Figure 1.

Virions produced in RHA–down-regulated cells have diminished HIV early reverse-transcription intermediates in MT4-infected cells. HEK293 cells were transfected with siRNA targeting DHX9 or NT for 8 h, followed by transfection of FLAG-RHA that is resistant to the siRNA. After overnight incubation, the cultures were transfected with HIV molecular clone pNL4–3. 24 h later, the cell lysates and cell-free medium were collected for immunoblotting or Gag ELISA. Equivalent Gag containing cell-free medium was used for infection of MT4 cells. A, representative immunoblot of cell lysates (20 μg) with antiserum to RHA, FLAG, Gag, or GAPDH. The antiserum detected the specific proteins on the immunoblots, as shown relative to the prestained protein standards. B, virion production from the HIV producer cells was measured by Gag ELISA on cell-free medium. The volume of medium corresponding to 200 ng of Gag was used for the MT4 infections. The data obtained from three independent experiments (blue, experiment 1; orange, experiment 2; gray, experiment 3) are summarized on the scatter plot showing standard deviation and significance, as determined by Student's t test (**, p ≤ 0.01). C, diagram to show the placement of primers (red thin lines) and PCR amplicons (thick red lines) on the minus strand DNA (dashed line) to measure strong stop DNA (early RT product) and the completed dsDNA intermediate (gray boxes and thick black lines) to measure the late RT product in MT4-infected cells. D, DNA was extracted from the infected MT4 cells and subjected to the qPCR using primer pairs in C. DNA copies were determined relative to standard curves. Statistically significant differences were measured by Student's t test: *** p ≤ 0.001; ** p ≤ 0.01. WB, Western blotting.