Figure 1

A system for identifying human RNAs bound by HIV Tat. A. HA or FLAG-tagged Tat was stably expressed from the HIV LTR as part of a bicistronic transcript with a G418 resistance gene in CEM T cells. Tat protein bound to the TAR stem-loop present at the 5’ end of the transcript. B. Western blotting for HA-Tat in CEM HA-Tat cells (1/1 and 1/5 dilutions) and in an equal number of C1866 cells 0, 5 and 7 days after infection with the SF2 strain of HIV engineered to encode HA-tagged Tat. C. Activity of untagged and HA-tagged Tat measured by GFP expression from the HIV LTR in the GHOST reporter line using flow cytometry. D. Enrichment (mean and SD) of TAR-Tat construct, 7SK and GAPDH RNA in HA-Tat and FLAG-Tat IPs compared to input RNA measured by Q-PCR. 7SK is enriched by 9-fold (SD 3.8) by IP for HA-tagged Tat and 3.6-fold (SD 0.73) by FLAG-tagged Tat. In comparison, GAPDH RNA is enriched by 0.2-fold (SD 0.007) and 1.14-fold (SD 0.25), respectively. Enrichment of 7SK compared to GAPDH is significant for both HA and FLAG IPs (p = 0.017 and 0.0051, respectively, t-test, n = 3). The enrichment in control IPs where the antibodies were switched are shown for comparison (white bars). E. Enrichment (mean and SD) of poly-adenylated TAR-Tat construct RNA in HA-Tat and FLAG-Tat IPs relative to input RNA measured using microarrays.