Fig. 1: Single cell characterization of HIV-1 gene expression, with and without Tat.

A Schematic of HIV-1 transcriptional regulation. Left: in the absence of Tat, pTEFb is not recruited and polymerases binds NELF and DSIF and pause near the promoter. Right: in the presence of Tat, pTEFb, composed of Cyclin T1 and Cdk9 associated with the super-elongation complex, is recruited to the nascent TAR RNA. Cdk9 phosphorylates NELF, DSIF, and RNA polymerase II, thereby triggering pausing exit and processive elongation. B Schematic of the HIV-1 reporter construct. SD1: major HIV-1 splice site donor; SA7: last HIV-1 splice site acceptor; ψ: packaging signal; RRE: Rev-responsive element; LTR: long terminal repeat. C–E. Expression of the 128xMS2 HIV-1 tagged reporter in cells expressing high levels of Tat. C- microscopy images of High Tat HeLa cells where the unspliced HIV-1 pre-mRNA is detected by smFISH with probes against the 128xMS2 tag. Cells bear a single copy of the reporter gene integrated with the Flp-in system. The bright spots in the nuclei correspond to nascent RNA at their transcription sites, while the dimmer spots correspond to single pre-mRNA molecules. Scale bar: 10 μm. This experiment has been done three times with similar results. D distribution of the number of released HIV-1 pre-mRNAs per cell, in High Tat cells. Experimental RNA distributions are from smFISH data. X-axis: number of HIV-1 pre-mRNA molecules per cell; y-axis: number of cells; inset: mean number of HIV-1 pre-mRNAs per cell. E distribution of the number of nascent HIV-1 pre-mRNAs per transcription site, in High Tat cells. Experimental RNA distribution is from smFISH data. X-axis: number of nascent HIV-1 pre-mRNA molecules per transcription site; y-axis: number of transcription sites; inset: mean number of nascent HIV-1 pre-mRNAs per cell. D, E source data are provided as a Source Data file. F–H Expression of the 128xMS2 HIV-1 tagged reporter in cells expressing low levels of Tat. Legend as in (C–E), except that experiments are from Low Tat cells. This experiment has been done three times with similar results. G, H source data are provided as a Source Data file. I–K Expression of the 128xMS2 HIV-1 tagged reporter in cells not expressing Tat. Legend as in (C–E), except that experiments are from No Tat cells. Image contrast adjustment is identical for panels C, F, and I. This experiment has been done four times with similar results. J, K Source data are provided as a Source Data file.