Figure 5.

Mutation G _I3 -2 increases vpr , but decreases vif mRNA and Vif protein levels. (A) RT-PCR analysis of RNA from HEK 293 T cells transiently transfected with pNL4-3 or its G_I3-2 mutant derivate. Compare with Additional file 1: Figure S1 for specific primer binding sites. RNA was isolated 48 h post transfection. Primer pairs are indicated at the bottom of each panel, transcript isoforms on the right. To compare total RNA amounts, separate RT-PCRs were performed by using primer pairs amplifying HIV-1 exon 7 and cellular GAPDH sequence. PCR amplicons were separated on a non-denaturing polyacrylamide gel (10%) and stained with ethidium bromide. (B-D) Quantitative RT-PCR of total RNA from (A) using primers indicated in Additional file 1: Figure S1. The NL4-3 splicing pattern (wt) was set to 100% and the relative splice site usage was normalized to exon 7 containing HIV-1 transcripts. (E) Immunoblot analysis of the indicated proteins employing lysates or pelleted virions from supernatant (sn) obtained from HEK 293 T cells that were transiently transfected with wild type or G_I3-2 mutant proviral DNA. Transfected cells were lysed in RIPA buffer and the lysates were collected 48 h post transfection. Cell-free supernatant was concentrated by sucrose centrifugation. (F) Quantification of Vif protein amounts from (E).