Identification of the initial nucleocapsid recognition element in the HIV-1 RNA packaging signal

Abstract

<jats:p>Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5ʹ-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. Here, we show that the dimeric 5′-leader contains more than two dozen NC binding sites with affinities ranging from 40 nM to 1.4 μM, and that all high-affinity sites (<jats:italic>K</jats:italic><jats:sub>d</jats:sub> ≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, Ψ<jats:sup>CES</jats:sup>). The four initial binding sites with highest affinity reside near two symmetrically equivalent three-way junction structures. Unlike the other high-affinity sites, which bind NC with exothermic energetics, binding to these sites occurs endothermically due to concomitant unwinding of a weakly base-paired [UUUU]:[GGAG] helical element. Mutations that stabilize base pairing within this element eliminate NC binding to this site and severely impair RNA packaging into virus-like particles. NMR studies reveal that a recently discovered small-molecule inhibitor of HIV-1 RNA packaging that appears to function by stabilizing the structure of the leader binds directly to the [UUUU]:[GGAG] helix. Our findings suggest a sequential NC binding mechanism for Gag-genome assembly and identify a potential RNA Achilles’ heel to which HIV therapeutics may be targeted.</jats:p>

NMR Studies of Retroviral Genome Packaging

“…Although NC is capable of binding tightly to the conserved GGAG tetraloop of Ψ [ 109 ] (see below), substitution of the GGAG loop by GCUA or AAGA did not significantly affect packaging or replication [ 233 ]. Recently, NMR and isothermal titration calorimetry (ITC) studies revealed that the weakly base-paired [UUUU]:[GGAG] helical region in the lower stem of the Ψ hairpin, serves as the initial high-affinity NC binding site (K d ~40 nM) and that structural rearrangements induced by NC binding are required for in vivo RNA packaging [ 236 ]. Although considerable effort focused on the structure and NC interactions of the Ψ-hairpin [ 237 , 238 ], it is now clear that competitive RNA packaging requires a much larger portion of the leader (see below) [ 40 ].…”

“…Extensive biochemical data demonstrated that the HIV-1 packaging signal contains more than two dozen NC binding sites, with varied affinities and different functional importance [ 40 , 41 , 71 ]. In particular, NMR-detected NC binding, in conjunction with ITC studies reveal that the [UUUU]:[GGAG] stem region of the Ψ-hairpin contains two very high-affinity binding sites (K d ~40 nM, compared with K d ~ 300 nM for the Ψ-hairpin apical loop, under physiologic-like ionic strength) [ 236 ]. The structural lability of this [UUUU]:[GGAG] helical region is shown to be required for both tight NC binding in vitro and efficient RNA packaging in transfected cells [ 236 ].…”

“…In particular, NMR-detected NC binding, in conjunction with ITC studies reveal that the [UUUU]:[GGAG] stem region of the Ψ-hairpin contains two very high-affinity binding sites (K d ~40 nM, compared with K d ~ 300 nM for the Ψ-hairpin apical loop, under physiologic-like ionic strength) [ 236 ]. The structural lability of this [UUUU]:[GGAG] helical region is shown to be required for both tight NC binding in vitro and efficient RNA packaging in transfected cells [ 236 ]. However, the molecular mechanism for such high-affinity binding remains unknown, and additional studies of NC bound to other RNA targets in the HIV-1 dimeric 5′-leader are clearly warranted.…”

“…In the same study, 1 H NMR spectroscopy showed that NSC specifically bound to the GGAG tetraloop (site-1 in Figure 19 B). However, a recent NMR study demonstrated that NSC targeted the structurally labile [UUUU]:[GGAG] stem region of the Ψ-hairpin, in the context of a larger construct of the HIV-1 packaging signal (site-2 in Figure 19 B) [ 236 ]. Using the full-length HIV-1 5′-leader, SHAPE analysis suggested that the function of NSC is to stabilize the Ψ-hairpin and subsequently stabilize a larger region of the RNA packaging signal [ 389 ].…”

Overview of the Nucleic-Acid Binding Properties of the HIV-1 Nucleocapsid Protein in Its Different Maturation States

“…The mature form of NC, NCp7, is a small protein of 55 residues, very basic (pI = 9.9), which contains two domains, each structured around a Zn 2+ ion ( Figure 1 A), named zinc-knuckle (ZK) domains, with the consensus sequence: CX 2 CX 4 HX 4 C. The electrostatic interactions represent an essential component of NC interactions with NAs, and NC binding is indeed dramatically dependent on the ionic strength [ 52 , 53 , 54 , 55 , 56 ]. Nevertheless, some NC interactions with NAs are more specific, with G-rich single-stranded sequences flanked by stable helices being the preferred sequence binding sites for NCp7 [ 20 , 31 , 57 , 58 , 59 , 60 , 61 , 62 , 63 ].…”