Recombinant Human Cytomegalovirus Expressing an Analog-Sensitive Kinase pUL97 as Novel Tool for Functional Analyses

Krämer N, Schütz M, Mato UG, Herhaus L, Marschall M, Zimmermann C (2022)

Publication Type: Journal article

Publication year: 2022

Journal

Viruses MDPI

Book Volume: 14

Article Number: 2285

Journal Issue: 10

DOI: 10.3390/v14102285

Abstract

The human cytomegalovirus (HCMV) is a member of the beta-herpesvirus family and inflicts life-long latent infections in its hosts. HCMV has been shown to manipulate and dysregulate many cellular processes. One major interactor with the cellular host is the viral kinase pUL97. The UL97 gene is essential for viral replication, and kinase-deficient mutants of pUL97 display a severe replication defect. Recently, another group established an analog-sensitive version of the pUL97 protein. This mutant kinase can be treated with a non-hydrolysable ATP analog, thereby inhibiting its kinase function. This process is reversible by removing the ATP analog by media change. We introduced this mutant version of the pUL97 protein into the laboratory strain Ad169 of HCMV, BADwt, creating a BAD-UL97-as1 viral mutant. This mutant virus replicated normally in infected cells in the absence of the ATP analog and maintained its ability to phosphorylate its cellular substrates. However, when treated with the ATP analog, BAD-UL97-as1 displayed a defect in the production of intra- and extracellular viral DNA and in the production of viral progeny. Furthermore, in the presence of 3MB-PP1, a well-established substrate of pUL97 was no longer hyperphosphorylated. This effect was detectable as early as 4 h post treatment, which allows for studies on pUL97 without the complication of low viral titers. Nevertheless, we observed off-target effects of 3MB-PP1 on several cellular processes, which should be considered with this approach.

Authors with CRIS profile

Martin Schütz Institute of Clinical and Molecular Virology Manfred Marschall Lehrstuhl für Klinische und Molekulare Virologie

Additional Organisation(s)

Interdisziplinäres Zentrum für Klinische Forschung (IZKF)

Related research project(s)

None - None A088: Regulatory interaction btw. cyclins and cytomegalovirus kinase pUL97: focus on pUL97 drug resistance mutations Feb. 1, 2020 - July 31, 2023

Involved external institutions

Goethe-Universität Frankfurt am Main DE Germany (DE) Universitätsmedizin der Johannes Gutenberg-Universität Mainz DE Germany (DE)

How to cite

APA:

Krämer, N., Schütz, M., Mato, U.G., Herhaus, L., Marschall, M., & Zimmermann, C. (2022). Recombinant Human Cytomegalovirus Expressing an Analog-Sensitive Kinase pUL97 as Novel Tool for Functional Analyses. Viruses, 14(10). https://doi.org/10.3390/v14102285

MLA:

Krämer, Nadine, et al. "Recombinant Human Cytomegalovirus Expressing an Analog-Sensitive Kinase pUL97 as Novel Tool for Functional Analyses." Viruses 14.10 (2022).

BibTeX: Download