RESUMO
The negative-sense asp open reading frame (ORF) positioned opposite to the human immunodeficiency virus type 1 (HIV-1) env gene encodes the 189 amino acid, membrane-associated ASP protein. Negative-sense transcription, regulated by long terminal repeat sequences, has been observed early in HIV-1 infection in vitro. All subtypes of HIV-1 were scanned to detect the negative-sense asp ORF and to identify potential regulatory sequences. A series of highly conserved upstream short open reading frames (sORFs) was identified. This potential control region from HIV-1(NL4-3), containing six sORFs, was cloned upstream of the reporter gene EGFP. Expression by transfection of HEK293 cells indicated that the introduction of this sORF region inhibits EGFP reporter expression; analysis of transcripts revealed no significant changes in levels of EGFP mRNA. Reverse transcriptase-polymerase chain reaction analysis (RT-PCR) further demonstrated that the upstream sORF region undergoes alternative splicing in vitro. The most abundant product is spliced to remove sORFs I to V, leaving only the in-frame sORF VI upstream of asp. Sequence analysis revealed the presence of typical splice donor- and acceptor-site motifs. Mutation of the highly conserved splice donor and acceptor sites modulates, but does not fully relieve, inhibition of EGFP production. The strong conservation of asp and its sORFs across all HIV-1 subtypes suggests that the asp gene product may have a role in the pathogenesis of HIV-1. Alternative splicing of the upstream sORF region provides a potential mechanism for controlling expression of the asp gene.
