NMR detection of intermolecular interaction sites in the dimeric 5′-leader of the HIV-1 genome

Abstract

HIV type-1 (HIV-1) contains a pseudodiploid RNA genome that is selected for packaging and maintained in virions as a noncovalently linked dimer. Genome dimerization is mediated by conserved elements within the 5′-leader of the RNA, including a palindromic dimer initiation signal (DIS) that has been proposed to form kissing hairpin and/or extended duplex intermolecular contacts. Here, we have applied a 2H-edited NMR approach to directly probe for intermolecular interactions in the full-length, dimeric HIV-1 5′-leader (688 nucleotides; 230 kDa). The interface is extensive and includes DIS:DIS base pairing in an extended duplex state as well as intermolecular pairing between elements of the upstream Unique-5′ (U5) sequence and those near the gag start site (AUG). Other pseudopalindromic regions of the leader, including the transcription activation (TAR), polyadenylation (PolyA), and primer binding (PBS) elements, do not participate in intermolecular base pairing. Using a 2H-edited one-dimensional NMR approach, we also show that the extended interface structure forms on a time scale similar to that of overall RNA dimerization. Our studies indicate that a kissing dimer-mediated structure, if formed, exists only transiently and readily converts to the extended interface structure, even in the absence of the HIV-1 nucleocapsid protein or other RNA chaperones.

HIV-1 leader RNA dimeric interface revealed by NMR

“…1B) (6). This approach confirmed formation of an extended duplex DIS dimer within the context of the dimeric leader.…”

“…In addition, gRNA dimerization has long been suspected to play structural roles, particularly during selective packaging of the viral gRNA (4,5). A recent study by Keane et al provides new insight into how the two ∼9-kb HIV gRNA copies are held together in the dimer and how dimerization involves a structural switch that regulates packaging and other steps during viral replication (6).…”

“…1A) (6). In this approach, a 1:1 mixture is prepared, consisting of two leader RNA constructs containing distinct NMR labeling patterns.…”

“…No evidence was obtained for intermolecular pairing involving PolyA, PBS, TAR, or the Ψ packaging element. However, Keane et al (6) point out that intermolecular pairing outside the probed region cannot be ruled out. For example, although no evidence was obtained for intermolecular pairing in the TAR hairpin below the bulge (Fig.…”

“…By revealing the structure of the dimeric interface of the HIV leader, the study by Keane et al (6) caps a series of NMR investigations that have illuminated a structural role for HIV gRNA dimerization as a molecular switch that converts a monomeric species competent for translation into a dimeric species stabilized by an extended interface that suppresses translation and splicing and that promotes selective packaging (Fig. 1B).…”