Integrase anchors viral RNA to the HIV-1 capsid interior

Nature News · Feb 18, 2026 · Collected from RSS

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MainIn addition to its canonical role of orchestrating the insertion of viral DNA into the host genome, HIV-1 integrase (IN) has an essential function in viral morphogenesis1,4,5 (reviewed previously6). The HIV-1 core contains viral ribonucleoproteins (RNPs; predominantly nucleocapsid (NC) protein bound to two copies of genomic RNA) encased within a closed lattice of capsid (CA) protein. Numerous examples of amino acid substitutions in IN, or allosteric IN inhibitors (ALLINIs), preclude the incorporation of viral RNPs into cores during maturation, yielding an eccentric virion morphology with RNPs situated outside of comparatively electron-lucent or ‘empty’ CA shells1,2,3,4,7. The failure to encapsulate RNPs results in a pronounced defect of reverse transcription, the signature class II phenotype associated with many HIV-1 IN mutant viruses5,8. Although HIV-1 IN has been shown to interact with viral genomic RNA in virio1,2, how the viral protein binds to and retains viral RNA inside mature cores remains unclear.Formation of ordered IN–RNA co-polymersWe tested recombinant HIV-1 IN protein for interaction with a range of RNA constructs using biolayer interferometry (BLI). In agreement with published observations1, HIV-1 IN bound to a 57-mer oligonucleotide spanning the HIV-1 transactivation response (RNATAR) element that folds into a long hairpin structure9, as well as to a range of shorter RNAs, including those composed solely of GA or UC dinucleotides that do not adopt stable tertiary structures (Extended Data Fig. 1). Elevated salt concentrations inhibit the IN–RNA interaction1, and HIV-1 IN quickly aggregates in vitro in buffers containing <1 M NaCl (Extended Data Fig. 2a). To overcome this technical hurdle, we screened a panel of primate lentiviral INs, identifying IN from simian immunodeficiency virus (SIV) from talapoin monkeys (SIVtal) as having favourable biochemical properties. Sharing around 52% amino acid sequence identity with HIV-1 IN, SIVtal IN displayed improved solubility (Extended Data Fig. 2a,b). Similar to HIV-1 IN2,10,11,12 and maedi-visna virus (MVV) IN13, SIVtal IN formed stable tetramers under a wide range of concentrations (Extended Data Fig. 2a). Finally, SIVtal IN showed robust interaction with RNA oligonucleotides in the BLI assay (Extended Data Fig. 1). To evaluate the effects of RNA binding on the IN structure, we used hydrogen–deuterium exchange coupled with mass spectrometry (HDX–MS). We incubated HIV-1 and SIVtal INs in the absence or presence of RNATAR and transferred the mixtures into deuterated buffer. RNA significantly diminished isotope exchange over extended portions of the IN amino acid sequences, including regions within the N-terminal domain (NTD), catalytic core domain (CCD) and C-terminal domain (CTD) (Extended Data Fig. 2c).Imaging SIVtal IN vitrified in the presence of RNATAR on a transmission electron microscope revealed the formation of elongated polymers (Extended Data Fig. 3a). Processing single-particle images enabled us to compute a three-dimensional (3D) reconstruction of the IN–RNA co-polymer at an overall resolution of 3.7 Å (Fig. 1a, Extended Data Fig. 3b and Extended Data Table 1). The averaged structure comprises two identical linear filaments running parallel to one another and joined by RNA duplexes shared between them (Fig. 1a and Supplementary Video 1). Each sister filament represents a chain of IN octamers formed around a pair of RNATAR chains. This assembly depends on the ability of RNATAR to adopt an extended duplex, multiple copies of which rigidly tether the parallel IN filaments. Concordantly, SIVtal IN formed single filaments (chains of octamers) in the presence of single-stranded oligo-GA ribooligonucleotides, while ordered IN structures did not form in the absence of RNA (Extended Data Fig. 3a). Although fortuitous formation of the RNATAR-tethered double-IN filament enabled high-resolution structure determination, we would not expect such extended long-range arrangements to occur with the 9 kb viral RNA genome.Fig. 1: The structure of the SIVtal IN–RNATAR complex.a, Cryo-EM reconstruction of the SIVtal IN–RNA complex shown in three orthogonal views. The structure comprises two IN filaments bridged by RNA duplexes (black). Octameric IN repeat units are distinguished by alternating colours and delineated with square brackets. b, Cartoon representation of a single octameric IN repeat unit bound to two RNA molecules. IN subunits are coloured green, cyan, magenta and yellow to indicate structurally equivalent chains within opposing tetramers. A-form duplex RNA chains are shown in the cartoon with an orange phosphodiester backbone. The NTDs from flanking octameric units are included in the model and shown in grey. Grey spheres represent Zn2+ ions coordinated within the NTDs. c, View of a single IN tetramer along its approximate two-fold symmetry axis. Canonical IN domains—NTD, CCD and CTD—are labelled. The two-fold symmetry is disrupted by the lateral displacement of the CTD dimer (formed by IN subunits in cyan and magenta). The images of cryo-EM maps (a) and structural models (b,c) were created using UCSF Chimera and PyMOL, respectively.Full size imageMasked refinement centred on a filament repeat unit resulted in 3D reconstruction of an IN octamer associated with two copies of RNATAR at an overall resolution of 3.3 Å. The local resolution ranged from 3 Å throughout the bulk of the protein regions to about 4 Å for the RNA component (Extended Data Fig. 3c), which was defined less well presumably due to averaging of the two opposing binding orientations. The IN octamer features two-fold symmetry and comprises two identical homotetramers sharing a pair of RNATAR chains (Fig. 1b). The tetramers feature the dimer-of-dimers architecture as has been observed in isolated lentiviral IN constructs10,14 and intasome assemblies15,16, yet deviates considerably from two-fold symmetry (Fig. 1c). The tetramers engage RNA chains solely through their CTDs. Within each tetramer, a pair of CTDs form the canonical clamshell dimer17, each binding across the major groove of the RNA duplex, clasping the minor groove in between (Fig. 2a). This binding mode effectively gauges the geometric parameters of the A-form duplex and may therefore enable the viral protein to recognize RNA duplex structures. Each of the two paired CTDs use a triad of positively charged residues—Arg228, Arg264 and Lys265 (corresponding to HIV-1 IN Arg228, Arg263 and Lys264, respectively)—to interact with the phosphodiester backbone. One of the remaining CTDs in each SIVtal IN tetramer interacts with the RNA backbone engaged by the opposing tetramer through the Lys244, Arg263 and Lys270 side chains (equivalent to HIV-1 IN Lys244, Arg262 and Arg269, respectively), effectively bridging the two halves of the octamer (Fig. 2b). The stacking of octameric filament repeat units is mediated by IN NTD–NTD interactions (Fig. 2c); here, residues Phe1, Val2, Ile5, Pro29 and Val32 (corresponding to HIV-1 IN Phe1, Leu2, Ile5, Pro29 and Val32, respectively) from neighbouring subunits form a compact hydrophobic interface. Moreover, the side chain of NTD residue Lys34 (Lys34 in HIV-1 IN) from one octamer interacts with the backbone of the RNA chain belonging to the neighbouring octamer (Fig. 2c).Fig. 2: IN–RNA and IN–IN interfaces within the SIVtal IN filament.a, Interaction of dimerized CTDs with an RNA duplex within the IN tetramer. b, Interactions involving CTD from one IN tetramer with an RNA duplex engaged by the opposing tetramer. c, Interactions involving neighbouring IN octamers. Protein and RNA chains are shown as cartoons and are coloured as in Fig. 1b. The side chains of residues discussed in the main text are shown as sticks and indicated. His12, His16, Cys40 and Cys43 comprise the zinc-coordinating motif conserved among retroviral INs. The diagrams were created using PyMOL.Full size imageThe structure of the SIVtal IN–RNATAR filament explains many previous observations made for HIV-1, including the importance of IN tetramerization for RNA binding2, the protection of Lys264 from modification by N-hydroxysuccinimide in the presence of RNA1, and the RNA-binding defects of virion-incorporated K34A, R262A/R263A and R269A/K273A IN tetramers1,2. Furthermore, in agreement with their involvement in RNA binding, HIV-1 IN mutant viruses K34A, R228A, K244A, R262A/R263A, R262A/K264A and R269A/K273A, as well as the disruption of the NTD structure by H12N, were shown to elicit the class II phenotype1,2,18,19,20, supporting the involvement of the IN filament assembly in RNA retention.IN filaments inside native HIV-1 coresOver 95% of the mature HIV-1 core shell is composed of CA hexamers that form a regular lattice with a centre-to-centre distance between neighbouring repeat units of around 91 Å (refs. 21,22,23). Notably, this parameter matches precisely the distance between consecutive IN octamer repeats within the SIVtal IN–RNATAR structure. Furthermore, the IN octamer repeat units are inclined relative to the main filament axis by around 120°, which allows a near-perfect overlap with the mature CA hexamer lattice (Extended Data Fig. 4a). This unexpected congruency of two structurally unrelated supramolecular assemblies suggested that they could have coevolved to associate during viral maturation. To test this hypothesis, we produced virus-like particles by co-transfection of HEK293T cells with a near-full-length HIV-1 proviral construct and a plasmid overexpressing IN in the form of a fusion with HIV-1 viral protein R (Vpr) and mNeonGreen (NG). The latter was included to increase the IN copy number by taking advantage of the ability of Vpr to direct fusion partners into viral particles24. For safety considerations, the proviral construct carried a deletion of tat25 and IN active-site mutations26, the latter of which were also included in the Vpr-NG-IN construct. The wild-type (WT) and mutant versions of Vpr-NG-IN impor