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Viruses 2014, 6(1), 354-370; doi:10.3390/v6010354

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

Department of Virology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
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Received: 14 December 2013 / Revised: 10 January 2014 / Accepted: 13 January 2014 / Published: 23 January 2014
(This article belongs to the Special Issue Recent CMV Research)
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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. View Full-Text
Keywords: human cytomegalovirus; bacterial artificial chromosomes; essential viral genes; transfection; adenovirus human cytomegalovirus; bacterial artificial chromosomes; essential viral genes; transfection; adenovirus
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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MDPI and ACS Style

Elbasani, E.; Gabaev, I.; Steinbrück, L.; Messerle, M.; Borst, E.M. Analysis of Essential Viral Gene Functions after Highly Efficient Adenofection of Cells with Cloned Human Cytomegalovirus Genomes. Viruses 2014, 6, 354-370.

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