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

Intragenic transcriptional cis-activation of the human immunodeficiency virus 1 does not result in allele-specific inhibition of the endogenous gene

<p>Abstract</p> <p>Background</p> <p>The human immunodeficiency virus type 1 (HIV-1) favors integration in active genes of host chromatin. It is believed that transcriptional interference of the viral promoter over the endogenous gene or vice versa might occur with implications in HIV-1 post-integrative transcriptional latency.</p> <p>Results</p> <p>In this work a cell line has been transduced with a HIV-based vector and selected for Tat-inducible expression. These cells were found to carry a single silent integration in sense orientation within the second intron of the <it>HMBOX1 </it>gene. The HIV-1 Tat transactivator induced the viral LTR and repressed <it>HMBOX1 </it>expression independently of vector integration. Instead, single-cell quantitative <it>in situ </it>hybridization revealed that allele-specific transcription of <it>HMBOX1 </it>carrying the integrated provirus was not affected by the transactivation of the viral LTR in <it>cis</it>.</p> <p>Conclusion</p> <p>A major observation of the work is that the HIV-1 genome has inserted in genes that are also repressed by Tat and this could be an advantage for the virus during transcriptional reactivation. In addition, it has also been observed that transcription of the provirus and of the endogenous gene in which it is integrated may coexist at the same time in the same genomic location.</p

A real-time view of the TAR:Tat:P-TEFb complex at HIV-1 transcription sites

HIV-1 transcription is tightly regulated: silent in long-term latency and highly active in acutely-infected cells. Transcription is activated by the viral protein Tat, which recruits the elongation factor P-TEFb by binding the TAR sequence present in nascent HIV-1 RNAs. In this study, we analyzed the dynamic of the TAR:Tat:P-TEFb complex in living cells, by performing FRAP experiments at HIV-1 transcription sites. Our results indicate that a large fraction of Tat present at these sites is recruited by Cyclin T1. We found that in the presence of Tat, Cdk9 remained bound to nascent HIV-1 RNAs for 71s. In contrast, when transcription was activated by PMA/ionomycin, in the absence of Tat, Cdk9 turned-over rapidly and resided on the HIV-1 promoter for only 11s. Thus, the mechanism of trans-activation determines the residency time of P-TEFb at the HIV-1 gene, possibly explaining why Tat is such a potent transcriptional activator. In addition, we observed that Tat occupied HIV-1 transcription sites for 55s, suggesting that the TAR:Tat:P-TEFb complex dissociates from the polymerase following transcription initiation, and undergoes subsequent cycles of association/dissociation

ZASP Interacts with the Mechanosensing Protein Ankrd2 and p53 in the Signalling Network of Striated Muscle

ZASP is a cytoskeletal PDZ-LIM protein predominantly expressed in striated muscle. It forms multiprotein complexes and plays a pivotal role in the structural integrity of sarcomeres. Mutations in the ZASP protein are associated with myofibrillar myopathy, left ventricular non-compaction and dilated cardiomyopathy. The ablation of its murine homologue Cypher results in neonatal lethality. ZASP has several alternatively spliced isoforms, in this paper we clarify the nomenclature of its human isoforms as well as their dynamics and expression pattern in striated muscle. Interaction is demonstrated between ZASP and two new binding partners both of which have roles in signalling, regulation of gene expression and muscle differentiation; the mechanosensing protein Ankrd2 and the tumour suppressor protein p53. These proteins and ZASP form a triple complex that appears to facilitate poly-SUMOylation of p53. We also show the importance of two of its functional domains, the ZM-motif and the PDZ domain. The PDZ domain can bind directly to both Ankrd2 and p53 indicating that there is no competition between it and p53 for the same binding site on Ankrd2. However there is competition for this binding site between p53 and a region of the ZASP protein lacking the PDZ domain, but containing the ZM-motif. ZASP is negative regulator of p53 in transactivation experiments with the p53-responsive promoters, MDM2 and BAX. Mutations in the ZASP ZM-motif induce modification in protein turnover. In fact, two mutants, A165V and A171T, were not able to bind Ankrd2 and bound only poorly to alpha-actinin2. This is important since the A165V mutation is responsible for zaspopathy, a well characterized autosomal dominant distal myopathy. Although the mechanism by which this mutant causes disease is still unknown, this is the first indication of how a ZASP disease associated mutant protein differs from that of the wild type ZASP protein

Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells

The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space

Endocytic reawakening of motility in jammed epithelia.

Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or rigidity transition. How cells control such phase transitions is, however, unknown. Here we show that RAB5A, a key endocytic protein, is sufficient to induce large-scale, coordinated motility over tens of cells, and ballistic motion in otherwise kinetically arrested monolayers. This is linked to increased traction forces and to the extension of cell protrusions, which align with local velocity. Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogates RAB5A-induced collective motility. A simple model based on mechanical junctional tension and an active cell reorientation mechanism for the velocity of self-propelled cells identifies regimes of monolayer dynamics that explain endocytic reawakening of locomotion in terms of a combination of large-scale directed migration and local unjamming. These changes in multicellular dynamics enable collectives to migrate under physical constraints and may be exploited by tumours for interstitial dissemination

Autophagy is defective in collagen VI muscular dystrophies and its reactivation rescues myofiber degeneration

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