Structural basis of HIV-1 capsid recognition by PF74 and CPSF6

Abstract

Upon infection of susceptible cells by HIV-1, the conical capsid formed by ∼250 hexamers and 12 pentamers of the CA protein is delivered to the cytoplasm. The capsid shields the RNA genome and proteins required for reverse transcription. In addition, the surface of the capsid mediates numerous host-virus interactions, which either promote infection or enable viral restriction by innate immune responses. In the intact capsid, there is an intermolecular interface between the N-terminal domain (NTD) of one subunit and the C-terminal domain (CTD) of the adjacent subunit within the same hexameric ring. The NTD-CTD interface is critical for capsid assembly, both as an architectural element of the CA hexamer and pentamer and as a mechanistic element for generating lattice curvature. Here we report biochemical experiments showing that PF-3450074 (PF74), a drug that inhibits HIV-1 infection, as well as host proteins cleavage and polyadenylation specific factor 6 (CPSF6) and nucleoporin 153 kDa (NUP153), bind to the CA hexamer with at least 10-fold higher affinities compared with nonassembled CA or isolated CA domains. The crystal structure of PF74 in complex with the CA hexamer reveals that PF74 binds in a preformed pocket encompassing the NTD-CTD interface, suggesting that the principal inhibitory target of PF74 is the assembled capsid. Likewise, CPSF6 binds in the same pocket. Given that the NTD-CTD interface is a specific molecular signature of assembled hexamers in the capsid, binding of NUP153 at this site suggests that key features of capsid architecture remain intact upon delivery of the preintegration complex to the nucleus.

Keywords: HIV-1 CA protein; X-ray crystallography; drug discovery; fluorescence polarization; isothermal calorimetry.

Fig. 1.

Structure of PF74 in complex with the HIV-1 CA hexamer. (A) Top view of the hexamer, with each subunit in a different color and the bound PF74 in white. (B) Unbiased mFo-DFc density at 3σ (magenta mesh) clearly defines the bound compound at the NTD–CTD interface. NTD is in cyan, and CTD is in green. (C) Chemical structure of PF74, with the R1, R2, and R3 rings labeled. (D) Superposition of hexamer-bound PF74 (NTD in cyan, compound in white) and NTD-bound PF74 (16) (NTD in dark blue, compound in cyan). (E) Close-up view of the R3 indole bound to its subpocket. Relevant residues are shown as spheres (NTD in blue and CTD in green). The hydrogen bond between the indole N3 amide and NTD Gln63 sidechain is shown in yellow. (F) Superposition of intersubunit NTD–CTD interfaces in the structure with bound PF74 (light shades) and unbound hexamer (dark shades). Key binding residues are shown as sticks and labeled.

Fig. 2.

Effect of PF74 on CA assemblies. (A) In vitro-assembled CA-NC tubes were incubated with either stabilization buffer (lane 1) or destabilization buffer with increasing amounts of PF74 (lanes 2–6), then subjected to centrifugation through a sucrose cushion. CA-NC proteins were detected by Western immunoblot analysis using anti-p24 antibodies. The CA-NC proteins in the pellet reflect the presence of intact assemblies. Similar results were obtained in three independent experiments, and a representative experiment is shown. (B) Human HeLa cells stably expressing rhesus TRIM5α (TRIM5α_rh) or containing the empty vector LPCX were challenged with similar amounts of HIV-1-GFP in the absence (lanes 1 and 3) or presence of 10 µM PF74 (lanes 2 and 4) for 12 h. Infected cells were used to prepare postnuclear supernatants (INPUT) that were layered onto a 50% sucrose cushion to separate soluble (SOLUBLE) from pelletable (PELLET) HIV-1 capsids. Fractions were analyzed by Western immunoblot analysis using antibodies against HIV-1 CA. The percentages of pelletable capsids relative to the infected control in the presence of DMSO are shown. Similar results were obtained in three independent experiments, and SDs are shown. There is a significant reduction in the TRIM5α signal versus LPCX (P = 0.02).

Fig. 3.

Structure of CPSF6 in complex with the HIV-1 CA hexamer. (A) Electron density of CPSF6_313–327 (orange mesh) and of the surrounding NTD (green) and CTD (cyan) residues from two adjacent CA monomers that form the binding pocket in mesh representation. CPSF6 is shown as sticks (orange), and the NTD (green) and CTD (cyan) as cartoons. Electron density at better-resolved binding sites suggests the possibility of a hydrogen bond between the sidechain of CTD Lys182 and the carbonyl oxygen atom of CPSF6 Gly318 (dotted black line). The mesh representations for CPSF6 residues on chain p, CTD chain c residues 165–190, and NTD chain d residues 95–109 were set at 0.9, 1.2, and 1.2 σ, respectively. (B) Side view of the CA hexamer with bound CPSF6 peptides (yellow). The CPSF6 binding site is located between the NTD (green) and CTD (cyan) of neighboring CA monomers. The remaining CA subunits in the hexamer are shown as a heat map depicting the degree of conservation based on the analysis of 97 unique HIV-1/HIV-2/other SIV sequences. Red is least conserved, and blue is most conserved. One of the bound CPSF6 peptides is shown as a backbone trace to reveal the conservation of the binding site. (C) A detailed view of the CPSF6_313–327 polypeptide (yellow) in the binding pocket formed by NTD α-helices 3 and 4 (green) and CTD α-helices 8 and 9 (cyan). (D) Weblogo representation of the sequence conservation of capsid CTD residues located in helices 8 and 9 and the linker between them. Ninety-seven independent primate lentiviral sequences were analyzed. The height of a particular residue indicates its degree of conservation. Lys182 is highly conserved, whereas Gln179 is not.

Fig. 4.

Analysis of CPSF6 and NUP153 binding to HIV-1 CA. (A) Binding isotherms of fluorescein-CPSF6 (fCPSF6) peptide to the isolated NTD (red) and the CA hexamer (blue), which were derived from van Holde–Wischet analysis of analytical ultracentrifugation data. (B) Representative van Holde–Wischet model-free analysis of the fCPSF6 sedimentation rates in the presence of increasing concentrations of NTD. The red-orange-yellow-green-blue-purple rainbow color series corresponds to NTD concentrations of 0 μM, 63 μM, 250 μM, 625 μM, 1460 μM, and 1800 μM, respectively. fCPSF6 concentration was 2.2 μM for the free peptide (red) and 12 nM in the rest of the titration series. The data show that the sedimentation coefficient of the free fCPSF6 (∼0.7S) does not depend on the concentration of the peptide. (C) van Holde–Wischet analysis of fCPSF6 binding to the CA hexamer. The red-orange-yellow-green-blue-purple rainbow color series corresponds to CA (monomer) concentrations of 0 μM, 45 μM, 65 μM, 108 μM, 305 μM, and 530 μM, respectively. (D) Fluorescence polarization measurements of fCPSF6 binding to the CA hexamer (blue), wild-type full-length CA (green), and isolated NTD (red). (E) Fluorescence polarization measurements of fCPSF6 binding to the wild-type (blue), K182A (red), and K182R (green) hexamers. (F) Fluorescence polarization measurements of fNUP153 binding to the CA hexamer (red) and NTD (green) is compared with fCPSF6 binding to the hexamer (blue). The fNUP153 FG peptide binds more tightly to the hexamer compared with the isolated NTD.