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Full-length human cytomegalovirus terminase pUL89 adopts a two-domain structure specific for DNA packaging.

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.

SUBMITTER: Theiß J

PROVIDER: S-EPMC6897398 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Full-length human cytomegalovirus terminase pUL89 adopts a two-domain structure specific for DNA packaging.

Theiß Janine J Sung Min Woo MW Holzenburg Andreas A Bogner Elke E

PLoS pathogens 20191206 12

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 ...[more]

PMID: 31809525

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Project description:The UL69 protein from human cytomegalovirus (HCMV) is a multifunctional regulatory protein and a member of the ICP27 protein family conserved throughout herpesviruses. UL69 plays many roles during productive infection, including the regulation of viral gene expression, nuclear export of intronless viral RNAs, and control of host cell cycle progression. Throughout the ICP27 protein family, an ability to self-associate is correlated with the functions of these proteins in transactivating certain viral genes. Here, we determined the domain boundaries of a globular ICP27 homology domain of UL69, which mediates self-association, and characterized the oligomeric state of the isolated domain. Size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) revealed that residues 200 to 540 form a stable homo-tetramer, whereas a shorter region comprising residues 248 to 536 forms a homo-dimer. Structural analysis of the UL69 tetramer by transmission electron microscopy (TEM) revealed a dimer-of-dimers three-dimensional envelope with bridge features likely from a region of the protein unique to betaherpesviruses. The data provide a structural template for tetramerization and improve our understanding of the structural diversity and features necessary for self-association within UL69 and the ICP27 family.IMPORTANCE Human cytomegalovirus (HCMV) infection is widespread in the human population but typically remains dormant in an asymptomatic latent state. HCMV causes disease in neonates and adults with suppressed or impaired immune function, as the virus is activated into a lytic state. All species of herpesvirus express a protein from the ICP27 family which functions as a posttranscriptional activator in the lytic state. In HCMV, this protein is called UL69. The region of sequence conservation in the ICP27 family is a folded domain that mediates protein interactions, including self-association and functions in transactivation. All members thus far analyzed homo-dimerize, with the exception of UL69, which forms higher-order oligomers. Here, we use biochemical and structural data to reveal that UL69 forms stable tetramers composed of a dimer of dimers and determine a region essential for cross-dimer stabilization.

Dataset Information

Full-length human cytomegalovirus terminase pUL89 adopts a two-domain structure specific for DNA packaging.

Publications

Full-length human cytomegalovirus terminase pUL89 adopts a two-domain structure specific for DNA packaging.

Similar Datasets

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