Structural changes in the SL5 and SL6 leader sequences of HIV-1 RNA following interactions with the viral mGag protein.

We previously identified sequences downstream of the SL4 region of HIV-1 RNA that are involved in the recognition of the 5' leader region of HIV-1 RNA by a minimal version of the HIV-1 Gag protein (mGag). These sequences increase the affinity of this interaction, promote Gag multimerization, and enhance formation of an early mGag-RNA complex. Now, we provide protein footprinting results on the +350 to +500 nucleotide region of viral RNA, based on use of different single-stranded and base-paired ribonucleases. Use of the mfold program confirmed the existence of both a stem-loop 5 (SL5), downstream of SL4, and a more complex multi-stem-loop structure (SL6). Footprinting analysis using mGag and single-stranded-specific nucleases showed almost complete protection of the single-stranded region. In contrast, results obtained with RNase V1, a double-stranded-specific nuclease, suggest that the RNA structure is changed upon protein binding, presumably because of formation of novel and longer stems. Furthermore, RNA footprinting, using viral nucleocapsid protein (NC) and RNase VI, indicate a highly double-stranded structure in several regions. These findings show that viral RNA structure is modified by interaction with proteins, and that NC may possess different chaperone activity in the context of the Gag precursor than in its mature form.