Analysis of Essential Viral Gene Functions After Highly Efficient Adenofection of Cells with Cloned Human Cytomegalovirus Genomes

Overview

Journal Viruses

Publisher MDPI

Specialty Microbiology

Date 2014 Jan 24

PMID 24452007

Citations 10

Authors

Endrit Elbasani

Ildar Gabaev

Lars Steinbruck

Martin Messerle

Eva Maria Borst

Affiliations

Soon will be listed here.

Abstract

Human cytomegalovirus (HCMV) has a large 240 kb genome that may encode more than 700 gene products with many of them remaining uncharacterized. Mutagenesis of bacterial artificial chromosome (BAC)-cloned CMV genomes has greatly facilitated the analysis of viral gene functions. However, the roles of essential proteins often remain particularly elusive because their investigation requires the cumbersome establishment of suitable complementation systems. Here, we show that HCMV genomes can be introduced into cells with unprecedented efficiency by applying a transfection protocol based on replication-defective, inactivated adenovirus particles (adenofection). Upon adenofection of several permissive cell types with HCMV genomes carrying mutations in essential genes, transfection rates of up to 60% were observed and viral proteins of all kinetic classes were found expressed. This enabled further analyses of the transfected cells by standard biochemical techniques. Remarkably, HCMV genomes lacking elements essential for viral DNA replication, such as the lytic origin of replication, still expressed several late proteins. In conclusion, adenofection allows the study of essential HCMV genes directly in BAC-transfected cells without the need for sophisticated complementation strategies.

Citing Articles

Establishment of a Luciferase-Based Reporter System to Study Aspects of Human Cytomegalovirus Infection, Replication Characteristics, and Antiviral Drug Efficacy.

Tillmanns J, Kicuntod J, Ehring A, Elbasani E, Borst E, Obergfall D Pathogens. 2024; 13(8).

PMID: 39204245 PMC: 11356942. DOI: 10.3390/pathogens13080645.

A virally encoded high-resolution screen of cytomegalovirus dependencies.

Finkel Y, Nachshon A, Aharon E, Arazi T, Simonovsky E, Dobesova M Nature. 2024; 630(8017):712-719.

PMID: 38839957 DOI: 10.1038/s41586-024-07503-z.

Molecular characterization of human cytomegalovirus infection with single-cell transcriptomics.

Schwartz M, Shnayder M, Nachshon A, Arazi T, Kitsberg Y, Levi Samia R Nat Microbiol. 2023; 8(3):455-468.

PMID: 36732471 DOI: 10.1038/s41564-023-01325-x.

A Unique Role of the Human Cytomegalovirus Small Capsid Protein in Capsid Assembly.

Borst E, Harmening S, Sanders S, Caragliano E, Wagner K, Lenac Rovis T mBio. 2022; 13(5):e0100722.

PMID: 36066102 PMC: 9600257. DOI: 10.1128/mbio.01007-22.

A Prominent Role of the Human Cytomegalovirus UL8 Glycoprotein in Restraining Proinflammatory Cytokine Production by Myeloid Cells at Late Times during Infection.

Perez-Carmona N, Martinez-Vicente P, Farre D, Gabaev I, Messerle M, Engel P J Virol. 2018; 92(9).

PMID: 29467314 PMC: 5899185. DOI: 10.1128/JVI.02229-17.

References

Nitzsche A, Steinhausser C, Mucke K, Paulus C, Nevels M . Histone H3 lysine 4 methylation marks postreplicative human cytomegalovirus chromatin. J Virol. 2012; 86(18):9817-27. PMC: 3446588. DOI: 10.1128/JVI.00581-12. View

Borst E, Benkartek C, Messerle M . Use of bacterial artificial chromosomes in generating targeted mutations in human and mouse cytomegaloviruses. Curr Protoc Immunol. 2008; Chapter 10:10.32.1-10.32.30. DOI: 10.1002/0471142735.im1032s77. View

Pari G, Kacica M, Anders D . Open reading frames UL44, IRS1/TRS1, and UL36-38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis. J Virol. 1993; 67(5):2575-82. PMC: 237578. DOI: 10.1128/JVI.67.5.2575-2582.1993. View

Murrell I, Tomasec P, Wilkie G, Dargan D, Davison A, Stanton R . Impact of sequence variation in the UL128 locus on production of human cytomegalovirus in fibroblast and epithelial cells. J Virol. 2013; 87(19):10489-500. PMC: 3807394. DOI: 10.1128/JVI.01546-13. View

Borst E, Wagner K, Binz A, Sodeik B, Messerle M . The essential human cytomegalovirus gene UL52 is required for cleavage-packaging of the viral genome. J Virol. 2007; 82(5):2065-78. PMC: 2258901. DOI: 10.1128/JVI.01967-07. View

Borst E, Messerle M . Analysis of human cytomegalovirus oriLyt sequence requirements in the context of the viral genome. J Virol. 2005; 79(6):3615-26. PMC: 1075693. DOI: 10.1128/JVI.79.6.3615-3626.2005. View

Borst E, Standker L, Wagner K, Schulz T, Forssmann W, Messerle M . A peptide inhibitor of cytomegalovirus infection from human hemofiltrate. Antimicrob Agents Chemother. 2013; 57(10):4751-60. PMC: 3811406. DOI: 10.1128/AAC.00854-13. View

Stern-Ginossar N, Weisburd B, Michalski A, Le V, Hein M, Huang S . Decoding human cytomegalovirus. Science. 2012; 338(6110):1088-93. PMC: 3817102. DOI: 10.1126/science.1227919. View

Cotten M, Baker A, Saltik M, Wagner E, Buschle M . Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary human cells in the presence of adenovirus. Gene Ther. 1994; 1(4):239-46. View

10.

Nevels M, Brune W, Shenk T . SUMOylation of the human cytomegalovirus 72-kilodalton IE1 protein facilitates expression of the 86-kilodalton IE2 protein and promotes viral replication. J Virol. 2004; 78(14):7803-12. PMC: 434104. DOI: 10.1128/JVI.78.14.7803-7812.2004. View

11.

Silva L, Loregian A, Pari G, Strang B, Coen D . The carboxy-terminal segment of the human cytomegalovirus DNA polymerase accessory subunit UL44 is crucial for viral replication. J Virol. 2010; 84(21):11563-8. PMC: 2953201. DOI: 10.1128/JVI.01033-10. View

12.

Nigro G, Adler S . Cytomegalovirus infections during pregnancy. Curr Opin Obstet Gynecol. 2010; 23(2):123-8. DOI: 10.1097/GCO.0b013e328342f1f6. View

13.

Bridge E, Ketner G . Redundant control of adenovirus late gene expression by early region 4. J Virol. 1989; 63(2):631-8. PMC: 247733. DOI: 10.1128/JVI.63.2.631-638.1989. View

14.

Dunn W, Chou C, Li H, Hai R, Patterson D, Stolc V . Functional profiling of a human cytomegalovirus genome. Proc Natl Acad Sci U S A. 2003; 100(24):14223-8. PMC: 283573. DOI: 10.1073/pnas.2334032100. View

15.

Tischer B, Smith G, Osterrieder N . En passant mutagenesis: a two step markerless red recombination system. Methods Mol Biol. 2010; 634:421-30. DOI: 10.1007/978-1-60761-652-8_30. View

16.

Baker A, Cotten M . Delivery of bacterial artificial chromosomes into mammalian cells with psoralen-inactivated adenovirus carrier. Nucleic Acids Res. 1997; 25(10):1950-6. PMC: 146693. DOI: 10.1093/nar/25.10.1950. View

17.

Cotten M, Saltik M . Intracellular delivery of lipopolysaccharide during DNA transfection activates a lipid A-dependent cell death response that can be prevented by polymyxin B. Hum Gene Ther. 1997; 8(5):555-61. DOI: 10.1089/hum.1997.8.5-555. View

18.

Yu D, Silva M, Shenk T . Functional map of human cytomegalovirus AD169 defined by global mutational analysis. Proc Natl Acad Sci U S A. 2003; 100(21):12396-401. PMC: 218769. DOI: 10.1073/pnas.1635160100. View

19.

Borst E, Messerle M . Development of a cytomegalovirus vector for somatic gene therapy. Bone Marrow Transplant. 2000; 25 Suppl 2:S80-2. DOI: 10.1038/sj.bmt.1702361. View

20.

Mohr H, Mohr C, Schneider M, Scrivano L, Adler B, Kraner-Schreiber S . Cytomegalovirus replicon-based regulation of gene expression in vitro and in vivo. PLoS Pathog. 2012; 8(6):e1002728. PMC: 3369935. DOI: 10.1371/journal.ppat.1002728. View