Fig. 6.

Increased HIV-p24 production from vDNA clones with wild-type 5′-MSD motif. a The GC-to-GT reversion at the 5′-MSD motif of residual vDNA clones improved viral p24 production. Equivalent amounts of vDNAs were transfected into TZM-bl cells, and 48h later, HIV-p24 levels in culture media were quantified by ELISA. C1P, a standard residual HIV-1 clone, was used as a positive control. mt-MSD and wt-MSD represent RV clones with mutated 5′-MSD and their wild-type counterparts, respectively. Error bars represent s.d., n = 4. The statistical significance of the data was calculated by using unpaired t-test; asterisk (**) and (***) indicate p < 0.002 and p < 0.0001, respectively. b Reduced HIV-p24 production from the standard HIV-JRCSF clone with 5′-MSD motif mutation. GT-to-GC mutation was incorporated at the 5′-MSD motif of HIV-JRCSF clone by site-directed mutagenesis. Equal amounts of the wild-type (JRCSF) and the mutant (JRCSF-MSD) vDNA clones were transfected into TZM-bl cells in triplicate, and 48h later, the levels of p24 production in culture media were measured by ELISA. Error bars represent s.d. The statistical significance of the data was calculated using unpaired t-test. Asterisks (***) denote p < 0.0005. c Replication defect of JRCSF-MSD mutant in stimulated CD4+ T-cells. HIV-JRCSF and JRCSF-MSD viruses were freshly prepared by transfecting their DNA clones into TZM-bl cells, followed by harvesting culture media after 48h. About 1x10⁶ of freshly stimulated CD4+ T-cells from a normal donor were infected overnight in triplicate in 24-well plate with the equivalent amounts of these viruses (1200 pg of p24 each). Next day, infected cells were washed three times and cultured in media containing 100 u/ml of IL-2. Culture supernatants were harvested on days indicated and tested for the levels of HIV-p24 by ELISA. Error bars represent s.d. d Low-levels of spliced vRNA produced by 5′-MSD mutated vDNAs. Total RNA isolated from TZM-bl cells transfected equivalently with various vDNA clones were used as template for RT-PCR amplification of HIV transcripts. RV-1 and RV-2a represent residual HIV clones with mutated and wild-type MSD motifs, respectively. Panels are labeled to the left with the corresponding amplified products. Lane M denotes GeneRuler plus DNA ladder