DNA template. As a control, the activity of 1,000 fmol of GST-UL44ΔC290 alone (▪) was also assayed. 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 radioactivity was counted. (B) Stimulation of UL54-mediated long-chain DNA synthesis by GST-full-length UL44 (FL) or GST-UL44ΔC290 (ΔC290) was assayed by measuring the incorporation of labeled TTP on a poly(dA)-oligo(dT) template. The reaction products were visualized by autoradiography following electrophoresis on a 4% alkaline agarose gel. Lane 1 contains 800 fmol of GST-UL44 (full-length) alone; lane 2 contains 800 fmol of GST-UL44ΔC290 alone; lanes 3 and 6 contain 200 fmol of UL54 alone; lanes 4 and 5 contain UL54 plus 400 and 800 fmol of GST-UL44 (full-length), respectively; lanes 7 and 8 contain UL54 plus 400 and 800 fmol of GST-UL44ΔC290, respectively.

FIG. 5.

UL44 mutants. The sequence of the UL44 connector loop (amino acids 129 to 140), in single-letter code, is reported on the top. A series of substitutions were engineered in this region of UL44 as described in Materials and Methods. For each mutant, the sequence of the region containing the mutation is shown, with the mutated residue indicated by an underlined boldface letter.

FIG. 6.

Binding of mutant UL44 proteins to UL54. The physical binding of wild-type (wt) and mutant GST-UL44ΔC290 proteins, as indicated at the tops of the figure panels, to UL54 was tested by GST pulldown assays as described in the legend to Fig. 3. I, input; E, eluted by glutathione. The position of UL54 is indicated by an arrow to the right of the panel. The mutants that exhibited reduced binding are indicated in boldface.

FIG. 7.

Results of ITC experiments binding wild-type and mutant UL44 to a synthetic peptide (UL54 peptide 1) corresponding to the C-terminal 22 residues of UL54. (A) Raw data for the titration of UL54 peptide 1 into a sample cell containing wild-type (wt) or mutant GST-UL44ΔC290 protein, as indicated on the top of each panel. (B) The heat of dilution of both protein and peptide in panel A was 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. The solid line is the best-fit curve for the data.

FIG. 8.

UL44 mutants are able to bind DNA. The DNA-binding activity of UL44 mutants was tested by comparing the elution profiles of wild-type (wt) and mutant GST-UL44ΔC290 proteins and GST, as indicated to the left of the panels, from a dsDNA-cellulose column as described in the legend to Fig. 2. The concentrations of NaCl used to elute the proteins are indicated above the panels. Input, unfractionated input protein.

FIG. 9.

Stimulation of long-chain DNA synthesis by UL54. Experiments were performed with a poly(dA) template and an oligo(dT) primer and labeled TTP as described in the legend to Fig. 4B. Lane 1, 200 fmol of UL54 alone; lane 2, 800 fmol of wild-type GST-UL44ΔC290 alone. The remaining lanes contained 200 fmol of UL54 plus 400 (lane 3) and 800 (lane 4) fmol of wild-type (wt) GST-UL44ΔC290, 400 (lane 5) and 800 (lane 6) fmol of GST-UL44ΔC290 Q133A, 400 (lane 7) and 800 (lane 8) fmol of GST-UL44ΔC290 D134A, 400 (lane 9) and 800 (lane 10) fmol of GST-UL44ΔC290 I135A, 400 (lane 11) and 800 (lane 12) fmol of GST-UL44ΔC290 V136A, and 400 (lane 134) and 800 (lane 14) fmol of GST-UL44ΔC290 R137A.

FIG. 10.

Structural features of UL44. (A) The peptide backbone of UL44 is shown in grey. The connector loop of UL44 (residues 129 to 140) is highlighted in black, with the four residues identified in this study as important for binding to UL54 (Q133, D134, I135, and V136) shown as ball-and-stick models and indicated by arrows. It should be noted that the connector loop appears to be flexible, as the individual atoms display higher average temperature factors than the rest of the molecule. Thus, the side chains may adopt multiple orientations in solution, which could differ from the positions displayed in this figure. This figure was created with Molscript and Raster3D (12, 19). (B) The solvent-accessible surfaces of the face containing the connector loop of UL44 (left) and of UL42 (right) are displayed with Grasp (20). The connector loop of each protein is indicated by arrowheads. In UL42, a deep groove, which accommodates the C-terminal helix of UL30, is present between the connector loop and a projection formed by residues D63 and R64 (indicated by arrows). In contrast, the analogous region of UL44 displays a much shallower groove, as it lacks a noticeable projection below the connector loop. The corresponding position in UL44 of the UL42 projection is indicated by an arrow and an asterisk.