Inhibition of human cytomegalovirus DNA polymerase by C-terminal peptides from the UL54 subunit

Abstract

In common with other herpesviruses, the human cytomegalovirus (HCMV) DNA polymerase contains a catalytic subunit (Pol or UL54) and an accessory protein (UL44) that is thought to increase the processivity of the enzyme. The observation that antisense inhibition of UL44 synthesis in HCMV-infected cells strongly inhibits viral DNA replication, together with the structural similarity predicted for the herpesvirus processivity subunits, highlights the importance of the accessory protein for virus growth and raises the possibility that the UL54/UL44 interaction might be a valid target for antiviral drugs. To investigate this possibility, overlapping peptides spanning residues 1161 to 1242 of UL54 were synthesized and tested for inhibition of the interaction between purified UL54 and UL44 proteins. A peptide, LPRRLHLEPAFLPYSVKAHECC, corresponding to residues 1221 to 1242 at the very C terminus of UL54, disrupted both the physical interaction between the two proteins and specifically inhibited the stimulation of UL54 by UL44. A mutant peptide lacking the two carboxy-terminal cysteines was markedly less inhibitory, suggesting a role for these residues in the UL54/UL44 interaction. Circular dichroism spectroscopy indicated that the UL54 C-terminal peptide can adopt a partially alpha-helical structure. Taken together, these results indicate that the two subunits of HCMV DNA polymerase most likely interact in a way which is analogous to that of the two subunits of herpes simplex virus DNA polymerase, even though there is no sequence homology in the binding site, and suggest that the UL54 peptide, or derivatives thereof, could form the basis for developing a new class of anti-HCMV inhibitors that act by disrupting the UL54/UL44 interaction.

FIG. 1.

Purified UL44 and UL54 preparations. 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 UL54 purification (A) and after each step of UL44 purification (B) were analyzed on a 5% or 10% polyacrylamide SDS-PAGE gel, respectively. Abbreviations: Mr, marker; Ext, protein extract from Sf9 cells infected with recombinant baculovirus P9; DC, after double-stranded DNA cellulose column; MQ, after MQ column; PS, after phenyl-Sepharose column. The position of the proteins (UL54 and UL44) and the molecular masses (in kilodaltons) of the markers are indicated on the right and left, respectively.

FIG. 2.

UL54/UL44 interaction assay. The UL44 protein was added to microtiter wells precoated with UL54 (0.2 μg) (•) or uncoated wells (○). Bound UL44 was detected with MAb YL1/2, which was in turn detected with an HRP-conjugated anti-rat antibody.

FIG. 3.

Inhibition of the physical interaction between UL54 and UL44 by C-proximal and C-terminal UL54 peptides as measured by an interaction ELISA. (A) Inhibitory activity of peptides corresponding to residues 1221 to 1242 (peptide 1 [•]), 1191 to 1210 (peptide 3 [▴]), and 1161 to 1180 (peptide 5 [▪]) of UL54. (B) Inhibitory activity of peptides corresponding to residues 1221 to 1242 (peptide 1 [•]), 1223 to 1242 (peptide 6 [○]), 1221 to 1241 (peptide 7 [⧫]), and 1221 to 1240 (peptide 8 [▾]) of UL54.

FIG. 4.

Stimulation of the activity of the catalytic subunit UL54 by the polymerase accessory protein UL44. The effect of UL44 on UL54 activity was examined by measuring incorporation of [³H]dTTP into two different templates: poly(dA)-oligo(dT) (A) and activated calf thymus DNA (B). Symbols: ▪, no protein; •, UL54; ▴, UL54 + UL44. Error bars show standard deviations.

FIG. 5.

Inhibition of the UL54-UL44 functional interaction by C-terminal UL54 peptides. Effect of synthetic peptides on incorporation of [³H]dTTP into a poly(dA)-oligo(dT) by UL54/UL44 complex was tested. The peptides correspond to residues 1221 to 1242 (▪) and residues 1161 to 1180 (•) of HCMV UL54. A peptide corresponding to C-terminal 27 residues of HSV-1 UL30 (aa 1209 to 1235 [▴]) was used as a control. The graph shows the average of data from three independent experiments together with the standard deviation (error bars).

FIG. 6.

Effect of the UL54 C-terminal peptide on activity of the catalytic subunit of HCMV DNA polymerase. The incorporation of [³H]dTTP into a poly(dA)-oligo(dT) template by UL54 alone was measured in the presence of different concentrations of peptide 1, corresponding to residues 1221 to 1242 of UL54. Symbols: ▴, no peptide; ▪, 12.5 μM; ▵, 25 μM; •, 50 μM; ○, 100 μM; □, 200 μM; ▿, 400 μM.

FIG. 7.

Analysis of the structure of UL54 inhibitory peptide. CD spectra of peptide 1 (A), corresponding to residues 1221 to 1242 of HCMV UL54, of a 27-mer peptide corresponding to residues 1209 to 1235 of HSV-1 UL30 (B) and of peptide 7 (C) and peptide 8 (D), corresponding to residues 1221 to 1241 and 1221 to 1240 of HCMV UL54, respectively. CD spectra of peptides in 50 mM phosphate buffer (pH 8.0) (dotted line), in 20% (vol/vol) TFE-water (dashed line), and in 95% (vol/vol) TFE-water (solid line) are reported. Double minima at 222 and 208 nm and a maximum at 192 nm are characteristic of α-helix spectra, while a weak maximum at 215 nm and an intense minimum at 195 nm are characteristic of random coils (26).