HIV-1 splicing is modulated by RNA structure

Abstract

Materials and methods The SD stem-loop structure in the HIV-1 leader RNA region was stabilized or destabilized through mutation in the context of the HIV-1 molecular clone pLAI. The effect of the SD hairpin mutation on viral gene expression was measured upon transfection of 293T cells and virus replication was measured upon infection of SupT1 cells. The mutations were also introduced into an LTRluciferase reporter plasmid that was designed to monitor SD splicing by causing reduced luciferase activity. Upon transfection of these constructs into 293T cells, the efficiency of splicing at the major SD site was analysed by luciferase activity assays and RNA analysis (RT-PCR). Results Further stabilization of the SD stem-loop structure significantly reduced HIV-1 gene expression and replication and splicing was inhibited in the LTR-luciferase assay. SD hairpin destabilization did not cause major virus replication effects, but LTR-luciferase experiments revealed a modest upregulation of the splicing efficiency. Conclusions The stability of the SD stem-loop structure modulates the frequency of splicing at the major SD site. These results suggest that the stability of this hairpin is finetuned to obtain the right balance between unspliced and spliced RNAs in HIV-infected cells.