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 (Kd ≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, ΨCES). 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.

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.

Human Immunodeficiency Virus Type‐1 (HIV‐1) is responsible for a pandemic that affects roughly 35 million people worldwide. To date, there is no cure, and the array of antiretroviral medications serves only to diminish the progression of the disease and prevent a more rapid deterioration of the immune system. Many antiretrovirals target four main stages of the viral life cycle: entry, reverse transcription, integration, and maturation. Genome selection for packaging, however, is relatively less clearly understood, thus no current therapies that target this process have been developed. HIV‐1 genome packaging has been shown to be a highly selective process in which only dimeric, unspliced, genomes are recognized by the nucleocapsid (NC) domain of the Gag polyprotein for encapsidation. The nature of dimerization, which is mediated by elements within the 5′ untranslated region (5′‐UTR) of the genome, is hypothesized to be either a “kissing” interaction involving the six nucleotide palindromic (GCGCGC) dimerization initiation site (DIS) sequence, or an extended dimer conformation featuring a more extensive intermolecular interface. Our studies aim to elucidate the dimerization mechanism using NMR spectroscopy. Previous studies in the Summers laboratory identified that the HIV‐1 5′‐UTR exists as an extended dimer at high concentrations and after long incubation times at 37 °C. The U5:AUG region, which is distal to the DIS, has been identified as one of the sites with an intermolecular interface. We hypothesize that dimer formation is initiated by kissing interactions and transitions to an extended conformation over time. We utilized NMR spectroscopy to test this hypothesis using a mutagenesis strategy know as long‐range Adenosine Interaction Detection (lr‐AID). 1D NMR data collected on small U5:AUG control RNAs containing either UUA/UAA or UUG/UAA lr‐AID signals revealed isolated chemical shifts at 6.5 ppm, and 6.7 ppm, respectively. We incorporated the UUA/UAA and the UUG/UAA sequences within the U5:AUG region of the 5′‐UTR and observed the appearance of a chemical shift at 6.5 ppm in a biologically relevant time frame. Currently, we are conducting time‐dependent dimerization studies using native gel electrophoresis techniques in order to further characterize this transition.Support or Funding InformationThis research was funded by NIH/NIGMS grant 1P50GM103297 and sponsored by NIH/NIGMS MARC U*STAR T34 08663 National Research Service award to UMBC. It was conducted at the Howard Hughes Medical Institute at UMBC, with support from the Summer Biomedical Training Program (SBTP). Supported in part by the Howard Hughes Medical Institute's Precollege and Undergraduate Science Education Program.

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