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.

Structure of P22 Headful Packaging Nuclease

“…Two additional sets of helices (␣ 1 -␣ 2 and ␣ 3 -␣ 4 ) surround the central core making contacts with the solvent. Overall, P22 headful nuclease is very similar to the nuclease domain of SPP1 (18), T4 (6), and HHV-5 (19). Despite the low sequence identity (16% with SPP1, 14% with T4, 12% with the T4-like phage RB49, and 12% with HHV-5), the four viral nucleases are structurally superimposable.…”

“…3A). Mg A lies in the active site of P22 nuclease at a position equivalent to the magnesium seen in the high resolution structure of T4/RB49 nuclease (6) or the manganese ion visualized in SPP1 (18) and HHV-5 nucleases (19). The second site, Mg B , is surrounded by four less defined water molecules (of which only one was included in the final model), likely consistent with a tetrahedral coordination (42) (Fig.…”

“…Atomic level structural information is presently available for a catalytically inactive construct of bacteriophage T4 L-terminase (6) and for the isolated L-terminase nuclease domains of SPP1 (18) and human cytomegalovirus (human herpesvirus 5 or HHV-5) (19). While no high resolution structure is available for a L-terminase holoenzyme, a pseudoatomic model of T4 L-terminase was proposed based on a 32 Å asymmetric cryo-electron microscopy reconstruction of T4 procapsid bound to L-terminase (6).…”

Structures of the phage Sf6 large terminase provide new insights into DNA translocation and cleavage

“…Crystallization of fulllength viral DNA-packaging motor proteins has been difficult as reported for phage T4 (13), phage P22 (18) and a herpesvirus (19), and it was only possible to crystallize and solve the structure of T4 gp17 with double mutations in the ATPase domain active center (D255E/E256D) and a 43-residue truncation at the C terminus, which rendered the protein inactive in ATP hydrolysis and DNA packaging (13). We have been able to crystallize the full-length, wild-type gp2.…”

“…Terminases contain a nuclease domain that is responsible for cleavage of viral DNA concatemer into genomelength units. Despite low sequence identity (<15%), the gp2 C-terminal domain exhibits a fold that not only is homologous to viral terminase nuclease domain structures (13,14,18,19), but also clearly resembles those of a large group of nucleases such as RNases H, topoisomerases, integrases, and DNA/RNA polymerases, which use similar metal-ion catalytic mechanisms (30,31). (Fig.…”