Structural studies of postentry restriction factors reveal antiparallel dimers that enable avid binding to the HIV-1 capsid lattice

David C. Goldstonea,b,1, Philip A. Walkera, Lesley J. Calderc, Peter J. Coombsa, Joshua Kirkpatrickb, Neil J. Balla, Laura Hilditchd,2, Melvyn W. Yapd, Peter B. Rosenthalc, Jonathan P. Stoyed,e, and Ian A. Taylora,1

Divisions of aMolecular Structure, cPhysical Biochemistry, and dVirology, National Institute for Medical Research, London NW7 1AA, United Kingdom; bSchool of Biological Sciences, University of Auckland, Auckland, New Zealand; and eFaculty of Medicine, Imperial College London, London SW7 2AZ, United Kingdom

Edited* by Stephen P. Goff, Columbia University College of Physicians and Surgeons, New York, NY, and approved May 27, 2014 (received for review February 10, 2014)

Restriction factors (RFs) form important components of host defenses a particular Trim5α can restrict (12, 13). The to retroviral infection. The Fv1, Trim5α, and TrimCyp RFs contain form of Trim5α inhibits HIV-1 replication, whereas the human N-terminal dimerization and C-terminal specificity domains that tar- homolog does not (14). However, a single change in get assembled retroviral capsid (CA) enclosing the viral the B30.2 domain of human Trim5α is sufficient to gain re- core. However, the molecular detail of the interaction between striction of HIV-1 (15, 16). Retrotransposition events have on RFs and their CA targets is unknown. Therefore, we have deter- several occasions resulted in fusion of the cis-trans prolyl isom- mined the crystal structure of the B-box and coiled-coil (BCC) region erase Cyclophilin A (CypA), a cellular factor capable of binding from Trim5α and used small-angle X-ray scattering to examine the to HIV-1 CA (17), to the RBCC motif of Trim5α, resulting in solution structure of Trim5α BCC, the dimerization domain of Fv1 TrimCyp RFs (18, 19). In a similar manner, fusion of CypA to (Fv1Ntd), and the hybrid restriction factor Fv1Cyp comprising the N-terminal dimerization domain of Fv1 yields an artificial Fv1NtD fused to the HIV-1 binding Cyclophilin A (CypA). RF, Fv1Cyp, that shares the characteristics of Fv1 restriction but These data reveal that coiled-coil regions of Fv1 and Trim5α form is capable of blocking infection by HIV-1 (20). extended antiparallel dimers. In Fv1Cyp, two CypA moieties are Central to the mechanism of postentry restriction is the rec- MICROBIOLOGY located at opposing ends, creating a molecule with a dumbbell ap- ognition of the intact retroviral capsid (21). Whereas structural pearance. In Trim5α, the B-boxes are located at either end of the and sequence comparisons demonstrate that the site of re- striction, for both Fv1 and Trim5α, is located across the apical coiled-coil, held in place by interactions with a helical motif from the – L2 region of the opposing monomer. A comparative analysis of surface of the retroviral CA protein (22 24), abrogation experi- Fv1Cyp and CypA binding to a preformed HIV-1 CA lattice reveals ments have clearly demonstrated that the intact retroviral capsid how RF dimerization enhances the affinity of interaction through lattice is required for recognition, rather than individual capsid proteins (25, 26). Assembly of MLV CA on lipid nanotubes has avidity effects. We conclude that the antiparallel organization of the demonstrated in vitro that a lattice is required for Fv1 binding NtD regions of Fv1 and Trim5α dimers correctly positions C-terminal and that perturbations in this lattice prevent recognition by Fv1 specificity and N-terminal effector domains and facilitates stable (27). Whereas the B30.2 domain determines the range of retro- binding to adjacent CA hexamers in viral cores. viruses a particular species’ Trim5α can restrict, an intact tri- partite motif is required for efficient restriction, in part because | MLV | SAXS | X-ray crystallography Significance n the course of evolution, mammals have developed systems to prevent and contain infection by retroviral pathogens (1). I Retroviral infection of cells can be blocked by the action of the These include the restriction factors (RFs) that target various postentry restriction factors. The Trim5α and Fv1 factors do so by stages of the retroviral lifecycle, including reverse transcription, targeting the capsid that surrounds the viral core. The nature of integration, and viral egress (2). A particular subset of these the interaction of these factors with the viral assembly is un- factors includes the postentry RFs Fv1, Trim5α, and TrimCyp clear. We show that these factors form antiparallel dimers that that act shortly after the retroviral core has entered the cyto- display specificity domains spaced to target motifs on the capsid plasm of the and prevent reverse transcription or integration lattice surface. In doing so Fv1 and Trim5α take advantage of the of the viral genome (3). The murine RF Fv1 blocks infection of murine leukemia virus regularly spaced array of binding sites on the capsid surface, (MLV) by a still poorly understood mechanism. Two major generating avidity to aid recognition of retroviral pathogens. alleles of Fv1 have been identified, Fv1n, which can restrict in- b Author contributions: D.C.G., P.J.C., J.K., N.J.B., L.H., M.W.Y., P.B.R., J.P.S., and I.A.T. de- fection by B-tropic MLV, and Fv1 , which restricts N-tropic MLV signed research; D.C.G., P.A.W., L.J.C., P.J.C., J.K., N.J.B., L.H., M.W.Y., P.B.R., and I.A.T. (4, 5). The Fv1 protein contains at least two functional regions, performed research; P.J.C., L.H., and M.W.Y. contributed new reagents/analytic tools; an N-terminal dimerization domain and a C-terminal domain that D.C.G., P.A.W., L.J.C., J.K., N.J.B., P.B.R., J.P.S., and I.A.T. analyzed data; and D.C.G., L.J.C., is required for capsid (CA) recognition (6). Previous biophysical P.B.R., J.P.S., and I.A.T. wrote the paper. studies have demonstrated that the N-terminal domain of Fv1 The authors declare no conflict of interest. α forms a tightly associated -helical elongated dimer (7). *This Direct Submission article had a prearranged editor. Whereas alleles of Fv1 are limited to different species of mice α Data deposition: Crystallography, atomic coordinates, and structure factors have been (8), Trim5 acts in a similar manner to restrict retroviral in- deposited in the , www.pdb.org (PDB ID code 4TN3). fection in and other mammalian species (9). The 1 α To whom correspondence may be addressed. E-mail: [email protected] or Trim5 protein contains an N-terminal tripartite motif consisting [email protected]. of a RING domain, a B-box2 domain, and a predicted coiled-coil 2Present address: Medical Research Council Centre for Medical Molecular Virology, Di- region (RBCC) (10). The C terminus of the protein contains a vision of Infection and Immunity, University College London, London WC1E 6BT, B30.2 or PRY/SPRY domain connected to the RBCC motif United Kingdom. through a linker region, L2 (11). Residues in the B30.2 domain This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. are largely responsible for determining the range of 1073/pnas.1402448111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1402448111 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 individual interactions between B30.2 and CA are of low affinity The MALLS analysis yields an invariant molecular weight of 42 (28). Avid binding of incoming virus therefore requires extensive kDa for Fv1NtD (concentration range of 1–12 mg/mL) and 80 Trim5α multimerization (29). Deletion of the coiled-coil region kDa for Fv1Cyp (concentration range of 1.5–3 mg/mL), in- prevents stable binding of the retroviral CA protein (21), whereas dicating both proteins form tight dimers in solution (Fig. 1A and the B-box promotes cooperative CA binding by mediating higher- Table S1). In a similar manner, SEC-MALLS analysis of this order Trim5α self-association (30). Disruption or removal of the RhT5 (88–296) EK/RD construct (Fig. 1A and Table S1) gives RING domain interferes with interactions, delaying a solution molecular mass of 54 kDa (concentration range of the block in virus replication until after reverse transcription has 0.5–5 mg/mL), revealing the core association domain of Trim5α occurred, but can also affect higher-order RF association (31). is also a tight dimer. To investigate the mechanism of restriction and the recogni- tion of the retroviral capsid by the postentry RFs we have ex- Small-Angle X-Ray Scattering. To determine low-resolution struc- amined the core self-association module of Trim5α, the N- tures for Fv1 and Trim5α RFs small-angle X-ray scattering terminal dimerization domain of Fv1, and the hybrid RF Fv1Cyp (SAXS) experiments were performed. SAXS data were recorded by X-ray crystallography, small-angle X-ray scattering, electron from samples of Fv1Ntd and Fv1Cyp over a q range of 0.006–0.4 − microscopy, and surface plasmon resonance. These experiments Å 1 and protein concentration of 1–4 mg/mL. Details of data reveal a strong structural similarity between all of the members collection parameters are presented in Table S1. Scattering of this class of RFs and show that they are molecules tailored to curves for Fv1Ntd recorded between 1 and 3 mg/mL were co- recognize the repeat spacing of CA in the retroviral capsid lat- incident across the entire q range and show no sign of aggrega- tice. Moreover, we demonstrate that multivalency, a general tion or any evidence of a structure factor in the very-low-q property of these factors, generates avidity effects that contribute region. Therefore, these data were combined and the merged to specificity and potency of retroviral restriction. scattering curve was used for further analysis (Fig. 1B). Guinier analysis of the merged scattering curve (Fig. 1B, Inset) shows Results good linearity and gives an estimated radius of gyration (Rg)of Fv1 and Trim5α RFs Form Tight Dimers. Size-exclusion chromatog- 43.7 ± 0.6Å (1 SD) for the particle. Evaluation of the forward raphy coupled to multiangle laser light scattering (SEC-MALLS) scatter compared with BSA gives a molecular mass of 46 kDa, was used to determine the oligomeric state of Fv1Ntd (Fv1 consistent with the molecular mass of 43 kDa for the Fv1NtD residues 20–200), Fv1Cyp (Fv1 residues 20–200 fused to CypA), dimer (Table S1). Scattering data collected for Fv1Cyp at >1 and RhT5 (88–296) EK/RD, consisting of residues 88–296 of mg/mL showed a concentration-dependent signal in the low-q Rhesus macaque Trim5α (RhT5) encompassing the B-box, coiled- region and signs of aggregation. Therefore, further analysis was coil, and L2 core self-association region and containing the (E120K/ limited to data collected at 1 mg/mL. Guinier plots of these low- R121D) aggregation suppression mutations (32). Fv1Ntd and concentration data were linear (Fig. 1B, Inset) and gave an es- Fv1Cyp elute from the SEC column as single symmetrical peaks. timated Rg of 55.6 Å and molecular mass 88.3 kDa, consistent

Fig. 1. SAXS analysis of Fv1Ntd, Fv1Cyp, and RhT5 (88–296) EK/RD. (A) SEC-MALLS analysis of Fv1-Ntd, Fv1-CypA, and RhT5 (88–296) EK/RD demonstrates that all proteins are dimers in solution. (B) Concentration-corrected merged SAXS curves. (Insets) Enlargement of the Guinier region and analysis. (C) Normalized pair-distribution functions. (D) Consensus ab initio models.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1402448111 Goldstone et al. Downloaded by guest on September 29, 2021 with an Fv1Cyp dimer. SAXS data were also recorded for RhT5 respectively). Details of data collection, phasing, and model re- − (88–296) EK/RD over a q range of 0–0.2 Å 1 at concentrations finement are presented in Table S2. from 0.4 to 1.54 mg/mL. Guinier analysis gave an estimate of the The structure (Fig. 2A) is dominated by two central 160-Å 30- α α Rg of 56.7 Å. The particle molar mass was derived from the turn antiparallel helices, 2A and 2B, that form a coiled-coil scattering contrast after normalization to the scattering of water structure running the length of the protein. The N-terminal (33), giving a value of 55.4 kDa, also consistent with a dimer. B-box domains are located at either end of the coil and pack against a pair of helices (α3–α4) from the L2 region of the op- Fv1 and Trim5α RFs Are Elongated Molecules. MALLS and Guinier posing monomer. The remainder of the L2 region, residues 260– analysis of scattering profiles from Fv1Ntd, Fv1Cyp, and RhT5 283, forms an extended chain (L2-E) that turns away from the (88–296) EK/RD reveals that these proteins are dimeric and have B-box and runs down the coil toward the twofold axis at the alargeRg compared with the molar mass, suggestive of an center of the dimer. The structure is well-defined for both chains elongated nature. Therefore, to gain insight into the distribution at residues comprising the B-boxes, , α3–α4 regions of of mass within these proteins the pair-distribution function [P(r)] L2, and the T4Ls (Fig. S4) but with differing sections of L2-E was calculated from the scattering data measured for each par- visible in the A and B molecules. The B-box structure is essen- ticle (Fig. 1C). The P(r) function for Fv1Ntd is distinctly bimodal, tially identical to that described for human Trim5α (32), com- containing a primary peak at 26 Å, a secondary peak at 95 Å, and prising a three-stranded antiparallel β-sheet and short α-helix, α1 β –β –α –β a maximum dimension (Dmax) of 145 Å. The real-space Rg de- connected with a 1 2 1 3 topology. Each B-box contains rived from the distribution is 46.7 Å, comparable to that from two zinc atoms coordinated in a tetrahedral fashion by residues the Guinier analysis (Table S1). Similarly, the pair-distribution function for Fv1Cyp is also bimodal with a Dmax at 195 Å and containing a primary peak at 32 Å and a secondary peak at 105 Å. The strong secondary maxima in the pair-distribution functions are characteristic of a dumbbell-shaped molecule where the secondary maxima represent the center-to-center distance of the lobes (34). The pair-distribution function for RhT5 (88–296) EK/RD is also characteristic of an elongated molecule with Dmax at 208 Å. However, the distribution more closely resembles that from a cylindrical particle (34) with a single major peak at 27 Å

and an asymmetrical fall-off to the maximum dimension of 208 Å MICROBIOLOGY that contains shoulders at 75 Å and 150 Å (Fig. 1C). Ab initio modeling of the molecular envelopes of Fv1Ntd and Fv1Cyp was undertaken and produced molecules with extended shapes (Fig. S1). Moreover, application of P2 symmetry in the modeling procedure generated models that best fit the data and have a high degree of convergence (Table S1). Given the twofold symmetry, the consensus models for Fv1NtD and Fv1Cyp are distinctly antiparallel, comprising narrow central regions with globular lobes located at each end of the structure (Fig. 1D). The Fv1Ntd model is 135 Å in length and contains a 60-Å central spacer. The globular end lobes are oriented ∼115° to the central region with a spacing that accounts for the secondary maxima at 95 Å in the P(r) function. Fv1Cyp is substantially larger, encompassing a central region comparable to the Fv1Ntd enve- lope but with additional volume at each globular lobe that locates the CypA domains to each end of the molecule (Fig. S2) and accordingly shifts the secondary maximum in the P(r) function to 105 Å. These data reveal that Fv1NtD and Fv1Cyp are dumbbell-shaped molecules and that the secondary maxima of the P(r) function derive from the peripherally located globular lobes that contain CypA domains in Fv1Cyp. A best-fit ab initio model for RhT5 (88–296) EK/RD was also generated with applied P2 symmetry (Table S1). The consensus model reveals an extended structure ∼210 Å in length comprising a 115-Å central spacer region and two small globular domains apparent at each end of the structure (Fig. 1D). Comparison of the globular domain at each end of the SAXS model with the B- box structures of Rhesus and human Trim5α (32) shows them to be of comparable molecular volume, suggesting that although not as pronounced as the dumbbell structure of the Fv1 factors the Fig. 2. Crystal structure of RhT5-T4L. (A) Cartoon representation of the B-boxes of Trim5 are also arranged in an antiparallel fashion. RhT5-T4L structure with zinc ions shown as spheres. Residues in the L2-E region (between α4 and α5) are missing in chain A (red) and are represented Crystal Structure of RhT5-T4 Lysozyme. To aid crystallization and as a dashed line but are present in the other monomer (chain B, blue). (B) determine the structure of a Trim5α homodimer, the sequence Zn1 is coordinated by C108, D111, H125, and H128 and Zn2 is coordinated by C97, H100, C116, and C119. The B-box of one monomer packs with the α3–α4 for bacteriophage T4 lysozyme (T4L) was fused C-terminally to region of the opposing monomer to form a hydrophobic core. (C)The“top” residue 291 in place of the CypA domains present RhT5Cyp but of the extended antiparallel helices has no distinct hydrophobic groove, still incorporating the 12-aa upstream RhT5Cyp linker sequence whereas the “bottom” has a clear hydrophobic groove (green) that spans (Fig. S3). Crystals of this chimera (RhT5-T4L) belong to the the length of the helices. (D) The L2-E region (orange sticks, labeled every spacegroup C2 and contain one dimer in the asymmetric unit. 10th residue) closely follows the hydrophobic groove on the surface of the The structure was determined using a combination of molecular extended coil. Hydrophobicity of residues was calculated using AAindex replacement and single-wavelength anomalous dispersion and (database code FASG890101) in PyMol where increasing hydrophobicity is the model refined at a resolution of 3.2 Å (R/Rfree of 26%/32%, proportional to the intensity of the green color.

Goldstone et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 H125, H128, C108, and D111 (Zn1) and C116, C119, C97, and H100 (Zn2) (Fig. 2B). The mutated residues, E120K/R121D, that aid solubility are located at the C terminus of α1 and have solvent-exposed side chains. At each end of the dimer, the an- tiparallel arrangement of the α2 helices places the B-box from one monomer in the proximity of the α3–α4 motifs from the opposing monomer. This arrangement facilitates the packing of α3–α4 against the opposing B-box and forms a hydrophobic core around the B-box β-sheet (Fig. 2B). The α2 helices also pack at only a shallow writhe angle. As a result the degree of twist along the entire coiled-coil is only small and gives rise to “top” and “bottom” surfaces. Analysis of the distribution of residue polarity along theses surfaces (Fig. 2C) reveals a top surface with a varie- gated residue distribution. However, the bottom surface contains a channel running the length of the structure lined with hydro- phobic residues that interact with residues in the L2-E region that traverses toward the center of the molecule (Fig. 2D). At the Fig. 3. EM and Biacore analysis of Fv1Cyp. (A) Gallery of negatively stained center of the molecule the sequence that links to the T4L domains EM images of Fv1Cyp. Images are the sums of three different images of the is largely disordered and only a single α5 helix from chain A of the same particle recorded in 0.5-μm steps (Fig. S5A), revealing thin connections structure is visible. This flexibility in the linker region is also ap- between the globular domains. (B) The interaction of Fv1Cyp and monomeric parent with respect to the T4L domains that do not obey the CypA with a HIV-1 CA-p2 lattice immobilized on a Ni-NTA tagged lipid twofold symmetry of the coiled-coil, adopting different orienta- monolayer analyzed by Biacore. Equilibrium affinity measurements derived tions with respect to the rest of the molecule (Fig. 2A). from sensorgram plateau values (Left) show Fv1Cyp exhibits an ∼18-fold higher affinity. Analysis of the kinetics for individual sensorgram binding Electron Microscopy Analysis of Fv1Cyp. To obtain an independent curves gives comparable values for equilibrium dissociation constants (Right). measurement of the shape and dimensions, negatively stained Fv1Cyp particles were analyzed by transmission electron mi- Discussion croscopy. In agreement with the SAXS analysis, the images (Fig. 3A and Fig. S5A) showed elongated particles with globular Recognition of the retroviral capsid protein in the context of the domains separated by greater than 100 Å (Fig. S5B). Thin con- intact capsid lattice is required for restriction by the postentry α nections of density between the globular domains were also ob- RFs Fv1 and Trim5 . Although not related by sequence or ho- served, consistent with a narrow flexible region between the mology both proteins share significant similarities in their mode C-terminal globular features that include the CypA domains. of action and recognition of the retroviral CA. Key features of Fv1 and Trim5α are the formation of dimers through N-terminal Surface Plasmon Resonance Analysis of Fv1Cyp–CA Interactions. coiled-coil regions and C-terminal specificity domains that de- Given the arrangement of the CypA recognition domains within termine the repertoire of viruses that can be restricted, B30.2 in Fv1Cyp the effect of dimerization on recognition and affinity for Trim5α (12, 13) and the Fv1CtD of Fv1n and Fv1b (6). In ad- the capsid lattice was investigated using Biacore surface plasmon dition, the Trim5α N-terminal region also contains RING and resonance (SPR). To do this a lattice of HIV-1 capsid was first B-box domains that promote higher-order associations required constructed on the surface of hydrophobic Biacore chip. We have for recognition and stable binding of the retroviral capsid (37). previously shown that the capsid protein from MLV forms a It is likely that Fv1 also contains one or more association- capsid-like hexagonal array when immobilized on lipid nanotubes promoting regions but located in the CtD (7). To understand how by a C-terminal his-tag (27). Repeating these experiments with the N-terminal regions of Fv1 and Trim5α contribute to recogni- the CA of HIV-1 demonstrated that a construct containing CA tion of the retroviral capsid we examined the size and shape of the followed by the P2 spacer peptide (CA-P2) also coated lipid self-association domains by SAXS and determined the structure of nanotubes and produced regular arrays (Fig. S6). To test binding the N-terminal B-box and coil region from Trim5α. These data of Fv1Cyp to an intact capsid lattice we reproduced this lattice by clearly show that the N-terminal domains of Fv1 and Trim5α com- doping 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes with prise antiparallel coiled-coil proteins that form elongated di- 30% 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)imi- mers with dimensions sufficient to span the interhexamer distan- nodiacetic acid)succinyl] (DGS-NTA) to create a Ni-NTA con- ces on the outer surface of the retroviral capsid. taining lipid layer on a hydrophobic Biacore SPR HPA chip. In the CypA-containing factors, Fv1Cyp and TrimCyp, the in- HIV-1 CA-P2 was then loaded onto the surface saturating the teraction with the HIV-1 capsid is mediated although binding of + Ni2 sites on the lipid monolayer, producing a stable baseline. To CypA domains to residues in the CA-Cyp loops displayed on the evaluate our approach we first tested the binding of free mono- outer surface of the capsid (17, 38). Moreover, mutations in the meric CypA. To account for potential nonspecific interaction with HIV-Cyp loop that diminish or abolish CypA binding restore cell the lipid layer and SPR chip a second channel containing B-tropic permissiveness by making HIV-1 resistant to restriction by the MLV was included as a non-CypA binding control. Analysis of CypA-containing factors (39). Our data reveal the Fv1Cyp mol- equilibrium binding curves from CypA results in a dissociation ecule contains two globular CypA-containing domains around constant of 11.9 μM (Fig. 3B), in good agreement with the 150 Å apart separated by a narrow spacer observed both directly reported affinity in solution from previous experiments (35, 36). in negative-stain EM experiments (Fig. 3A) and as the 2° maxima The experiment was then repeated with Fv1Cyp over a concen- in the distance distribution function derived from SAXS data tration range of 75 nM–10 μM. Analysis of these equilibrium (Fig. 1C). Calculation of the distribution of interatomic vectors binding curves gave an equilibrium dissociation constant of 0.68 between pairs of residues residing in HIV-1 Cyp loops across the μM. As expected for a weak interaction, the CypA-binding ki- surface of the reconstructed HIV CA shell (38) reveals that pair netics were rapid and could not be analyzed reliably. However, distances of 130–170 Å are highly represented (Fig. 4A). This kinetic analysis of the Fv1-Cyp binding curves yielded an on rate striking correlation between the lobe spacing in Fv1Cyp and the − − − of 0.097 μM 1·s 1 and off rate of 0.069 s 1 for the 1.25 μM in- spacing of Cyp loops in the HIV-1 capsid suggests a model where α jection of Fv1Cyp, resulting in a Kd of 0.71 μM (Fig. 3B). These the extended Fv1 and Trim5 dimers have evolved to facilitate data reveal a dimerization-conferred avidity where dimerization the spacing of specificity domains to optimize their interaction and spacing of CypA monomers by the Fv1NtD results in a 17.5- with many distal binding sites on the retroviral CA. In this model, fold increase in apparent affinity of CypA for an HIV-1 CA array. an Fv1Cyp dimer with a CypA domain located on one CA is able

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1402448111 Goldstone et al. Downloaded by guest on September 29, 2021 to sample greater than 30 other Cyp loops located in adjacent or more distal CA hexamers at ∼150 Å spacing to make a pro- ductive interaction (Fig. 4B). This notion is further supported by our Biacore analysis of the Fv1Cyp–CA lattice interaction that reveals strong avidity effects likely resulting from the many ways Fv1Cyp can simultaneously associate with pairs of Cyp-loop binding sites on the CA lattice. Moreover, it has been demon- strated that TrimCyp efficiently inhibits HIV-1 even when only 25% of the CA in the capsid is competent to bind to CypA (40), suggesting that only one to two Cyp-loop binding sites per hex- amer are actually required for a productive RF engagement. The SAXS analysis of RhT5 (88–296) EK/RD and crystal structure of the RhT5-T4L demonstrates that, like Fv1, Trim5α also comprises an antiparallel extended dimer but with the B-box effector domains displayed at either end. Examination of the B-box spacing gives a distance of 172 Å (distance between the equivalent Zn atoms). Notably, this is equivalent to one edge of the Trim5α hexagonal lattice observed in previous EM studies and proposed to contain four Trim5α molecules arranged as two pairs of parallel dimers (37). Our data now suggest an alternative model (Fig. 4C) that places a single antiparallel Trim5α dimer on each hexagonedgewithB-box–(α3–α4) and RING domains located at each threefold vertex and B30.2 domains connected by L2-E regions located toward the dimer twofold axis. Given that the 170-Å length constraint is already accommodated by the B-box and coil regions, the model predicts the B30.2 and RING domains would locate outside the plane of the assembly, plausibly with the B30.2 domain below facing toward the capsid lattice and RING

domain above to be accessible to components of the ubiquitina- MICROBIOLOGY tion machinery. This model is supported by the recently published structure of the coiled-coil domain from the related protein Trim25 (41). This structure shows the same elongated antiparallel assembly of two central helices with low writhe, and the similarity with the Trim5α structure now suggests antiparallel flattened coil structures are likely a general feature of the whole Trim family. However, in Trim25 structure the B-boxes are absent, so it is unclear whether the packing we observe between B-box and op- posing (α3–α4) regions is also found in other Trims or is specific to Trim5α to support a B-box conformation required for Trim5α higher-order assembly and capsid recognition. – At the present time we can only speculate about the posi- Fig. 4. RF CA lattice interactions. (A) A layer of CA lattice taken from 3J34. α Red spheres mark the Cyp loops and the gray region highlights those that tioning of the CA binding domains of Trim5 and Fv1. However, are spaced at 130–170 Å from the central Cyp loop (yellow) and accessible to it seems reasonable to hypothesize that the Fv1 C-terminal bind the second CypA of Fv1Cyp. (B) The SAXS envelope of Fv1Cyp dimer is domains would locate similarly to the CypAs in Fv1Cyp. In this positioned with one CypA domain located on the Cyp loop of a central case as well as recognition of the MLV capsid further Fv1-CtD hexamer and the second spaced between 140–150 Å located on the Cyp loop self-association could mediate higher-order assembly similar to of an adjacent CA hexamer. (C) A schematic representation of the HIV-CA the B-boxes in Trim5α in Fig. 4C. In contrast, given the location lattice (green) with the Trim5-21R lattice, from ref. 37, is overlaid as dashed of the Trim5α L2-E C terminus in the crystal structure and the lines (black). The SAXS model for the RhT5 (88–296) EK/RD dimer shown in electron density observed at the center of the coil in the Trim5α orange surface representation has dimensions equating to the length of hexagonal assembly (37) it is likely the B30.2 domains locate a single edge of the hexamer in the Trim5-21R lattice and sufficient to span close to the twofold axis of the coiled-coil. In support of this adjacent hexamers in the CA lattice. The B-boxes are shown as cyan circles at notion, alignment of the Trim5α B30.2 structure (PDB ID code the interface between Trim5α dimers and the B30.2 domains are repre- 43BN) with equivalent residues in the short helix α5 of Trim5α sented by the ovals toward the Trim5-21R twofold axis. BCC places the SPRY domain at the twofold axis of the coiled- coil and orients the variable capsid recognition loops in an ap- propriate position to recognize the retroviral capsid (Fig. S7). orientation for specificity domains to sample and promote a In TrimCyp factors, there are additional residues resulting productive interaction with the CA lattice and for RING and from CypA fusion into helix α5 (18). These form an unstructured B-box effector domains to recruit the components of ubiquiti- linker between α5 and T4L in the Rh5-T4L structure. Modeling nation and immune innate signaling pathways. Notably, com- of TrimCyp by replacement of the T4L with CypA (Fig. S7) then parison of the structure, evolution, and mode of action of positions the CypA domains around 120 Å apart and given the Trim5α and the antiviral proteins MxA/MxB (42) also reveals a local disorder around α5 affords them a large degree of con- number of common design features. These include (i) a virus formational flexibility. In this way, TrimCyp would have the ca- interaction domain showing signatures of positive selection, (ii) pacity to sample multiple hexamers, as seen in Fv1Cyp, for a one or more multimerization domains, and (iii) an effector do- productive interaction with the HIV-1 capsid lattice. main. Together they allow recognition and restriction of multiple In summary, recognition of the retroviral capsid by Trim5α agents with repeated structures characteristic of viruses and may and Fv1 factors is mediated by a combination of avidity gener- describe the properties of multiple IFN-stimulated . ated by primary dimerization/higher-order association and the weak intrinsic CA-binding site affinity of individual B30.2 or Materials and Methods CypA domains. The elongated antiparallel structure of these Protein Expression and Purification. Fv1Ntd (residues 20–200 of Fv1) and factors plays a key role in providing the optimal spacing and Fv1Cyp consisting of Fv1-Ntd fused to CypA were expressed with N-terminal

Goldstone et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 His-tags in Escherichia coli BL21(DE3). HIV CA-P2 and MLV CA were expressed preparation, experimental procedures, data processing, and analysis are with C-terminal His-tags. Proteins were purified by immobilized nickel af- provided in SI Materials and Methods. finity and SEC. CypA was expressed as a GST fusion in E. coli BL21(DE3) and purified by glutathione affinity and SEC. RhT5 (88–296) EK/RD and Rh5-T4L Crystallization and Structure Determination. Crystals of Rh5-T4L were grown were expressed from pET47b with an N-terminal His-tags and purified by by vapor diffusion at 18 °C in drops consisting of 1 μL of Rh5-T4L at a con- immobilized nickel affinity chromatography and SEC. For structure de- centration of 3 mg/mL and 0.5 μL of reservoir solution (10% PEG8000, 20% termination, selenium was incorporated into Rh5-T4L by supplementing ethylene glycol, and 0.1 M bicine/Tris·HCl, pH 8.5). X-ray diffraction data culture media with seleno-methionine combined with inhibition of methi- were collected on Australian Synchrotron beamline MX2 at a wavelength onine biosynthesis. Details of protein expression constructs and protein of 0.9793 Å. Details of data processing, structure determination, and re- production procedures are provided in SI Materials and Methods. finement are provided SI Materials and Methods.

SEC-MALLS. SEC-MALLS was used to determine the solution molecular weight Negative-Stain Electron Microscopy. Samples were absorbed to carbon-coated – of Fv1NtD, Fv1Cyp, and RhT5 (88 296) EK/RD and to assess protein hetero- grids and negatively stained with 1% sodium silicotungstate, pH 7.0. The grids geneity. Data for Fv1NtD and Fv1Cyp were recorded using a Superdex 200 were viewed with an FEI Spirit TWIN microscope operated at 120 kV with 10/300 GL column mounted on a Jasco HPLC. Scattered light intensities and a tungsten filament source. Images were recorded on an Eagle 2K camera protein concentration data were measured using a DAWN HELEOS laser (FEI) at a magnification of 52K (4.3Å per pixel) using a range of defocuses. photometer and an OPTILAB-rEX differential refractometer (Wyatt Instru- ments). SEC-MALLS analysis of RhT5 (88–296) EK/RD was carried out using SPR. Binding of Fv1Cyp and CypA to a preformed HIV-1 CA lattice was per- Superdex 200 10/300 GL column, on a Dionex HPLC with a PSS SLD7000 7 formed using SPR. Data were recorded on a Biacore 2000 instrument using angle MALLS detector and Shodex RI-101 differential refractive index de- the hydrophobic HPA biosensor (GE Healthcare). Details of sample and chip tector. Details of experimental procedures and data analysis are provided in SI Materials and Methods. preparation, experimental procedure, and data analysis are provided in SI Materials and Methods. SAXS. SAXS data for Fv1NtD and Fv1Cyp were recorded on European Syn- chrotron Radiation Facility beamline 14-3 on a PILATUS 1M detector at a ACKNOWLEDGMENTS. We gratefully acknowledge the European Synchro- tron Radiation Facility (Grant MX1153) and the New Zealand synchrotron wavelength of 0.931Å and camera length of 2.43 m covering a momentum group for access to small-angle X-ray scattering beamlines. This work was < < −1 = π θ λ transfer of 0.006 q 0.6Å [q 4 sin( )/ ]. Data for RhT5 (88-296) EK/RD supported by UK Medical Research Council Grants U117565647 (to I.A.T.), were collected at the Australian Synchrotron SAXS/WAXS beamline at a U117581334 (to P.B.R.), and U117512710 (to J.P.S.) and by a Rutherford Dis- wavelength of 1.13 Å with a camera length of 3 m covering a momentum covery Fellowship from the New Zealand government administered by the − transfer range of 0.0 < q < 0.3Å 1 [q = 4πsin(θ)/λ]. Details of sample Royal Society of New Zealand (to D.C.G.).

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