ABSTRACT
Terminases are a class of proteins which catalyze the generation of unit-length genomes during DNA packaging. These essential proteins are conserved throughout the herpesviruses and many double-stranded DNA bacteriophages. We have determined the structure of the large terminase subunit pUL56 of human cytomegalovirus, a highly pathogenic virus, to 2.6 nm resolution. Image analysis of purified pUL56 suggests that the molecule exists as a dimer formed by the association of two ring-like structures positioned on top of each other and connected by a pronounced density on one side. The 3D reconstruction of pUL56 provides first structural insights into the active protein.
Subject(s)
Adenosine Triphosphatases/metabolism , Cytomegalovirus/enzymology , Endodeoxyribonucleases/chemistry , Protein Subunits/chemistry , Viral Proteins/chemistry , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/drug effects , Baculoviridae/genetics , Benzimidazoles/pharmacology , Blotting, Western , Cross-Linking Reagents/metabolism , Cytomegalovirus/ultrastructure , Dimerization , Electrophoresis, Polyacrylamide Gel , Endodeoxyribonucleases/isolation & purification , Endodeoxyribonucleases/metabolism , Endodeoxyribonucleases/ultrastructure , Glutaral/metabolism , Glutathione Transferase/metabolism , Humans , Imaging, Three-Dimensional , Molecular Weight , Protein Binding , Protein Subunits/metabolism , Recombinant Proteins/metabolism , Ribonucleosides/pharmacology , Structure-Activity Relationship , Time Factors , Viral Proteins/isolation & purification , Viral Proteins/metabolism , Viral Proteins/ultrastructureABSTRACT
Herpesvirus DNA packaging involves binding and cleavage of DNA containing the specific DNA-packaging motifs. Here we report a first characterization of the terminase subunits pUL56 and pUL89 of human cytomegalovirus (HCMV). Both gene products were shown to have comparable nuclease activities in vitro. Under limiting protein concentrations the nuclease activity is enhanced by interaction of pUL56 and pUL89. High amounts of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole partially inhibited the pUL89-associated nuclease activity. It was demonstrated that pUL56 is able to bind to nucleocapsids in vivo. Electron microscopy (EM) and image analysis of purified pUL56 revealed that the molecules occurred as a distinct ring-shaped structure with a pronounced cleft. EM analysis of purified pUL89 demonstrated that this protein is also a toroidal DNA-metabolizing protein. Upon interaction of pUL56 with linearized DNA, the DNA remains uncut while the cutting event itself is mediated by pUL89. Using biochemical assays in conjunction with EM pUL56 was shown to (i) bind to DNA and (ii) associate with the capsid. In contrast to this, EM analysis implied that pUL89 is required to effect DNA cleavage. The data provide the first insights into the terminase-dependent viral DNA-packaging mechanism of HCMV.
