The sequence complementarity between HIV-1 5 ' splice site SD4 and U1 snRNA determines the steady-state level of an unstable env pre-mRNA

HIV-1 env expression from certain subgenomic vectors requires the viral reg ulatory protein Rev, its target sequence RRE, and a 5 ' splice site upstrea m of the env open reading frame, To determine the role of this splice site in the 5 ' -splice-site-dependent Rev-mediated env gene expression, we have subjected the HIV-1 5 ' splice site, SD4, to a mutational analysis and hav e analyzed the effect of those mutations on env expression. The results dem onstrate that the overall strength of hydrogen bonding between the 5 ' spli ce site, SD4, and the free 5 ' end of the U1 snRNA correlates with env expr ession efficiency, as long as env expression is suboptimal, and that a cont inuous stretch of 14 hydrogen bonds can lead to full env expression, as a r esult of stabilizing the pre-mRNA, The U1 snRNA-mediated stabilization is i ndependent of functional splicing, as a mismatch in position +1 of the 5 ' splice site that led to loss of detectable amounts of spliced transcripts d id not preclude stabilization and expression of the unspliced env mRNA, pro vided that Rev enables its nuclear export. The nucleotides capable of parti cipating in U1 snRNA:pre-mRNA interaction include positions -3 to +8 of the 5 ' splice site and all 11 nt constituting the single-stranded 5 ' end of U1 snRNA, Moreover, env gene expression is significantly decreased upon the introduction of point mutations in several upstream GAR nucleotide motifs, which are mediating SF2/ASF responsiveness in an in vitro splicing assay. This suggests that the GAR sequences may play a role in stabilizing the pre -mRNA by sequestering U1 snRNP to SD4.