Insight into the structure of the pUL89 C‐terminal domain of the human cytomegalovirus terminase complex

Couvreux, Anthony; Hantz, Sébastien; Marquant, Rodrigue; Champier, Gaël; Alain, Sophie; Morellet, Nelly; Bouaziz, Serge

doi:10.1002/prot.22669

Cited by 11 publications

(14 citation statements)

References 53 publications

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“…Their terminases have been intensively studied, in particular those of phages T4 and RB49 (10), λ (11), SPP1 (12), P22 (13), and Sf6 (14), and the structure of the nuclease domains of the large terminase subunits gp17 of RB49 (15) and G2P of SPP1 (16) and the full-length large terminase subunit gp17 of T4 (15) have been determined. A theoretical model for the structure of the C-terminal domain of UL89 has been proposed recently (17).…”

mentioning

confidence: 99%

Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain

Nadal

Mas

Blanco

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

105

182

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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.

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mentioning

confidence: 99%

Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain

Nadal

Mas

Blanco

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

105

182

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“…1A, construct 2), the first 1,928 nucleotides (out of 2,553) of the UL56 ORF were deleted, and for the UL89 knockout genome (Fig. 1A, construct 3), most of exon 2 (1,049 nucleotides out of 1,137) was removed, including the sequences encoding the proposed interaction domain for pUL56 (6,47). Care was taken to retain putative regulatory sequences of neighboring essential ORFs.…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, conformational changes are a universal prerequisite for the generation of force in molecular nanomotors (2,65,66). Structural analyses of the isolated pUL56 and pUL89 terminase subunits have been reported (5)(6)(7)67); however, in view of the folding-upon-binding principle that presumably also applies to the HCMV terminase, it remains an important goal to determine the structure of the complex as a whole and to elucidate conformational changes of the individual subunits. Further conformational alterations likely also occur upon binding to the viral genome and the capsid portal.…”

Section: Discussionmentioning

confidence: 99%

“…For HCMV, several lines of evidence indicate that the viral terminase comprises the proteins pUL56 and pUL89, with pUL56 binding to the capsid portal protein pUL104 as well as to the packaging signals on the concatemeric viral DNA (3,4) and pUL89 mediating cleavage after exactly one genome copy has been encapsidated (5). Analyses of the pUL89 structure revealed similarities between herpesvirus and bacteriophage terminases and showed that the endonuclease domain is contained within the pUL89 C terminus (6)(7)(8). Modeling of the herpesviral pUL56 orthologs disclosed structural similarity to human topoisomerase I, suggesting a role in processing of the viral DNA as proposed by Visalli et al (9).…”

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confidence: 99%

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Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation

Neuber

Wagner

Goldner

et al. 2017

J Virol

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Human cytomegalovirus (HCMV) genome encapsidation requires several essential viral proteins, among them pUL56, pUL89, and the recently described pUL51, which constitute the viral terminase. To gain insight into terminase complex assembly, we investigated interactions between the individual subunits. For analysis in the viral context, HCMV bacterial artificial chromosomes carrying deletions in the open reading frames encoding the terminase proteins were used. These experiments were complemented by transient-transfection assays with plasmids expressing the terminase components. We found that if one terminase protein was missing, the levels of the other terminase proteins were markedly diminished, which could be overcome by proteasome inhibition or providing the missing subunit in trans. These data imply that sequestration of the individual subunits within the terminase complex protects them from proteasomal turnover. The finding that efficient interactions among the terminase proteins occurred only when all three were present together is reminiscent of a folding-upon-binding principle leading to cooperative stability. Furthermore, whereas pUL56 was translocated into the nucleus on its own, correct nuclear localization of pUL51 and pUL89 again required all three terminase constituents. Altogether, these features point to a model of the HCMV terminase as a multiprotein complex in which the three players regulate each other concerning stability, subcellular localization, and assembly into the functional tripartite holoenzyme.IMPORTANCE HCMV is a major risk factor in immunocompromised individuals, and congenital CMV infection is the leading viral cause for long-term sequelae, including deafness and mental retardation. The current treatment of CMV disease is based on drugs sharing the same mechanism, namely, inhibiting viral DNA replication, and often results in adverse side effects and the appearance of resistant virus strains. Recently, the HCMV terminase has emerged as an auspicious target for novel antiviral drugs. A new drug candidate inhibiting the HCMV terminase, Letermovir, displayed excellent potency in clinical trials; however, its precise mode of action is not understood yet. Here, we describe the mutual dependence of the HCMV terminase constituents for their assembly into a functional terminase complex. Besides providing new basic insights into terminase formation, these results will be valuable when studying the mechanism of action for drugs targeting the HCMV terminase and developing additional substances interfering with viral genome encapsidation.

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“…1 , its secondary structure is predicted as an alpha helix. Previous studies demonstrated that the peptide pUL89(580-600) implicated in the pUL56-pUL89 interface 13 adopts an alpha helix secondary structure 14 , 15 . Moreover, wide protein-protein interfaces analyses revealed a preferential interaction of an helix of one protein with one of its counterpart 16 , 17 .…”

Section: Resultsmentioning

confidence: 99%

Identification of a short sequence in the HCMV terminase pUL56 essential for interaction with pUL89 subunit

Ligat

Jacquet

Chou

et al. 2017

Sci Rep

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The human cytomegalovirus (HCMV) terminase complex consists of several components acting together to cleave viral DNA into unit length genomes and translocate them into capsids, a critical process in the production of infectious virions subsequent to DNA replication. Previous studies suggest that the carboxyl-terminal portion of the pUL56 subunit interacts with the pUL89 subunit. However, the specific interacting residues of pUL56 remain unknown. We identified a conserved sequence in the C-terminal moiety of pUL56 (671WMVVKYMGFF680). Overrepresentation of conserved aromatic amino acids through 20 herpesviruses homologues of pUL56 suggests an involvement of this short peptide into the interaction between the larger pUL56 terminase subunit and the smaller pUL89 subunit. Use of Alpha technology highlighted an interaction between pUL56 and pUL89 driven through the peptide 671WMVVKYMGFF680. A deletion of these residues blocks viral replication. We hypothesize that it is the consequence of the disruption of the pUL56-pUL89 interaction. These results show that this motif is essential for HCMV replication and could be a target for development of new small antiviral drugs or peptidomimetics.

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Insight into the structure of the pUL89 C‐terminal domain of the human cytomegalovirus terminase complex

Cited by 11 publications

References 53 publications

Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain

Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain

Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation

Identification of a short sequence in the HCMV terminase pUL56 essential for interaction with pUL89 subunit

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