The 5´-leader of the HIV-1 genome regulates multiple functions during viral replication by mechanisms that have yet to be established. We developed an NMR approach that enabled direct detection of structural elements within the intact leader (712 nucleotide dimer) that are critical for genome packaging. Residues spanning the gag start codon (AUG) form a hairpin in the monomeric leader and base pair with residues of the Unique-5´ region (U5) in the dimer. U5:AUG formation promotes dimerization by displacing and exposing a dimer-promoting hairpin, and enhances binding by the nucleocapsid protein (NC), the cognate domain of the viral Gag polyprotein that directs packaging. Our findings support a packaging mechanism in which translation, dimerization, NC binding, and packaging are regulated by a common RNA structural switch.

The 5′-leader of the HIV-1 genome contains conserved elements that direct selective packaging of the unspliced, dimeric viral RNA into assembling particles. Using a 2H-edited NMR approach, we determined the structure of a 155-nucleotide region of the leader that is independently capable of directing packaging (Core Encapsidation Signal; ΨCES). The RNA adopts an unexpected tandem three-way junction structure, in which residues of the major splice donor and translation initiation sites are sequestered by long-range base pairing, and guanosines essential for both packaging and high-affinity binding to the cognate Gag protein are exposed in helical junctions. The structure reveals how translation is attenuated, Gag binding promoted, and unspliced dimeric genomes selected, by the RNA conformer that directs packaging.

Like all retroviruses, the Human Immunodeficiency Virus (HIV) selectively packages two copies of its unspliced RNA genome, both of which are utilized for strand-transfer mediated recombination during reverse transcription – a process that enables rapid evolution under environmental and chemotherapeutic pressures. The viral RNA appears to be selected for packaging as a dimer, and there is evidence that dimerization and packaging are mechanistically coupled. Both processes are mediated by interactions between the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins and RNA elements within the 5′-untranslated region (5′-UTR) of the genome. A number of secondary structures have been predicted for regions of the genome that are responsible for packaging, and high-resolution structures have been determined for a few small RNA fragments and protein-RNA complexes. However, major questions remain open regarding the RNA structures, and potentially the structural changes, that are responsible for dimeric genome selection. Here we review efforts that have been made to identify the molecular determinants and mechanism of HIV-1 genome packaging.

Assembly of Human Immunodeficiency Virus Type-1 (HIV-1) particles is initiated in the cytoplasm by the formation of a ribonucleoprotein complex comprising the dimeric RNA genome and a small number of viral Gag polyproteins. Genomes are recognized by the nucleocapsid (NC) domains of Gag, which interact with packaging elements believed to be located primarily within the 5´-leader of the viral RNA. Recent studies revealed that the native 5´-leader exists as an equilibrium of two conformers, one in which dimer-promoting residues and NC binding sites are sequestered and packaging is attenuated, and one in which these sites are exposed and packaging is promoted. To identify the elements within the dimeric 5´-leader that are important for packaging, we generated HIV-1 5´-leader RNAs containing mutations and deletions designed to eliminate substructures without perturbing the overall structure of the leader and examined effects of the mutations on RNA dimerization, NC binding and packaging. Our findings identify a 159 residue RNA packaging signal that possesses dimerization and NC binding properties similar to those of the intact 5´-leader and contains elements required for efficient RNA packaging.

The Biological Magnetic Resonance Data Bank contains NMR chemical shift depositions for 132 RNAs and RNA-containing complexes. We have analyzed the 1H NMR chemical shifts reported for non-exchangeable protons of residues that reside within A-form helical regions of these RNAs. The analysis focused on the central base pair within a stretch of three adjacent base pairs (BP triplets), and included both Watson–Crick (WC; G:C, A:U) and G:U wobble pairs. Chemical shift values were included for all 43 possible WC-BP triplets, as well as 137 additional triplets that contain one or more G:U wobbles. Sequence-dependent chemical shift correlations were identified, including correlations involving terminating base pairs within the triplets and canonical and non-canonical structures adjacent to the BP triplets (i.e. bulges, loops, WC and non-WC BPs), despite the fact that the NMR data were obtained under different conditions of pH, buffer, ionic strength, and temperature. A computer program (RNAShifts) was developed that enables convenient comparison of RNA 1H NMR assignments with database predictions, which should facilitate future signal assignment/validation efforts and enable rapid identification of non-canonical RNA structures and RNA-ligand/protein interaction sites.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-012-9683-9) contains supplementary material, which is available to authorized users.

A B S T R A C TAnti-HIV-1 drug design has been notably challenging due to the virus' ability to mutate and develop immunity against commercially available drugs. The aims of this project were to develop a series of fleximer base analogues that not only possess inherent flexibility that can remain active when faced with binding site mutations, but also target a non-canonical, highly conserved target: the nucleocapsid protein of HIV (NC). The compounds were predicted by computational studies not to function via zinc ejection, which would endow them with significant advantages over non-specific and thus toxic zinc-ejectors. The target fleximer bases were synthesized using palladium-catalyzed cross-coupling techniques and subsequently tested against NC and HIV-1. The results of those studies are described herein.