NMR analysis of intra- and inter-molecular stems in the dimerization initiation site of the HIV-1 genome

Two positive-strand HIV-1 genomic RNAs form a dimer in virion particles thr ough interaction of the dimerization initiation sites (DIS), The DIS RNA fr agment spontaneously formed a "loose-dimer" and was converted into a "tight -dimer" by supplementation with nucleocapsid protein NCp7, This two-step di merization reaction requires the whole DIS sequence [Takahashi ct al, (2000 ) RNA 6, 96-102], In the present study, we measured imino proton resonances to investigate the secondary structures of the two types of dimers in a 39 -mer RNA covering the entire DIS (DIS39), including discrimination between intra- and inter-molecular base pairing, Both the presence and absence of i ntermolecular NOE between N-15-labeled and unlabeled DIS39 were unambiguous ly detected in an equimolar mixture of N-15-labeled and unlabeled DIS39, Th e stem-bulge-stem structures in both dimers were confirmed and found to be very close to each other from clear superimposition of the NMR spectra in t he two dimeric states. Nevertheless, the modes of base paring in the stems of the loose- and tight-dimers were intra- and inter-molecular, respectivel y. Our results suggest a large structural alteration of genomic RNA occurs during virion maturation.