FIG. 3.

Quantification of transcripts initiated at HIV or host gene promoters. (A) Relative quantification of transcripts generated at the HIV integration site in clone J-Lat E27. J-Lat E27 cells were untreated or treated with TNF-α (10 ng/ml) for 24 h, RNA was extracted, and gene expression was measured by reverse transcription followed by real-time PCR of cDNA (RT-qPCR). Amplicons corresponding to 5′ LTR (R-gag, primers 8 and 9 [Fig. 2A]), Tat, GFP, and 3′ LTR (U3, primers 17 and 18) were used to measure HIV expression. Additionally, amplicons to measure UBXD8 expression at exon 5 (primers 1 and 2) or expression through the boundaries of intron 8 and 5′ LTR (primers 5 and 6) or 3′ LTR and intron 8 (primers 8 and 10) were used. GAPDH expression was measured for normalization. In order to compare different amplicons, qPCR was performed in parallel from genomic DNA (gDNA). Data are expressed as relative units (RU) of (cDNA amplification/GAPDH)/gDNA amplification. Values represent the mean and range of a representative experiment performed in duplicate. (B) HIV transcripts are generated mainly from the viral promoter. J-Lat E27 cells were untreated or treated with PMA (10 nM) for 16 h, RNA was extracted, and transcription through the 5′ LTR was measured by RT-qPCR with the oligonucleotides indicated. GAPDH expression and qPCR from gDNA were used for normalization as in panel A. Data are expressed as in panel A. (C) Downstream transcripts are mainly originated from 3′ LTR. PMA treatment of J-Lat E27 cells was performed as in panel B, and transcription through the 3′ LTR was measured with the qPCR primers indicated. (D) Expression of downstream gene exons is induced upon HIV activation. J-Lat E27 and A2 cells were untreated or treated with TNF-α (10 ng/ml) and HMBA (10 mM) for 24 h, RNA was extracted, and transcription through UBXD8 exon 9 was measured by RT-qPCR. GAPDH expression was measured for normalization. Data are expressed as RU of UBXD8 exon 9/GAPDH expression. In panels B to D, values represent the mean and SD of representative experiments performed in triplicate.