Single-cell and real-time analysis of transcription rates from integrated HIV-1 provirus

Viral RNA biogenesis is a crucial step in the replication of retroviruses that require both the production of a genomic RNA as well as of translation templates. Cellular and viral factors concur in the biogenesis of RNA at the specific sub-nuclear chromatin site where the reaction takes place. The possibility of tracking viral RNA in living cells gives the unique possibility of measuring the kinetic parameters of RNA biogenesis as well as defining the dynamic recruitment of host and viral factors to the site of replication. In order to study the activation of HIV-1 gene expression from the integrated viral promoter we exploited a method that allows the visualization of newly transcribed RNA in living cells through the specific recognition of an RNA consensus sequence for the bacteriophage MS2 coat protein tagged with an autofluorescent protein. We observed that transcription of HIV-1 occurred in discrete foci within the nucleus of cells carrying several tandem arrays of the HIV-1 construct. These foci, representing newly transcribed RNA, co-localized with the viral Tat transactivator as well as members of the positive transcription elongation factor (P-TEFb) and RNA polymerase II (RNAPII). This experimental setting was used to measure the dynamic of HIV-1 RNA transcription in living cells. By fluorescence recovery after photobleaching (FRAP) we were able to demonstrate that following photobleaching the process reaches a steady state with a negligible immobile fraction allowing precise kinetic measurements of RNA polymerase elongation rates. We found that elongation proceeded at approximately 2 kb/min, and that 3'-end formation and release took another minute to complete. In addition we also analyzed the dynamic of RNAPII and the TAR:Tat:pTEFb complex at the site of HIV-1 transcription in living cells. Our data suggest that, while the residence time of RNAPII exceeds the time required for elongation through the viral template, the complex dissociates from the polymerase following transcription initiation, and may undergo subsequent cycles of association/dissociation. This approach was extended to the analysis of single integrated HIV- 1 transcription units by transduction of a HIV-based lentiviral vector into a human cell line and subsequent selection for Tat-induction from a latent state. Nascent RNAs from single integrated transcription units were detectable in living cells by MS2 RNA-tagging. At steady state a constant number of RNAs was measured at the transcription site corresponding to a minimal density of polymerases with negligible fluctuations over time both in space and intensity of the signal. Recovery of fluorescence after photobleaching of the transcription site was complete within seconds, much faster than what was observed previously. However, the necessity of taking into account also the diffusion of the tagged MS2 protein required the development of novel analytical tools. To this end we developed a model that describes each polymerase sliding along the DNA like the peak of a positive progressive traveling wave (TranWave) to predict the number of MS2 RNA repeats at the transcription site in function of time. The outcome of this approach and its following improvements are being discussed. This work provides for the first time a kinetic framework to analyze HIV-1 RNA biogenesis and RNA/protein dynamics in living cells.