A Novel Bent Intermediate in the Dimerization of HIV-1 DIS RNA

Abstract

Dimerization Initiation Sequence (DIS) plays a crucial role in genome dimerization through formation of a “kissing complex” intermediate between two homologous DIS sequences. DIS is a conserved hairpin loop motif on the 5' UTR of the HIV-1 genome. This bimolecular kissing complex ultimately leads to the formation of an extended RNA duplex. Studying the mechanism and kinetics of this RNA interaction is the key to ex-ploiting DIS as a possible HIV drug target.Here, we report a novel study that makes an important contribution to understanding the dimerization mechanism of HIV-1 RNA in vitro. This work employed single-molecule fluorescence resonance energy transfer (smFRET) to monitor the dimerization of minimal HIV-1 RNA sequence containing DIS. Most significantly, we observe a previously uncharacterized folding intermediate that plays a critical role in the dimerization mechanism. Our data reveal that dimerization involves three distinct steps in dynamic equilibrium and the equilibrium between the steps are regulated by Mg2+ ions. Two of the steps are identified as previously proposed structures: the kissing complex and the extended duplex. In addition to these, our data reveal a previously unobserved folding intermediate, consistent with a bent kissing complex conformation, similar to the TAR-TAT complex. Mutations of the highly conserved purines flanking the DIS loop destabilize this intermediate which indicates that these purines may play an important role in the HIV-1 RNA dimerization in vivo. The mechanistic insights gained from these experiments would represent significant progress in understanding the HIV-1 dimerization mechanism.