Identification of NF90 as HIV-1TAR binding protein. a: Autoradiogram of a NorthWestern blot in which 25ug of HeLa cell nuclear protein from each purification step was probed with 7.5 × 10⁶ cpm of radiolabeled TAR RNA for 2 hours, washed, and exposed to autoradiographic film for 2 hours. b: Immobilon-P transferred proteins were probed with polyclonal anti-NF90 at 1 ug/mL.

NF90ctv impacts HIV-1 replication at the transcription level. Total cell RNA extracted from NF90ctv expressing cells (pOZNF90, lanes 4,5) or control cells transduced with empty vector (pOZ, lanes 2,3) infected with HIV-1pNL4-3 or pseudotyped VSVG-HIV-1 for single round infection (lanes 6,7), was fractionated on a 1% forlmadehyde-agarose gel and probed with [32p]-labeled HIV-1LTR probe. Shown in Figure 4A is a Northern blot of HIV-1 p NL4-3 and VSVG-HIV infected and non-infected cells (designated as plus (+) or minus (-) respectively. Lane 1 is positive control HIV RNA from ACH2 cells. Figure 4B shows the same gel probed with β-actin. Figure 4C illustrates the ethidium bromide-stained gel shown in Figure 4A and 4B.

Competition of Tat/TAR interaction by NF90ctv interferes with HIV-1 chromatin activation: Changes in Histone H3 K4 and H3K9 methylation were measured by ChIP assays in integrated HIV-1 chromatin in OM10.1 cells. Comparison of H3Kme3 in uninduced (lane, 5) and TNF-α induced cells (lane, 10) shows marks of transcriptional activation of HIV-1 chromatin. In cells expressing NF90ctv however, the H3K4me3 methylation, a mark of transcriptionally active gene is inhibited (lane 15). Lanes 1, 6, and 11 represent IP controls. The empty vector (pCI-neo) was used as transfection control for NF90ctv transfected OM10.1 cells (lanes 11–15). H3K9 methylations (lanes 2–4, 7–9 and 12–14) show a reduction in TNF-α induced cells (compare lanes 2–4 and 7–9). Note the lack of inhibition of H3K9 methylation (lanes 4, 9, and 14) in TNF-α induced cells in the presence of NF90ctv (lane 14), suggesting a long-term memory of transcriptional inhibition in NF90ctv expressing cells.

Competition Analysis of RNA Binding Specificity. A: The structure of the wild type TAR RNA and the TAR mutants used in this assay are illustrated. B: 500 ng of protein from the TAR Fraction containing NF90 was incubated with 0.2 pmole radiolabeled TAR RNA (lanes 2, 6, 10, 14, 18). Competition for radiolabeled TAR RNA binding was done with increasing amounts of unlabeled TAR RNA (lanes 3, 5), TM12 RNA (lanes 7, 9), TM18 RNA (lanes 11–13), TM27 RNA (lanes 15–17), or TM12+TM27 RNAs (lanes 19–21). Samples were run on a 10%PAGE.

NF90 inhibits Tat- trans-activation in concentration-dependent manner. The effect of NF90ctv on basal and Tat-mediated transactivation of HIV-1 LTR was assessed in both HeLa and Jurkat cells using CaCl₂/Hepes transfection as follow. Duplicate 6 well- plates of HeLa cells received 2.5 ug of pHIV-β gal per 5 × 10⁶ cells and increasing amounts (0.0 ug, 2.0 ug or 10.0 ug) of pCMV-NF90ctv. One set of plate (Transactivation received constant amounts (5.0 ug) of pSV₂-Tat72. In each assay, 0.2 μg of pCMVCAT was cotransfected to monitor transfection efficiency. Following the transfection, cell extracts were prepared and standardized for total protein; colorimetric β-galactosidase and CAT assays were performed as before [37).

Competition of TAR/TAR complex formation by NF90c protein. One microgram of purified biotin-labeled TAR RNA was mixed with one microgram of purified Tat protein for 10 minutes on ice. Next, 100 μl of 30% strepavidin sepharose beads in binding buffer (50 mM Tris-HCl, pH7.8; 5 mM DTT, 100 μg of BSA, 60 mM KCl and 5 mM MgCl₂) were added to the reaction for a final volume of 200 μl. The TAR/Tat complex was incubated with beads for an additional 1 hr on ice. Next, purified NF90c at various concentrations (0.1, 1, and 5 μg) were added to the mixture. All samples were further incubated on ice for additional one hour. Finally, samples were centrifuged at 4°c for 5 minutes, and washed (3×) with TNE₃₀₀ + 0.1% NP-40 (50 mM Tris-HCl, pH7.8, 300 mM NaCl, 1 mM ETDA, plus 0.1% NP-40). A final wash was with TNE₅₀ + 0.1% NP-40 was performed. Bound complexes were separated on a 4–20% SDS/PAGE and western blotted with anti-Tat mAb or anti-NF90c antibodies. Same Blot was cut in half for either Tat or NF90c western blot. Lane 1: ¹⁴C protein molecular weight marker, Lane 2 is with no Tat, Lane 3 with Tat, Lane 4 and 5 are with addition of five microgram of either wild type TAR or a mutant TAR RNA (TM26) as specific and non-specific competitors, respectively. Lanes 6–8 represent addition of purified NF90ctv protein in presence of constant amount of Tat.