Exploring the Human Cytomegalovirus Core Nuclear Egress Complex as a Novel Antiviral Target: A New Type of Small Molecule Inhibitors

Alkhashrom, Sewar; Kicuntod, Jintawee; Häge, Sigrun; Schweininger, Johannes; Muller, Yves A.; Lischka, Peter; Marschall, Manfred; Eichler, Jutta

doi:10.3390/v13030471

Cited by 11 publications

(22 citation statements)

References 31 publications

(70 reference statements)

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“…Such an antiviral MoA might be achieved by the identification of small molecules that either block the pUL97 recognition of specific substrates or interfere with pUL97–cyclin binding. As far as the latter point is concerned, our initial screening experiments, using small molecules derived from the Prestwick Chemical Library ® of approved drugs, provided experimental evidence that inhibitors with the potential to block HCMV-specific protein interactions can display strong antiviral activity [ 60 , 61 ]. This strategy might similarly be adapted to the screening of sterical inhibitors of pUL97–cyclin assembly.…”

Section: Discussionmentioning

confidence: 99%

Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H

Schütz¹,

Müller²,

Socher

et al. 2022

IJMS

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The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97–cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97–cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97–cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97–cyclin interaction. High selective pressure on the formation of pUL97–cyclin complexes was identified by the use of clinically relevant mutants.

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Section: Discussionmentioning

confidence: 99%

Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H

Schütz¹,

Müller²,

Socher

et al. 2022

IJMS

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“…The validation of the NEC as an efficient target for small molecules exerting antiviral activity, has been achieved by several ways. These experiments included deletion and replacement mutants in NEC binding studies, recombinant viruses, experimental knock-down models, inhibitory peptides, NEC-directed small molecules, and some more [ 42 , 103 , 123 , 124 , 125 ]. A key point in this direction may have been the screening analysis using the Prestwick Chemical Library ® that contains a selection of pharmacologically valuable compounds and approved drugs [ 123 , 124 ].…”

Section: Validation Of the Nec As A Unique Target Of Anti-herpesviral...mentioning

confidence: 99%

“…These experiments included deletion and replacement mutants in NEC binding studies, recombinant viruses, experimental knock-down models, inhibitory peptides, NEC-directed small molecules, and some more [ 42 , 103 , 123 , 124 , 125 ]. A key point in this direction may have been the screening analysis using the Prestwick Chemical Library ® that contains a selection of pharmacologically valuable compounds and approved drugs [ 123 , 124 ]. The results of these studies provided a proof-of-concept that an inhibitory compound such as merbromin (MBM) that prevents the formation of the HCMV core NEC has the potential to block further processing of nuclear egress, eventually resulting in an intranuclear trapping of viral capsids.…”

Section: Validation Of the Nec As A Unique Target Of Anti-herpesviral...mentioning

confidence: 99%

‘Come together’—The Regulatory Interaction of Herpesviral Nuclear Egress Proteins Comprises Both Essential and Accessory Functions

Häge

Marschall

2022

Cells

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Herpesviral nuclear egress is a fine-tuned regulatory process that defines the nucleocytoplasmic release of viral capsids. Nuclear capsids are unable to traverse via nuclear pores due to the fact of their large size; therefore, herpesviruses evolved to develop a vesicular transport pathway mediating the transition across the two leaflets of the nuclear membrane. The entire process involves a number of regulatory proteins, which support the local distortion of the nuclear envelope. In the case of the prototype species of β-Herpesvirinae, the human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the core proteins pUL50 and pUL53 that oligomerize, form capsid docking lattices and mediate multicomponent assembly with NEC-associated viral and cellular proteins. The NEC-binding principle is based on the hook-into-groove interaction through an N-terminal hook-like pUL53 protrusion that embraces an α-helical pUL50 binding groove. Thus far, the function and characteristics of herpesviral core NECs have been well studied and point to the groove proteins, such as pUL50, as the multi-interacting, major determinants of NEC formation and egress. This review provides closer insight into (i) sequence and structure conservation of herpesviral core NEC proteins, (ii) experimentation on cross-viral core NEC interactions, (iii) the essential functional roles of hook and groove proteins for viral replication, (iv) an establishment of assay systems for NEC-directed antiviral research and (v) the validation of NEC as putative antiviral drug targets. Finally, this article provides new insights into the conservation, function and antiviral targeting of herpesviral core NEC proteins and, into the complex regulatory role of hook and groove proteins during the assembly, egress and maturation of infectious virus.

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“…It should also be mentioned, however, that an effect similar to MBV was not reproducibly obtained for other known inhibitors of viral nuclear egress, such as the recently identified core NEC-interfering small molecule merbromin (MBM; [ 13 , 39 ]). For MBM, the data obtained with the qPCR-based nuclear egress assay did not show unequivocal and robust indication for NEC-inhibitory activity (data not shown).…”

Section: Resultsmentioning

confidence: 99%

The Oligomeric Assemblies of Cytomegalovirus Core Nuclear Egress Proteins Are Associated with Host Kinases and Show Sensitivity to Antiviral Kinase Inhibitors

Kicuntod

Häge

Hahn

et al. 2022

Viruses

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The nucleo-cytoplasmic capsid egress of herpesviruses is a unique regulated process that ensures the efficiency of viral replication and release. For human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50–pUL53 heterodimer that is able to oligomerize and thus to build hexameric lattices. These structures determine capsid binding and multicomponent protein interaction including NEC-associated host factors. The underlying characteristic of the core NEC formation is based on the N-terminal hook structure of pUL53 that binds into an alpha-helical groove of pUL50, and is thus described as a hook-into-groove interaction. This central regulatory element has recently been validated as a target of antiviral strategies, and first NEC-targeted prototypes of inhibitory small molecules were reported by our previous study. Here, we further analyzed the oligomerization properties of the viral NEC through an approach of chemical protein cross-linking. Findings were as follows: (i) a cross-link approach demonstrated the oligomeric state of the HCMV core NEC using material from HCMV-infected or plasmid-transfected cells, (ii) a Western blot-based identification of NEC-associated kinases using the cross-linked multicomponent NECs was successful, and (iii) we demonstrated the NEC-inhibitory and antiviral activity of specific inhibitors directed to these target kinases. Combined, the results strongly underline the functional importance of the oligomerization of the HCMV-specific NEC that is both phosphorylation-dependent and sensitive to antiviral kinase inhibitors.

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Exploring the Human Cytomegalovirus Core Nuclear Egress Complex as a Novel Antiviral Target: A New Type of Small Molecule Inhibitors

Cited by 11 publications

References 31 publications

Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H

Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H

‘Come together’—The Regulatory Interaction of Herpesviral Nuclear Egress Proteins Comprises Both Essential and Accessory Functions

The Oligomeric Assemblies of Cytomegalovirus Core Nuclear Egress Proteins Are Associated with Host Kinases and Show Sensitivity to Antiviral Kinase Inhibitors

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