FIG. 5.

Depletion of chromatin reassembly factors reactivates intron-integrated latent HIV. (A and B) shRNA-mediated depletion of Spt6 and Chd1 in clones J-Lat E27 and A2. Cells of the indicated latently infected clones were infected with control, Spt6, or Chd1 shRNA expression lentiviruses (pLKO.1-Puro), and 7 days after puromycin selection, HIV-GFP reactivation was measured by fluorescence-activated cell sorting and expressed as the percentage of cells that became GFP positive (%GFP). Depletion of these factors was tested by immunoblotting and RT-qPCR with specific antibodies (Spt6 [ab32820; Abcam], Chd1 [H00001105-A01; Abnova]) or primers, respectively, and is shown for clone J-Lat E27 as an example (A). (C) HIV activation by TSA or TNF-α is enhanced in Spt6-depleted J-Lat cells. J-Lat E27 and A2 cells infected with control or Spt6 shRNA expression vectors and puromycin selected for 6 days were treated or not with TSA (400 nM) or TNF-α (10 ng/ml) for 24 h. HIV-GFP expression was measured by fluorescence-activated cell sorting and was expressed as percentage of GFP-positive cells (%GFP) or mean fluorescence intensity (MFI). (D) Spt6 depletion in nonintronic J-Lat clones. Cells of J-Lat clones H2 and A1 and of the constitutively expressing clone J-Act C9 were infected with control or Spt6 shRNA expression lentiviruses and selected in puromycin as in panel A, and HIV-GFP expression was measured by fluorescence-activated cell sorting. A portion of the cells were treated with TNF-α (10 ng/ml) for 24 h prior to fluorescence-activated cell sorting analysis. HIV-GFP expression is expressed as percent GFP-positive cells and as MFI for clone J-Act C9, as percent GFP-positive cells is saturated. (E) Analysis of HIV transcription in J-Lat clones upon Spt6 depletion. Cells of J-Lat E27 and A2 clones depleted of Spt6 as in panel A were RNA extracted, and HIV expression was measured by RT-qPCR with different amplicons covering the HIV genome as in Fig. 3A.