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.
Highlights
Human cytomegalovirus (HCMV) is still a serious threat for immunocompromised individuals such as AIDS patients and transplant recipients [1,2]
Since primary human foreskin fibroblasts (HFF) originate from various sources and may vary in their properties, we instead used commercially available primary fetal lung fibroblasts (MRC-5), which in addition are easier to transfect than HFF [29]
We present a novel approach to analyze essential HCMV gene functions by transfecting cells with bacterial artificial chromosomes (BAC) that are deleted for the gene of interest
Summary
Human cytomegalovirus (HCMV) is still a serious threat for immunocompromised individuals such as AIDS patients and transplant recipients [1,2]. Successful examples include a few cell lines expressing the essential protein of interest [12,13,14,15,16], as well as inducible systems based on tetracycline-regulated transcription [17,18], or the fusion of essential HCMV proteins to a destabilizing domain [19,20,21,22,23] Each of these procedures has limitations, such as low virus productivity on the complementing cells, occurrence of escape mutants (rescuants), insufficient tightness of conditional gene regulation, or impairment of viral protein function upon fusion to regulatory domains. By using the adenofection technique, essential viral gene functions can be assessed immediately after transfection of the HCMV BAC mutants, which obviates the requirement to develop individual complementation strategies
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