ATPase Activity of the Terminase Subunit pUL56 of Human Cytomegalovirus

Abstract

Herpesviral DNA packaging is a complex process resulting in unit-length genomes packed into preformed procapsids. This process is believed to be mediated by two packaging proteins, the terminase subunits. In the case of double-stranded DNA bacteriophages, the translocation of DNA was shown to be an energy-dependent process associated with an ATPase activity of the large terminase subunit. In the case of human cytomegalovirus it was not known which protein has the ability to hydrolyze ATP. In this study we expressed human cytomegalovirus terminase subunits, pUL89 and the carboxyl-terminal half of pUL56, as GST fusion proteins and purified these by affinity chromatography. ATPase assays demonstrated that the enzymatic activity is exclusively associated with pUL56. The characterization of the ATP hydrolysis showed that the enzymatic reaction is a fast process, whereas the spontaneous ATP decay followed slow kinetics. Interestingly, although pUL89 did not show any ATPase activity, it was capable of enhancing the UL56-associated ATP hydrolysis. Furthermore, a specific association of in vitro translated pUL89 with the carboxyl-terminal half of GST-UL56C was detected. This interaction was confirmed by co-immunoprecipitations of infected cells. Our results clearly demonstrated that (i) both terminase subunits interact with each other and (ii) the subunit pUL56 has an ATPase activity. Human cytomegalovirus (HCMV)1 DNA replication results in the formation of large head-to-tail DNA concatemers. The subsequent maturation into unit-length molecules involves site-specific cleavage at sequence motifs (pac motifs) located within the a sequence of the terminal and internal repeat segments (1-4). Unit-length genomes are then encapsidated into preassembled capsids. DNA-packaging is a complex biological process. While it is commonly accepted that ATP hydrolysis is the driving force behind it, the molecular mechanisms of DNA translocation and genome packaging remain unclear. In general, the following five steps are involved. (i) The recognition of concatemeric DNA by a specific protein (complex) able to (ii) bind and cut the DNA at specific sequence motifs (packaging signals, e.g. pac1 and pac2), (iii) translocation of the DNAprotein complex into the procapsid, (iv) packaging of one unitlength genome, and (v) completion of the packaging process by cutting off excess DNA at the portal region (5-7).Recently, we have demonstrated that the HCMV pUL56 gene product (pUL56), the homolog of the HSV-1 open reading frame UL28, is associated with sequence-specific binding of DNA containing packaging motifs leading to the suggestion that pUL56 plays a key role in DNA packaging (7-9). Comparable results were reported of the HSV-1 pUL28, demonstrating a direct interaction of pUL28 with DNA containing the pac1 motif (10). Furthermore, by the use of viral mutants it was demonstrated that the deletion of pUL28 leads to nuclear accumulation of naked nucleocapsids and uncleaved concatemeric DNA (11, 12). In addition, it has been noted that the HCMV UL...

Human Cytomegalovirus TRS1 Protein Is Required for Efficient Assembly of DNA-Containing Capsids

“…The concatemers must then be cleaved into unit-length pieces as the DNA is packaged into preassembled procapsids. This cleavage has been shown to utilize ATP and proceed concomitantly with insertion of the DNA into the capsid (12). In HSV-1 infections, at least seven proteins function at this point, including the large UL28 and small UL15 terminase subunits and the UL6 portal protein (18,38,39).…”

Assembly of monomeric human cytomegalovirus pUL104 into portal structures

“…The human cytomegalovirus (HCMV) terminase consists of a large and a small subunit, called viral protein pUL56 and pUL89, respectively. The large subunit endows the terminase with ATPase activity and the small subunit is required for cleavage of concatemeric DNA (Bogner, 2002;Hwang & Bogner, 2002;Scheffczik et al, 2002;Scholz et al, 2003). In addition to the terminase, the other critical packaging component is the portal protein.…”

Dramatic Effects of 2-Bromo-5,6-Dichloro-1-β- d -Ribofuranosyl Benzimidazole Riboside on the Genome Structure, Packaging, and Egress of Guinea Pig Cytomegalovirus

“…A functionally equivalent herpesvirus terminase has been postulated to be comprised of two subunits; in HSV-1, these are the products of the UL15 and UL28 genes. These proteins or their homologs in other herpesviruses have been shown to associate with one another (1,5,29,34,35), bind in vitro to packaging sequences (2, 10), and possess in vitro nucleolytic (10, 58) and ATPase activities (29,59) (the later is a requirement for DNA translocation in bacteriophage). Additional clues to their activities have come from the study of compounds that block the cleavage and packaging process, which include the halogenated benzimidazoles BDCRB (2-bromo-5,6-dichloro-1-␤-D-riborfuranosyl benzimidazole riboside) and TCRB (2,5,6-trichloro-1-␤-D-ri-* Corresponding author.…”