Abstract
A key step in replication of human cytomegalovirus (HCMV) in the host cell is the generation and packaging of unit-length genomes into preformed capsids. The enzymes involved in this process are the terminases. The HCMV terminase complex consists of two terminase subunits, the ATPase pUL56 and the nuclease pUL89. A potential third component pUL51 has been proposed. Even though the terminase subunit pUL89 has been shown to be essential for DNA packaging and interaction with pUL56, it is not known how pUL89 mechanistically achieves sequence-specific DNA binding and nicking. To identify essential domains and invariant amino acids vis-a-vis nuclease activity and DNA binding, alanine substitutions of predicted motifs were analyzed. The analyses indicated that aspartate 463 is an invariant amino acid for the nuclease activity, while argine 544 is an invariant aa for DNA binding. Structural analysis of recombinant protein using electron microscopy in conjunction with single particle analysis revealed a curvilinear monomer with two distinct domains connected by a thinner hinge-like region that agrees well with the predicted structure. These results allow us to model how the terminase subunit pUL89’s structure may mediate its function.
Highlights
The human cytomegalovirus (HCMV) genome consists of a double stranded linear DNA that immediately circularizes after nuclear import [1, 2]
HCMV is a member of the herpesvirus family and represents a major human pathogen causing severe disease in newborns and immunocompromised patients for which the development of new non-nucleosidic antiviral agents are highly important
This manuscript focuses on DNA packaging, which is a target for development of new antivirals
Summary
The human cytomegalovirus (HCMV) genome consists of a double stranded linear DNA that immediately circularizes after nuclear import [1, 2]. The HCMV genome is approximately 235 kb in size, containing repetitive and unique components. Viral replication of this covalently closed circular DNA leads to the formation of concatemers, which must be resolved into unitlength genomes during packaging [3, 4]. A group of specific enzymes called terminases, first described for doublestranded DNA bacteriophages [5], mediates cleavage and by binding to the portal during packaging of viral DNA. Terminases represent multifunctional heterooligomers in which one subunit provides ATP for translocation into preformed capsids and cleaves concatemers into unit-length genomes while the other is required for sequence specific DNA binding [5,6,7,8]. Similarities in sequence specific cleavage are found in Herpesviridae and cos-containing dsDNA bacteriophages (e.g. λ) [9]
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