Figure 4

HIV-1 infected T-cell lines produce a miRNA derived from the TAR element. A) Graphical representation of what region of TAR is detected by the probe the probe used for RPA. TAR 5' probe detects sequence from the 5' area of the stem (~30 bases), while TAR 3' detects the 3' side of the stem. B) Thirty micrograms of total RNA from ACH2 was hybridized to a radiolabeled RNA probe specific for 5' (lanes 3 and 4) region of the TAR stem loop and then treated with RNase A and T1. As a control the probes were incubated alone with RNase A and T1 (lanes 3). Molecular weight marker corresponding to 20 nucleotides is shown (lane 1) as well as a 22 nucleotide standard (lane 2). C) Thirty micrograms of total RNA from THP-1, CEM, OM10.1, (lanes 3 and 4) ACH2, OM10.1 and HLM-1 (lanes 5–7) and from OM10.1, ACH2 and HLM-1 stimulated with TSA for 24 hours (lanes 8–10) were hybridized to a radiolabeled TAR 5' probe and then treated with RNase A and T1. As a control probe was incubated alone with RNase A and T1 (lane 2). Molecular weight markers corresponding to 20 and 30 nucleotides are shown (lane 1). Arrows indicate the probe protected by TAR at 27 nucleotides and probe protected by a TAR miRNA at approximately 22. D) T-cell blasts were cultured from PBMCs using IL-2 and PHA for 48 hours. T-cells were infected with HIV-1 (LAV) and cultured in RPMI with IL-2 for 6, 12 and 18 days. RNA from uninfected cells at day 6, 12 and 18 (8.3, 3.7 and 16.8 micrograms respectively – lanes 4–6) and infected cells at 6,12 and 18 (9.2, 16.5 and 22.9 micrograms – lanes 7–9) was probed for presence of TAR derived miRNA using the 5'TAR probe. A sample containing no RNA was included as an additional negative control as were 15 and 30 micrograms of ACH2 RNA (lanes 1–3). E) HLM-1 cells were transfected with siLuc or siDicer for 72 hours. RNA extract was used to perform RPA for HIV-1 miRNA. F) Expression of HIV-1 TAR miRNA was determined by densitometry and normalized to 5S ribosomal RNA.