Fig 4.
Coexpression of a modified U1 snRNP with full complementarity to 5′ss D3 induces HIV-1 exon 3 splicing and vpr mRNA expression. (A) Schematic drawing of a 5′-end-modified U1 snRNA (right) perfectly matching the 5′ss D3 sequence. Mutated nucleotides are indicated by gray capital letters. Additional base pairing interactions between 5′ss D3 and the optimized 5′ end of the U1 snRNA are indicated by vertical gray lines. (B) HEK 293T cells (2.5 × 105) were transiently cotransfected with 1 μg of both a proviral plasmid and a U1 snRNA expression plasmid. Total RNA was isolated and subjected to RT-PCR analyses. PCR products were resolved by PAGE and stained with ethidium bromide. RT-PCR samples are shown at the top. The main viral mRNA species are indicated on the right. Viral supernatants were collected as well and analyzed for viral p24gag concentrations by immunoblotting (bottom). E, extended exon; dm, double mutation; sn, supernatant.