Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain

Abstract

During viral replication, herpesviruses package their DNA into the procapsid by means of the terminase protein complex. In human cytomegalovirus (herpesvirus 5), the terminase is composed of subunits UL89 and UL56. UL89 cleaves the long DNA concatemers into unit-length genomes of appropriate length for encapsidation. We used ESPRIT, a high-throughput screening method, to identify a soluble purifiable fragment of UL89 from a library of 18,432 randomly truncated ul89 DNA constructs. The purified protein was crystallized and its three-dimensional structure was solved. This protein corresponds to the key nuclease domain of the terminase and shows an RNase H/integrase-like fold. We demonstrate that UL89-C has the capacity to process the DNA and that this function is dependent on Mn(2+) ions, two of which are located at the active site pocket. We also show that the nuclease function can be inactivated by raltegravir, a recently approved anti-AIDS drug that targets the HIV integrase.

Fig. 1.

(A) Overall structure of UL89-C in a ribbon representation. The metal ions are indicated by yellow spheres. UL56-interacting helix α4 is highlighted in blue, α helices in cyan, 3₁₀ helices in green, and β strands in magenta. (B) Detailed view of the active site area. One Mn²⁺ ion is shown coordinated by Asp463 and Glu534 and a second one is coordinated by Asp463 and Glu651. (C) Topology diagram of UL89. Color code as in (A).

Fig. 2.

UL89-C surface representations. (A) Conservation surface of UL89-C based on a Risler matrix calculation. Residue color is shown on the basis of conservation score within the eight human herpesvirus: orange, more than 80% similarity; green, full conservation. Metal ions are shown in yellow. (B) Electrostatic potential surface of UL89-C in the same view as (A). The positive potential is shown in blue whereas the negative potential is in red.

Fig. 3.

UL89-C in vitro nuclease assay. (A) Effect of divalent cations on UL89-C linear dsDNA nuclease activity. Lane 1: linear (digested with HindIII) pUC18 plasmid in the absence of the nuclease. Lane 2: nuclease reaction in the absence of divalent cations. Lane 3: nuclease reaction in the presence of Mg²⁺. Lane 4: nuclease reaction in the presence of Ca²⁺. Lane 5: nuclease reaction in the presence of Mn²⁺. (B) Effect of divalent cations on UL89-C circular dsDNA nuclease activity. Lane 1: circular pUC18 plasmid in the absence of the nuclease. Lane 2: linear (digested with HindIII) pUC18 plasmid in the absence of the nuclease. Lane 3: nuclease reaction in the absence of divalent cations. Lane 4: nuclease reaction in the presence of Mg²⁺. Lane 5: nuclease reaction in the presence of Ca²⁺. Lane 6: nuclease reaction in the presence of Mn²⁺. (C) Nuclease assays with the UL89-C WT and mutant proteins with linear dsDNA. Lane 1: linear (digested with HindIII) pUC18 plasmid in absence of the nuclease. Lane 2: nuclease assay with the WT proteins. Lane 3: nuclease assay with the D463A mutant. Lane 4: nuclease assay with the D651A mutant. Lane 5: nuclease assay with the D463A E534A mutant. (D) Nuclease assay with the UL89-C WT and mutant proteins with ds-circular DNA. Lane 1: circular pUC18 plasmid in absence of the nuclease. Lane 2: nuclease assay with the WT protein. Lane 3: nuclease assay with the D463A mutant. Lane 4: nuclease assay with the D651A mutant. Lane 5: nuclease assay with the D463A E534A mutant. Mutant proteins show only residual nuclease function. Labels: sc, supercoiled circular plasmid DNA; ni-oc, nicked open circular plasmid DNA; li, linear plasmid DNA.

Fig. 4.

Inhibition of UL89-C nuclease activity by raltegravir. Lane 1: Circular pUC18 plasmid in the absence of the nuclease. Lane 2: Linear (digested with HindIII) pUC18 plasmid in the absence of the nuclease. Lane 3: Nuclease assay with the UL89-C WT protein. Lanes 4–14: Nuclease assays in the presence of a range of concentrations of raltegravir (0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256 and 512 μM).