PMID- 37591314 OWN - NLM STAT- MEDLINE DCOM- 20230831 LR - 20230906 IS - 1872-7492 (Electronic) IS - 0168-1702 (Print) IS - 0168-1702 (Linking) VI - 335 DP - 2023 Oct 2 TI - Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency. PG - 199200 LID - S0168-1702(23)00162-4 [pii] LID - 10.1016/j.virusres.2023.199200 [doi] LID - 199200 AB - Human cytomegalovirus (HCMV) infection is shaped by a tightly regulated interplay between viral and cellular proteins. Distinct kinase activities, such as the viral cyclin-dependent kinase ortholog (vCDK) pUL97 and cellular CDK7 are both crucial for efficient viral replication. Previously, we reported that both kinases, vCDK/pUL97 and CDK7, interact with cyclin H, thereby achieving an enhanced level of kinase activity and overall functionality in viral replication. Here we provide a variety of novel results, as generated on a methodologically extended basis, and present a concept for the codetermination of viral replication efficiency through these kinase activities: (i) cyclin H expression, in various human cell types, is substantially upregulated by strains of HCMV including the clinically relevant HCMV Merlin; (ii) vCDK/pUL97 interacts with human cyclin H in both HCMV-infected and plasmid-transfected cell systems; (iii) a doxycycline-inducible shRNA-dependent knock-down (KD) of cyclin H significantly reduces pUL97 activity (qSox in vitro kinase assay); (iv) accordingly, pUL97 in vitro kinase activity is seen significantly increased upon addition of recombinant cyclin H; (v) as a point of specific importance, human CDK7 activity shows an increase by vCDK/pUL97-mediated trans-stimulation (whereas pUL97 is not stimulated by CDK7); (vi) phosphosite-specific antibodies indicate an upregulated CDK7 phosphorylation upon HCMV infection, as mediated through a pUL97-specific modulatory effect (i.e. shown by pUL97 inhibitor treatment or pUL97-deficient viral mutant); (vii) finally, an efficient KD of cyclin H in primary fibroblasts generally results in an impaired HCMV replication efficiency as measured on protein and genomic levels. These results show evidence for the codetermination of viral replication by vCDK/pUL97, cyclin H and CDK7, thus supporting the specific importance of cyclin H as a central regulatory factor, and suggesting novel targeting options for antiviral drugs. CI - Copyright (c) 2023. Published by Elsevier B.V. FAU - Schutz, Martin AU - Schutz M AD - Institute for Clinical and Molecular Virology, Friedrich-Alexander-Universitat Erlangen-Nurnberg (FAU), Schlossgarten 4, Erlangen 91054, Germany. Electronic address: martin.schuetz@uk-erlangen.de. FAU - Wangen, Christina AU - Wangen C AD - Institute for Clinical and Molecular Virology, Friedrich-Alexander-Universitat Erlangen-Nurnberg (FAU), Schlossgarten 4, Erlangen 91054, Germany. FAU - Sommerer, Mona AU - Sommerer M AD - Institute for Clinical and Molecular Virology, Friedrich-Alexander-Universitat Erlangen-Nurnberg (FAU), Schlossgarten 4, Erlangen 91054, Germany. FAU - Kogler, Melanie AU - Kogler M AD - Institute for Clinical and Molecular Virology, Friedrich-Alexander-Universitat Erlangen-Nurnberg (FAU), Schlossgarten 4, Erlangen 91054, Germany. FAU - Eickhoff, Jan AU - Eickhoff J AD - Lead Discovery Center GmbH, Dortmund, Germany. FAU - Degenhart, Carsten AU - Degenhart C AD - Lead Discovery Center GmbH, Dortmund, Germany. FAU - Klebl, Bert AU - Klebl B AD - Lead Discovery Center GmbH, Dortmund, Germany. FAU - Naing, Zin AU - Naing Z AD - Serology and Virology Division, NSW Health Pathology Microbiology, Prince of Wales Hospital, and Schools of Women's and Children's Health, Medicine and Biotechnology and Biomolecular Sciences, University of New South Wales, High Street, Sydney, Australia. FAU - Egilmezer, Ece AU - Egilmezer E AD - Serology and Virology Division, NSW Health Pathology Microbiology, Prince of Wales Hospital, and Schools of Women's and Children's Health, Medicine and Biotechnology and Biomolecular Sciences, University of New South Wales, High Street, Sydney, Australia. FAU - Hamilton, Stuart T AU - Hamilton ST AD - Serology and Virology Division, NSW Health Pathology Microbiology, Prince of Wales Hospital, and Schools of Women's and Children's Health, Medicine and Biotechnology and Biomolecular Sciences, University of New South Wales, High Street, Sydney, Australia. FAU - Rawlinson, William D AU - Rawlinson WD AD - Serology and Virology Division, NSW Health Pathology Microbiology, Prince of Wales Hospital, and Schools of Women's and Children's Health, Medicine and Biotechnology and Biomolecular Sciences, University of New South Wales, High Street, Sydney, Australia. FAU - Sticht, Heinrich AU - Sticht H AD - Division of Bioinformatics, Institute of Biochemistry, FAU, Erlangen, Germany. FAU - Marschall, Manfred AU - Marschall M AD - Institute for Clinical and Molecular Virology, Friedrich-Alexander-Universitat Erlangen-Nurnberg (FAU), Schlossgarten 4, Erlangen 91054, Germany. Electronic address: manfred.marschall@fau.de. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20230819 PL - Netherlands TA - Virus Res JT - Virus research JID - 8410979 RN - 0 (Antiviral Agents) RN - 0 (Cyclin H) RN - EC 2.7.11.22 (Cyclin-Dependent Kinases) RN - 0 (CCNH protein, human) RN - 0 (CDK7 protein, human) SB - IM MH - Humans MH - Antiviral Agents MH - Cyclin H MH - *Cyclin-Dependent Kinases/genetics MH - *Cytomegalovirus/genetics MH - Phosphorylation PMC - PMC10445456 OTO - NOTNLM OT - Codetermination of viral replication OT - Cyclin H knock-down OT - Functional importance of cyclin H OT - Human cytomegalovirus OT - Modulation of vCDK/pUL97 activity in vitro OT - Options for antiviral drug targeting OT - Viral cyclin-dependent kinase (CDK) ortholog OT - pUL97-mediated CDK7 trans-stimulation OT - vCDK/pUL97-cyclin binding COIS- Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. EDAT- 2023/08/18 00:42 MHDA- 2023/08/29 12:42 PMCR- 2023/08/19 CRDT- 2023/08/17 19:17 PHST- 2023/06/15 00:00 [received] PHST- 2023/08/11 00:00 [revised] PHST- 2023/08/14 00:00 [accepted] PHST- 2023/08/29 12:42 [medline] PHST- 2023/08/18 00:42 [pubmed] PHST- 2023/08/17 19:17 [entrez] PHST- 2023/08/19 00:00 [pmc-release] AID - S0168-1702(23)00162-4 [pii] AID - 199200 [pii] AID - 10.1016/j.virusres.2023.199200 [doi] PST - ppublish SO - Virus Res. 2023 Oct 2;335:199200. doi: 10.1016/j.virusres.2023.199200. Epub 2023 Aug 19.