CPSF6 defines a conserved capsid interface that modulates HIV-1 replication

Abstract

The HIV-1 genome enters cells inside a shell comprised of capsid (CA) protein. Variation in CA sequence alters HIV-1 infectivity and escape from host restriction factors. However, apart from the Cyclophilin A-binding loop, CA has no known interfaces with which to interact with cellular cofactors. Here we describe a novel protein-protein interface in the N-terminal domain of HIV-1 CA, determined by X-ray crystallography, which mediates both viral restriction and host cofactor dependence. The interface is highly conserved across lentiviruses and is accessible in the context of a hexameric lattice. Mutation of the interface prevents binding to and restriction by CPSF6-358, a truncated cytosolic form of the RNA processing factor, cleavage and polyadenylation specific factor 6 (CPSF6). Furthermore, mutations that prevent CPSF6 binding also relieve dependence on nuclear entry cofactors TNPO3 and RanBP2. These results suggest that the HIV-1 capsid mediates direct host cofactor interactions to facilitate viral infection.

Figure 1. Exposed CA mutations affecting HIV-1 infectivity.

Location of CA mutations that are solvent-exposed in the hexameric CA structure and which are found to result in decreased HIV-1 infectivity . CA mutations are labeled and represented as red spheres. The CA hexamer structure was derived from pdb: 3H47 by generating symmetry-related copies in PyMOL. Left: view looking down onto the hexamer, right: view from the side, with CypA-binding loops at the top.

Figure 2. CPSF6 binds diverse lentiviral capsids.

Isothermal titration calorimetry (ITC) of CPSF6_313–327 against CA^N domains of lentiviral capsids. CPSF6_313–327 binding to HIV-1 is specific (A), being abolished by CA mutation N74D (B). CPSF6_313–327 also binds to diverse primate lentiviral capsids, HIV-2, FIV and SIVmac (C). The stoichiometry (N), affinity (K_d), enthalpy (ΔH) and entropy (ΔS) of interaction are shown.

Figure 3. Crystal structure of HIV-1 CA^N in complex with CPSF6_313–327.

(A) Crystal structure of the HIV-1 CA^N:CPSF6_313–327 complex. HIV-1 CA^N is shown as cartoon representation and CPSF6 as sticks. Secondary structure elements in HIV-1 CA^N are labeled. The electron density for the CypA-binding loop was poor, so this region is represented by a dashed line. (B) Close-up view of the HIV-1 CA^N:CPSF6_313–327 interface, showing HIV-1 CA^N as surface representation. The three CPSF6 residues that fill the centre of the channel in HIV-1 are indicated. (C) The CPSF6-binding interface on HIV-1 CA^N. Residues involved in binding to CPSF6_313–327 are labeled and shown in green. (D) Intramolecular interactions in CPSF6_313–327. Residues involved in intramolecular hydrogen bonding interactions are labeled and the interactions shown as dashed lines. The sequence of CPSF6_313–327 is shown for reference. (E) Stereo figure showing overview of the HIV-1 CA^N:CPSF6_313–327 interaction. The N- and C-termini of CPSF6_313–327 (labeled) project out of the binding channel in HIV-1 CA^N.

Figure 4. Interactions in the HIV-1 CA^N:CPSF6_313–327 complex.

(A–D) Detailed views of the HIV-1 CA^N:CPSF6_313–327 interface. HIV-1 CA^N is shown as grey cartoon, HIV-1 residues that bind CPSF6 are in green and CPSF6_313–327 in yellow. Water molecules involved in water-mediated interactions in the complex are shown as cyan spheres. (A) Overview of the HIV-1 CA^N:CPSF6_313–327 interface, showing all interacting residues and intermolecular hydrogen-bonding interactions. Views of the close-ups shown in (B), (C) and (D) are indicated. (B–D) HIV-1 CA^N α-helices are labeled to aid orientation. Interacting residues are labeled (CPSF6 in normal font; HIV-1 in italics).

Figure 5. The CPSF6-binding interface is accessible and highly conserved in HIV-1 virions.

(A) Model of CPSF6_313–327 binding to HIV-1 CA hexamer. The hexamer structure was derived from pdb: 3H47 by generating symmetry-related copies in PyMOL. The model was composed by superposition of CA^N chains from HIV-1 CA^N:CPSF6_313–327 on the hexamer using secondary structure matching. HIV-1 CA helices are shown as cylinders and CPSF6_313–327 as spheres. Left: top view, right: side view. N-terminal and C-terminal CA domains (NTD and CTD) are labeled. (B) Close-up of boxed region in (A). CAs are shown as cartoons and CPSF6_313–327 as sticks. The region between CTD positions 175 and 188 was disordered and so is represented by a dashed line. (C) Same view as in (B), showing exposed CA mutations (labelled and shown as red spheres) that result in decreased HIV-1 infectivity (see Figure 1 ). (D) Model of CPSF6_313–327 binding at a hexamer-hexamer interface. Neighbouring hexamers were derived from pdb: 3H47 by generating extended symmetry-related copies in PyMOL. HIV-1 CA^N:CPSF6_313–327 was superposed on the hexamer using secondary structure matching. The model shows that CPSF6_313–327 binding is likely to be accommodated at neighbouring CTD-mediated hexamer-hexamer junctions.

Figure 6. The CPSF6-binding interface determines HIV-1 nuclear entry requirements.

(A–B) CPSF6 residue F321 is critical for interaction with HIV-1 CA^N. (A) HeLa cells expressing empty vector (white bar), HA-tagged CPSF6-358 (black bar) or CPSF6-358 bearing mutation F321N (striped bar) were infected with GFP-encoding VSV-G pseudotyped HIV-1 vector. F321N abolished restriction by CPSF6-358, confirming the importance of this residue in the HIV-1 CA^N:CPSF6 interaction. (B) Western blot to show CPSF6-358 and CPSF6-358 F321N expression levels, with actin as loading control. (C) ITC of CPSF6_313–327 against mutant HIV-1 CA^Ns. All mutations at the CPSF6-binding interface resulted in reduced affinity to CPSF6_313–327. The stoichiometry (N), affinity (K_d), enthalpy (ΔH) and entropy (ΔS) of interaction are shown. (D) Titres of VSV-G pseudotyped GFP-encoding HIV-1 vectors bearing wild type or mutant CA on HeLa cells expressing empty vector (EV), CPSF6-358, control knockdown cells (shC) and cells depleted for TNPO3 (shTNPO3) or RanBP2 (shRanBP2). The data are representative of two independent experiments, each using three different virus doses. Mutation of HIV-1 CA^N residues involved in binding to CPSF6 resulted in the loss of dependence on TNPO3 and RanBP2, suggesting a link between CPSF6 binding and normal nuclear import of HIV-1. (E) Western blot to show knockdown of TNPO3 and RanBP2, with cyclophilin A (CypA) as loading control.

Figure 7. The CPSF6 interface is conserved in HIV-1, HIV-2, SIVmac CA^N.

(A) The CPSF6-binding interface is highly conserved within HIV-1 viruses. The ConSurf Server , was used to map ∼100 unique HIV-1 CA^N sequences onto the HIV-1 CA^N:CPSF6_313–327 structure. HIV-1 CA^N is shown as surface representation and CPSF6_313–327 as yellow sticks. The level of conservation at each position in CA^N is shown by the colour; dark blue = most conserved, red = least conserved. Residues at the CPSF6-binding interface are among the most highly conserved of all, suggesting that this is a functionally important interface. The figure was generated using the PyMOL script output by ConSurf, with conservation grades replacing the B-factor column. (B) Sequence alignment of HIV-1, HIV-2, SIVmac CA^N. (C) Titres of VSV-G pseudotyped GFP-encoding HIV-2 and SIVmac vectors bearing wild type or mutant CA on HeLa cells expressing empty vector (EV) or CPSF6-358.

Figure 8. Drug PF-3450074 binds to part of the CPSF6-binding interface in HIV-1 CA^N.

(A) Superposition of HIV-1 CA^N:PF-3450074 structure (pdb: 2XDE) on HIV-1 CA^N:CPSF6_313–327 using secondary structure matching of the HIV-1 CA^N domains. Drug PF-3450074 is shown in cyan, CPSF6_313–327 in yellow. The three CPSF6 residues that fill the centre of the channel in HIV-1 are indicated. One of the phenyl rings in PF-3450074 superposes almost exactly with the phenyl ring of F321 in CPSF6_313–327. (B) Crystal structure of HIV-1 CA^N:PF-3450074 (pdb: 2XDE) showing residues involved in binding to CPSF6 (green sticks). The yellow sphere represents a water molecule involved in a water-mediated interaction in the complex. (C) ITC of PF-3450074 against wild type and mutant HIV-1 CA^Ns. (D) Titration of 1 mM CPSF6_313–327 into 80 µM HIV-1 CA^N was carried out in the absence or presence of 100 µM PF-3450074 (‘control’ and ‘+ drug’ respectively). PF-3450074 completely inhibits binding of CPSF6_313–327 to HIV-1 CA^N.