Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3' splice site and its cis-regulatory element: possible involvement in RNA splicing

The HIV-1 transcript is alternatively spliced to over 30 different mRNAs, W hether RNA secondary structure can influence HIV-1 RNA alternative splicing has not previously been examined. Here we have determined the secondary st ructure of the HIV-1/BRU RNA segment, containing the alternative A3, A4a, A 4b, A4c and A5 3' splice sites. Site A3, required for tat mRNA production, is contained in the terminal loop of a stem-loop structure (SLS2), which is highly conserved in HIV-1 and related SIVcpz strains. The exon splicing si lencer (ESS2) acting on site A3 is located in a long irregular stem-loop st ructure (SLSS), Two SLS3 domains were protected by nuclear components under splicing condition assays. One contains the A4c branch points and a putati ve SR protein binding site. The other one is adjacent to ESS2, Unexpectedly , only the 3' A residue of ESS2 was protected, The suboptimal A3 polypyrimi dine tract (PPT) is base paired. Using site-directed mutagenesis and transf ection of a mini-HIV-l cDNA into HeLa cells, we found that, in a wild-type PPT context, a mutation of the A3 downstream sequence that reinforced SLS2 stability decreased site A3 utilization. This was not the case with an opti mized PPT, Hence, sequence and secondary structure of the PPT may cooperate in limiting site A3 utilization.