Figure 5

DBR1 siRNA suppresses HIV-1 reverse transcription after minus-strand strong-stop DNA formation. (A) Location of primers. The oligonucleotide primers M667/AA55 specific for the R/U5 region of the LTR were used to detect early reverse transcripts (strong-stop DNA) (B) and env primers Env1/Env2 to were used detect intermediate reverse transcripts (C). LTR/gag primers M667/M661 were chosen to detect complete first-strand viral cDNA (D). In B, C and D, GHOST-R5X4 cells were transfected with DBR1 siRNA pHyper-D123 (black bars, siRNA), control vector pHyper (white bars, siRNA) or CDM9-DBR1 (black bars, cDNA), control vector CDM9 (white bars, cDNA) or co-transfected with CDM9-DBR1 and DBR1 siRNA pHyper-D123 (black bars, siRNA + cDNA), control vectors CDM9 and pHyper (white bars, siRNA + cDNA) for forty-eight hours, followed by infection with Dnase-treated HIV-1 (JR-CSF). Twenty-four hours later, the cells were lysed and DNA was isolated for real time quantitative PCR to evaluate the synthesis of HIV-1 cDNA. Copies of HIV-1 DNA were normalized against copies of β-globin. Error bars indicate standard deviations. Asterisks denote p < 0.05 by Student's t-test.