Figure 5
Effect of modulating NKA expression, [Ca2+]i, and PI3K-AKT on HIV-1 gene expression. Depletion of the NKA perturbs HIV-1 RNA processing (n ≥ 3, mean, s.e.m.). HeLa rtTA-HIV(Gag-GFP) cells were transduced with two shRNAs (E6/E10) to knockdown expression of the NKA α1 subunit and their effect on HIV-1 gene expression analyzed. After positive selection of cells with puromycin for 24–48 h, HIV-1 expression was Dox induced for 20 h, and cells harvested for analyses. Total mRNA (and protein) was isolated from cells, reverse transcribed, and analyzed by qRT-PCR and data normalized to β-actin (a,c). Results are shown relative to Stuffer (+). (a) Expression levels of NKA α1 mRNAs assayed by qRT-PCR. (b) HIV-1 Gag expression assayed by p24CA ELISA of cell lysates (40 μg). (c) Accumulation of HIV-1 US (black), SS (white), and MS (gray) mRNAs quantified by qRT-PCR. (d–g) CSs suppress HIV-1 gene expression independent of intracellular Ca2+ flux/signaling and PI3K-AKT activation. HeLa rtTA-HIV(Gag-GFP) cells were pre-treated with either a chelator of [Ca2+]i (5 µM BAPTA-AM, [Ca2+]ii), inhibitors of NCX Ca2+-entry (5 µM KB-R7943, NCXi) or PI3K (10 μM LY294002, PI3Ki), or untreated (DMSO, no pathway inhibitor) for ~2 h prior to treatment with ~IC80s of CSs or DMSO for 4 h, and Dox induced for ~20 h. The pathway used by a CS to inhibit HIV-1 expression was determined by monitoring for recovery of Gag-GFP expression by measuring GFP fluorescence in cell lysates (and initially by plate scans of live/fixed cells, Supplementary Fig. S7a and S8c). (d) Levels of [Ca2+]i measured by Fura Red AM™ (n ≥ 4, mean, s.e.m.). (e–f) Quantification of Gag-GFP expression by reducing SDS-PAGE in lysates (35 μg) of cells which were pre-treated with/without KB-R7943 (n ≥ 3–4 mean, s.e.m.). Graph (e) and representative gel (f) of these results. Tubulin immunoblots serve as internal loading control and for normalization of this data. (g) Graph quantitating Gag expression by p24CA ELISA in lysates of cells (35 μg) that were pre-treated with/without LY294002 (n ≥ 3, mean, s.e.m.). Statistical comparisons in (a–g) were performed as illustrated (black/gray dashed lines) or described in Methods.
