A quantitative nuclear egress assay to investigate the nucleocytoplasmic capsid release of human cytomegalovirus.

Autor: Häge S; Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-Nürnberg (FAU), Schlossgarten 4, Erlangen, Germany. Electronic address: sigrun.haege@fau.de., Horsch D; Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-Nürnberg (FAU), Schlossgarten 4, Erlangen, Germany. Electronic address: deborahhorsch@web.de., Stilp AC; Institute for Virology, Ulm University Medical Center, Albert-Einstein-Allee 11, Ulm, Germany. Electronic address: anne-charlotte.stilp@uniklinik-ulm.de., Kicuntod J; Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-Nürnberg (FAU), Schlossgarten 4, Erlangen, Germany. Electronic address: jintawee.kicuntod@extern.uk-erlangen.de., Müller R; Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-Nürnberg (FAU), Schlossgarten 4, Erlangen, Germany. Electronic address: mueller.regina@uk-erlangen.de., Hamilton ST; Serology and Virology Division, NSW Health Pathology Microbiology, Prince of Wales Hospital, Schools of Women's and Children's Health, Medicine and Biotechnology and Biomolecular Sciences, University of New South Wales, High Street, Sydney, Australia. Electronic address: stuart.hamilton@health.nsw.gov.au., Egilmezer E; Serology and Virology Division, NSW Health Pathology Microbiology, Prince of Wales Hospital, Schools of Women's and Children's Health, Medicine and Biotechnology and Biomolecular Sciences, University of New South Wales, High Street, Sydney, Australia. Electronic address: ece.egilmezer1@health.nsw.gov.au., Rawlinson WD; Serology and Virology Division, NSW Health Pathology Microbiology, Prince of Wales Hospital, Schools of Women's and Children's Health, Medicine and Biotechnology and Biomolecular Sciences, University of New South Wales, High Street, Sydney, Australia. Electronic address: w.rawlinson@unsw.edu.au., Stamminger T; Institute for Virology, Ulm University Medical Center, Albert-Einstein-Allee 11, Ulm, Germany. Electronic address: thomas.stamminger@uniklinik-ulm.de., Sonntag E; Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-Nürnberg (FAU), Schlossgarten 4, Erlangen, Germany. Electronic address: ericsonntag@web.de., Marschall M; Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-Nürnberg (FAU), Schlossgarten 4, Erlangen, Germany. Electronic address: manfred.marschall@fau.de.
Jazyk: angličtina
Zdroj: Journal of virological methods [J Virol Methods] 2020 Sep; Vol. 283, pp. 113909. Date of Electronic Publication: 2020 Jun 13.
DOI: 10.1016/j.jviromet.2020.113909
Abstrakt: Nuclear egress is a rate-limiting step of herpesviral replication, restricting the nucleocytoplasmic transport of viral capsids. The process is regulated by two viral nuclear egress proteins (core NEC pUL50-pUL53), which recruit additional cellular and viral proteins. The multicomponent NEC mediates disassembly of the nuclear lamina barrier and the docking of nuclear capsids. The quantitation of nuclear egress has been accomplished by electron microscopic analysis, but is generally hampered by the low number of detectable cytoplasmic capsids. A newly established method for the quantitation of viral nuclear egress improves the characterization of viral mutants, host cell permissiveness and antiviral drug efficacy. In this study, various strains of human cytomegalovirus (HCMV) were used to measure the replication efficiencies in primary human fibroblasts, applying methods of cell fractionation, DNase digestion, sucrose cushions and quantitative PCR. Several stages of optimization led to a reliable quantitative assay that allowed the characterization of viral nuclear egress efficacy. Using this assay, recovery of the nuclear egress of a NEC-defective HCMV mutant was quantitatively assessed by applying an inducible NEC-expressing fibroblast culture for trans-complementation. This novel assay system can be further used to accurately quantitate and characterize the functionality of nuclear egress of HCMV or other herpesviruses.
Competing Interests: Declaration of Competing Interest The authors declare no competing interests.
(Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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