Figure 8

A single G run is sufficient to maintain the HIV-1 splicing pattern. (A) RT-PCR analysis of RNA isolated from HEK 293 T cells transiently transfected with pNL4-3 or its mutant derivates 48 h post transfection. The used primer pairs are illustrated in Additional file 1: Figure S1. Transcript isoforms are indicated on the right. Separate RT-PCRs were performed by using primer pairs amplifying HIV-1 exon 7 to compare total RNA amounts. PCR amplicons were separated on a non-denaturing polyacrylamide gel (10%) and stained with ethidium bromide. (B-C) Quantitative RT-PCR of total RNA obtained from panel (A). 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. Compare with Additional file 1: Figure S1 for specific primer binding sites. (D) Immunoblot analysis of the indicated proteins employing lysates from HEK 293 T cells (cellular) and their supernatants (sn) transiently transfected with the indicated proviral DNAs. Transfected cells were lysed in RIPA buffer and lysates were collected 48 h post transfection. Virions were pelleted by sucrose centrifugation.