Real-time imaging of the HIV-1 transcription cycle in single living cells
Résumé
The dynamic nature of cellular processes is emerging as an important modulator of physiological and pathological events. The key event in the life cycle of the human immunodeficiency virus type 1 (HIV-1) is transcription: it controls both viral gene expression and the latent phenotype. The basal transcription machinery and cellular and viral regulatory elements are dynamically recruited to the proviral DNA embedded into chromatin and to newly synthesized viral RNA. Their interactions determine fundamental steps, such as RNA polymerase recruitment, initiation, elongation, splicing, termination, and processing of pre-mRNA. The study of these events requires a novel armamentarium of techniques for live-cell imaging and fluorescence tagging of proteins and nucleic acids. The final outcome should not be only a descriptive view of the process but, most importantly, a quantitative analysis of the kinetics involved. Here, we provide an overview of the methodologies available for fluorescent labeling proteins and nucleic acids in live-cell imaging. We also describe the concept of fluorescent recovery after photobleaching (FRAP) and how it can be used to obtain information about HIV RNA transcription dynamics in living cells.