Figure 5
Quantification of HIV-1 RNA content in the virion. (A) Efficiency of HIV-1 genomic RNA packaging. RNA was isolated from equivalent amounts of p24 from NL4-3, NLΔSL1, NLΔSL1-913 and NLΔSL1-1907, reverse-transcribed and measured by qPCR with a primer/probe set specific to the HIV-1 unspliced genomic RNA. The amount of NL4-3 genomic RNA was set at 100%. Copy numbers ranged from 2.0 × 106 to 2.9 × 106 in four independent experiments. *, indicates p < 10-4 and significant deviation from the wild-type copy number as determined by Student's t test. (B) Efficiency of spliced HIV-1 RNA packaging. cDNA was subjected to qPCR targeting the env mRNA or rev mRNA sequence as described above. The amount of NL4-3 spliced mRNA was set at 100%. Copy numbers of env mRNA ranged from 24,042 to 28,865, and rev mRNA from 8,387 to 14,335 in four independent experiments. *, indicates significant deviation from the wild-type copy number as determined by Student's t test; p < 10-4, except for NLΔSL1-913, p < 10-3. (C) Relative amounts of HIV-1 genomic RNA and mRNA in the virion. The copy numbers of HIV-1 genomic RNA and env and rev mRNA were used to calculate the relative amount of mRNA in the virion [(mRNA copy/genomic RNA copy) × 100] and normalized to genomic RNA level. (D) Determination of viral RNA expression in producer cells. Total RNA was isolated from 293T cells transfected with the corresponding vectors and reverse transcribed. The cDNA was quantified by qPCR with primer/probe sets specific for the HIV-1 genomic RNA, env mRNA and rev mRNA sequences. The copy number in each sample was adjusted for input by the level of PBGD mRNA and for transfection efficiency by GFP expression from a co-transfected reporter construct. The amount of NL4-3 RNA was set at 100%.