Characterizing the interactions between the nucleocapsid protein and the 5'-utr of HIV-1: Implications for diploid genome packaging

During assembly of the human immunodeficiency virus type 1 (HIV-1), two copies of the intact, unspliced viral RNA genome are efficiently incorporated into virions, whereas host RNAs and spliced viral RNAs are excluded. Packaging is mediated by interactions between the nucleocapsid (NC) domain of the viral Gag polyproteins and the packaging segments located in the highly conserved 5'-untranslated region (5'-UTR) of the RNA genome. Current understanding of the structural determinants of the diploid genome selection and packaging by HIV-1 remains limited, in part due to the lack of high-resolution structural information about the 5'-UTR. We hypothesized that diploid genome selection is mediated by a dimerization-dependent RNA structural switch mechanism, in which the NC binding sites are sequestered in the monomeric RNA, and become exposed upon dimerization. In our efforts to test this hypothesis, we found that RNA dimerization through the dimer-promoting hairpin (DIS) per se does not alter the RNA structure and expose more NC binding sites. Instead, our findings suggest that long-range interactions between the Gag open reading frame (AUG) and the upstream element (U5) regulates the monomer-dimer equilibrium of the 5'-UTR. The structural changes upon dimerization expose approximately 10 high affinity NC binding sites that were sequestered in the monomer. Mutations that stabilize the monomer conformation inhibit RNA packaging in vivo, whereas those that stabilize the dimer promote packaging. Our findings support a nucleotide displacement RNA switch mechanism for diploid genome selection, in which the U5:AUG interactions expose the DIS and a critical number of NC binding sites required for packaging. We also studied the role of Tar, PolyA and PBS elements in the dimeric 5'-UTR structure, and found that they do not affect the U5:AUG long-range interaction mediated monomer-dimer equilibrium. Both in vitro NC binding studies and in vivo packaging assays of the Tar, PolyA and PBS deleted mutants have enabled us to identify a 156-nucleotide RNA segment spanning from U5 through AUG, that appears to serve as the core packaging signal of HIV-1.