Rhesus TRIM5α Disrupts the HIV-1 Capsid at the InterHexamer Interfaces

TRIM proteins play important roles in the innate immune defense against retroviral infection, including human immunodeficiency virus type-1 (HIV-1). Rhesus macaque TRIM5α (TRIM5αrh) targets the HIV-1 capsid and blocks infection at an early post-entry stage, prior to reverse transcription. Studies have shown that binding of TRIM5α to the assembled capsid is essential for restriction and requires the coiled-coil and B30.2/SPRY domains, but the molecular mechanism of restriction is not fully understood. In this study, we investigated, by cryoEM combined with mutagenesis and chemical cross-linking, the direct interactions between HIV-1 capsid protein (CA) assemblies and purified TRIM5αrh containing coiled-coil and SPRY domains (CC-SPRYrh). Concentration-dependent binding of CC-SPRYrh to CA assemblies was observed, while under equivalent conditions the human protein did not bind. Importantly, CC-SPRYrh, but not its human counterpart, disrupted CA tubes in a non-random fashion, releasing fragments of protofilaments consisting of CA hexamers without dissociation into monomers. Furthermore, such structural destruction was prevented by inter-hexamer crosslinking using P207C/T216C mutant CA with disulfide bonds at the CTD-CTD trimer interface of capsid assemblies, but not by intra-hexamer crosslinking via A14C/E45C at the NTD-NTD interface. The same disruption effect by TRIM5αrh on the inter-hexamer interfaces also occurred with purified intact HIV-1 cores. These results provide insights concerning how TRIM5α disrupts the virion core and demonstrate that structural damage of the viral capsid by TRIM5α is likely one of the important components of the mechanism of TRIM5α-mediated HIV-1 restriction.


Introduction
TRIM5a is an important component of the innate immune defense against retroviral infection, including human immunodeficiency virus type -1 (HIV-1) [1,2], and numerous studies suggest that TRIM5a interacts with assembled capsids and induces premature capsid disassembly (uncoating), before reverse transcription takes place [3][4][5][6]. TRIM5a is a 56 kD protein comprising a tripartite motif (TRIM; with RING, B-box 2, and coiled-coil (CC) domains) followed by a C-terminal B30.2/SPRY domain [7][8][9]. Each of these domains plays distinct roles in the antiviral function of TRIM5a. The B30.2/SPRY domain binds to the viral capsid and determines the specificity of restriction, with sequence variation within this domain greatly impacting binding specificity [6,[10][11][12][13][14][15][16]. For example, a single amino acid change in human TRIM5a (TRIM5a hu ), R332P, renders the protein capable of binding the HIV-1 capsid, causing it to behave like rhesus TRIM5a (TRIM5a rh ) with regard to HIV-1 restriction [11,17]. The CC domain is necessary and sufficient for TRIM5a homo-dimerization, and this is important for capsid binding and restriction [12,[18][19][20]. In vitro, specific recognition and binding to a hexagonal CA lattice requires both the CC and SPRY domains [19]. The B-box 2 domain is thought to be involved in higherorder structure formation and self-association, and its presence in the protein enhances TRIM5a binding to the capsid, compared to the CC-SPRY domains alone [21,22]. Several mutations in the Bbox 2 domain abrogate HIV-1 restriction by TRIM5a rh [22][23][24]. The N-terminal RING domain is the least explored domain of TRIM5a. In general, RING domains are components of a particular class of E3 ubiquitin ligases that are involved in proteasome-mediated protein degradation (reviewed in [25]). TRIM5a exhibits E3 activity, but the role of the ubiquitin ligase activity in retrovirus restriction is unclear. Deletion of the Nterminal RING domain reduces, but does not abolish antiviral restriction [23,26], and treatment of cells with proteasome inhibitors does not prevent restriction by TRIM5a [27]. However, proteasome activity is necessary for the TRIM5a-mediated block to reverse transcription [27], and engagement of restrictionsensitive virus cores results in proteasome-dependent degradation of TRIM5a [28]. Together, these data suggest that TRIM5a action in host restriction of retroviruses involves all of its domains.
The negative influence of TRIM5a on viral reverse transcription is well established [1,3,4,6,29,30], however, the detailed mechanism of restriction has not been elucidated. TRIM5a binds to assembled complexes composed of the CA-NC region of Pr55 gag , but does not significantly interact with monomeric or soluble CA protein [31]. Furthermore, mutations in CA that decrease capsid stability appear to reduce TRIM5a binding in target cells, as HIV-1 particles with unstable cores are less effective at saturating TRIM5a-mediated restriction [5]. Finally, recent studies using a recombinant TRIM5a rh chimera, containing the RING domain of TRIM21, demonstrated that the hybrid protein binds to CA-NC tubular assemblies and causes shortening of the tubes [32,33], or selfassembles into higher-order structures, enhanced by binding to a preformed CA-NC hexagonal template [34].
Here, we employed cryoEM to investigate the direct interactions of tubular HIV-1 capsid assemblies and purified HIV-1 cores with the TRIM5a rh CC-SPRY protein and the structural consequences of TRIM5a rh CC-SPRY binding. We demonstrate that TRIM5a rh binding disrupts the tubes and creates nonrandom fragments. Specific inter-hexamer interfaces are preferentially broken, resulting in strings of subunits that are held together by the CA-CTD dimer. We further demonstrate that disruption by TRIM5a rh of purified HIV-1 cores also occurred preferentially at the inter-hexamer interfaces. Our data suggest that TRIM5a rh -mediated HIV-1 restriction involves direct engagement of the viral capsid, and structural damage to the capsid is likely one of the key components in this event.

Results and Discussion
Expression, purification, and biophysical characterization of recombinant TRIM5a CC-SPRY To investigate the direct interactions between TRIM5a rh and the HIV-1 capsid, we generated purified recombinant proteins.
Full length, wild-type TRIM5a rh has been quite difficult to obtain in sufficient quantities for biophysical and structural studies [32,35]. Therefore, we tested the expression and solubility of a number of different TRIM5a rh constructs, including one that comprises the CC-SPRY portion, by performing transient expression in SF9 insect cells. TRIM5a rh CC-SPRY (residues 134-497) and TRIM5a hu CC-SPRY (residues of 132-493) exhibited sufficient protein levels and solubility and, therefore, were selected for production in SF21 insect cells, using recombinant baculoviruses. The quaternary state of the purified recombinant human and rhesus TRIM5a CC-SPRY proteins was assessed by size exclusion chromatography in conjunction with inline multi-angle light scattering, confirming that these proteins were dimers. The observed molecular masses extracted from the light scattering analyses are 92 kDa and 89 kDa, respectively (Fig. 1A), compared to the theoretical values of 46.1 kDa and 45.6 kDa, respectively, based on amino acid sequences. Both proteins gave rise to almost identical CD spectra with a predominantly a-helical signature (Fig. 1B).
TRIM5a rh CC-SPRY binds to HIV-1 CA and CA-NC tubular assemblies in a dose-dependent manner It is widely accepted that the restriction specificity of TRIM5a resides in its SPRY domain and that this domain interacts with retroviral capsids [1,3,11,14,15,36]. However, only recently has direct binding been demonstrated for a TRIM5-21R fusion chimera with CA-NC assemblies [32,34]. We used recombinant TRIM5a CC-SPRY proteins to examine direct binding to CA and CA-NC assemblies. Incubation of preassembled HIV-1 CA or CA-NC tubes with TRIM5a rh resulted in co-sedimentation of TRIM5a rh CC-SPRY/CA or TRIM5a rh CC-SPRY/CA-NC complexes, respectively, in the pelleted fractions (Fig. 2, Fig. S1). More TRIM5a rh was observed bound to CA assemblies than to CA-NC assemblies (Figs. 2 & 3). In contrast, we observed negligible binding of TRIM5a hu CC-SPRY to HIV-1 CA or CA-NC complexes under the same assay conditions (Fig. 2). These data are consistent with previous results that demonstrated the inability of TRIM5a hu to bind and restrict HIV-1, but a capacity for the same protein to recognize N-tropic murine leukemia virus (MLV) capsid [3,4,12].

Author Summary
The cellular protein TRIM5a is a host cell restriction factor that blocks HIV-1 infection in Rhesus macaque cells by targeting the viral capsid. Here, we show that direct binding of a TRIM5a protein, consisting of the coiled-coil and B30.2/SPRY domains, to the viral capsid results in disruption of the surface lattice and fragmentation of the capsid, specifically at inter-hexamer interfaces. Our results reinforce the notion that structural damage of the viral capsid by TRIM5a is central to the mechanism of TRIM5amediated HIV-1 restriction. A more quantitative analysis of TRIM5a rh binding was carried out by measuring molar ratios of CA and CA-NC-bound TRIM5a rh CC-SPRY over a range of TRIM5a rh concentrations. Dosedependent binding was observed for both CA and CA-NC assemblies (Fig. 3). Consistently, at all concentrations, TRIM5a rh CC-SPRY bound CA more efficiently than CA-NC. This could be due to differences in CA and CA-NC structures on the surfaces of the assemblies, or differences in the flexibility of these assemblies, as CA-NC tubes were assembled in the presence of oligonucleotide. The binding ratios were 0.41 for TRIM5a rh CC-SPRY/CA and 0.21 for TRIM5a rh CC-SPRY/CA-NC, respectively, for the highest concentration of TRIM5a rh CC-SPRY (18 mM). When a lower concentration of TRIM5a rh CC-SPRY (1 mM) was used for binding to the CA-NC tubular assemblies (10 mM), a molar ratio of 0.034 was obtained. This ratio is somewhat lower than the value reported by Langelier et al. for TRIM5-21R by immunoblotting [32]. The lower binding ratio for TRIM5a rh CC-SPRY is expected, since it lacks the self-associating B-box 2 domain, compared to the TRIM5-21R fusion protein. Furthermore, incubation with CC-SPRY rh did not alter the fraction of pelletable CA and CA-NC, even at the highest TRIM5a rh CC-SPRY concentrations (Figs. 2&3). These results are in accord with those reported for TRIM5-21R [32] and a binding study with CA-NC assemblies using TRIM5a rh -containing lysates [37]. Taken together, the data indicate that dimeric TRIM5a rh CC-SPRY directly interacts with tubular CA and CA-NC assemblies and that binding of TRIM5a rh does not dissociate these assemblies into soluble monomeric CA protein.
Binding of TRIM5a rh CC-SPRY to tubular CA assemblies releases discrete, linear fragments Although no dramatic effect of purified TRIM5a rh on uncoating has been observed in vitro using CA-NC assemblies [32], a substantial decrease in intact CA-NC tubes was noted when TRIM5a rhcontaining cell lysates were mixed with CA-NC tubular assemblies [37]. To investigate this apparent dichotomy, we carried out cryoEM structural analyses of the samples that were used in the TRIM5a CC-SPRY/CA tubular assembly binding assays (Figs. 2A&3A). CryoEM micrographs showed well-ordered CA tubular structures after incubation with binding buffer only (Fig. 4A) or TRIM5a hu CC-SPRY (Fig. 4B), similar to our previously described assemblies [38]. In contrast, incubation of CA tubular assemblies with TRIM5a rh CC-SPRY (18 mM) resulted in a massive structural break-down of the tubes (Fig. 4C), accompanied by the appearance of distinct fragments composed of strings of hexamers (Fig. 4C inset) [38]. The remaining tubes had generally lost the regularity of the hexagonal lattice. Some TRIM5a rh CC-SPRY densities apparently remained on several of the fragments (Fig. 4C inset). Gold-labeling of TRIM5a rh CC-SPRY in complex with CA tubular assemblies confirmed that TRIM5a rh CC-SPRY bound to the CA assemblies (Fig. S2). These break-down fragments were primarily present in the pellet fraction after centrifugation (Fig. 3A), confirmed by cryoEM imaging of the pellet samples ( Fig. S3), explaining why no effect on uncoating was detected in assays that measure soluble CA [32,37]. These results suggest that the predominant effect of TRIM5a rh is the break down of HIV-1 capsids into fragments and not the dissociation into soluble monomers.
We further examined the effect of CA mutations on TRIM5a rh disruption. Several CA mutants, including A92E, which was used in our previous structural study [38], and the E45A mutant, which produces hyperstable capsids, were analyzed. The effect of TRIM5a rh CC-SPRY binding to A92E CA tubular assemblies was similar to that observed with wild-type CA (Fig. S4A&B). The CA tubular assemblies carrying the capsid-stabilizing E45A mutation [46] also experienced structural damage by TRIM5a rh , but to a lesser degree (Fig. S4C&D). This suggests that the overall stability of HIV-1 capsid assemblies may modulate or interfere with TRIM5a rh function, consistent with findings that hyperstable capsid core mutants effectively saturate TRIM5a-mediated restriction [5].

Cross-linking of the inter-hexamer CA interface prevents TRIM5a rh disruption
To determine which interface in the capsid lattice is disrupted by CC-SPRY rh , we tested the effect of TRIM5a rh CC-SPRY on cross-linked CA tubular assemblies. In previous work, we showed  PLoS Pathogens | www.plospathogens.org that introduction of a pair of cysteines, P207C/T216C, at the pseudo three-fold inter-hexamer interface, efficiently cross-linked three neighboring CA molecules into trimers upon oxidation (Fig. 5A&B). The interactions at this interface are mediated by the CA-CTD, predominantly helices H10 and H11 [38]. Such crosslinked P207C/T216C CA tubular assemblies are expected to contain stronger hexamer-hexamer interactions, stabilizing the lattice. The P207C/T216C mutant assembles into tubular structures very similar to the wild-type CA (Fig. S5). Both oxidized and non-oxidized P207C/T216C CA tubular assemblies bound TRIM5a rh CC-SPRY, without any significant difference between them (Fig. 5B, lanes 1-4). However, cryoEM analysis revealed that TRIM5a rh CC-SPRY exerted very little structural damage onto the cross-linked tubes, whereas the non-oxidized tubular assemblies exhibited similar structural breakdown as seen for wild type CA tubes (Fig. 5C&D). These data suggest that TRIM5a rh CC-SPRY engages in inter-hexamer binding, most likely pulling apart the trimer interface, thereby disrupting the assembled tubes. We further tested this possibility by measuring the cross-linking efficiency of P207C/T216C CA assembly after TRIM5a rh CC-SPRY treatment. As can be seen from the results illustrated in Fig. 5B (lanes 5&6), the level of cross-linked trimers was significantly reduced after incubation with TRIM5a rh CC-SPRY. The percentage of the cross-linked CA trimer over total CA in the reduced sample is 3-fold less in the TRIM5a rh CC-SPRY treated sample, compared to untreated sample, confirming that the trimer interface between three neighboring hexamers is disrupted by TRIM5a rh CC-SPRY.
An alternative scenario could involve binding of the TRIM5a CC-SPRY dimer within a CA hexamer, with TRIM5a rh CC- SPRY dimers pushing apart the hexamers. However, simple geometric considerations make this a very unlikely scenario if TRIM5a rh SPRY binds near the cyclophilin A binding loop in CA [39], since the distance between two sites (.110 Å ) is too large for the TRIM5a CC-SPRY dimer protein to span. Nonetheless, we tested for this possibility using a A14C/E45C CA double cysteine mutant, which can cross-link CA within hexamers [40]. Following incubation with TRIM5a rh CC-SPRY, crosslinked A14C/E45C CA assemblies exhibited only a slight reduction in CA hexamers (Fig. S6, compare lanes 2 & 5), compared to the dramatic reduction of the trimer in the P207C/T216C CA assemblies (Fig. 5B, right  panel). This small effect on the CA hexamer could be caused by minor perturbations at the intra-hexamer CA interfaces upon TRIM5a rh CC-SPRY binding. Small amounts of CA dimer (,50kD, Fig. S6, lanes 1, 3, 5, 7&9) in the non-oxidized assemblies and dimer of hexamers (,280kDa, Fig. S6, lanes 2&8) in the oxidized A14C/E45C CA assemblies were observed by SDS-PAGE, possibly due to the CA CTD dimer interaction. Interestingly, the amount of hexamer dimers was greatly diminished in the TRIM5a rh CC-SPRY treated sample (Fig. S6, lane 5 compared to lane 2&8). Again, these data further support that TRIM5a rh CC-SPRY binding perturbs the CA inter-hexamer interface.

TRIM5a rh disrupts isolated HIV-1 cores similar to the in vitro capsid assemblies
To extend the above in vitro studies to biological HIV-1 capsids, we examined the effect of TRIM5a rh CC-SPRY on isolated HIV-1 cores. For this purpose, we purified cores from the HIV-1 CA mutants A14C/E45C and P207C/T216C for two reasons; first, the mutant cores appeared to be more stable through the isolation procedure, and second, A14C/E45C and P207C/T216C cores bear the same cysteine mutations that we used for the in vitro analysis described in the previous section. A14C/E45C and P207C/T216C cores were isolated from virions in high yield (average of 44% of virion-associated CA, vs. ,15% typically observed for wild type) by brief detergent treatment and sucrose gradient sedimentation. The CA protein in A14C/E45C cores was readily cross-linked into hexamers, as shown by non-reducing SDS-PAGE analysis (Fig. S7). Despite the extensive CA hexameric crosslinking in A14C/E45C cores, incubation with TRIM5a rh CC-SPRY resulted in a dramatic loss of intact cores observed by cryoEM, compared to the samples treated with the same amount of human TRIM5a CC-SPRY ( Fig. 6A-C). In contrast, no significant reduction in the number of P207C/T216C cross-linked cores was seen upon TRIM5a rh incubation ( Fig. 6E and F, Fig. S7, +oxidizer samples). However, without ensuring effective crosslinking at the trimer interface (Fig. S7, -oxidizer), a four-fold decrease in the number of P207C/T216C cores was seen upon TRIM5a rh treatment, compared to incubation with TRIM5a hu (Fig. 6D and F, -oxidizer samples). Although very few, a small number of P207C/T216C cores were observed in TRIM5a rh treated samples, presumably due to low levels of spontaneous crosslinking of isolated P207C/T216C cores at the trimer interface. Furthermore, similar protofilament fragments as seen for the in vitro assemblies were also observed after TRIM5a rh treatment of cores (Fig. 6D, arrows and inset). The above data demonstrate, for the first time, that TRIM5a rh CC-SPRY is capable of exerting direct structural damage on the isolated HIV-1 cores and TRIM5a rh binding preferentially disrupts the interhexamer interfaces in the HIV-1 capsid.

A model for TRIM5a rh action on HIV-1 capsid
Examination of the fragments present in the cryoEM images revealed predominantly curved linear structures (Fig. 4C). These structures resemble fragments of protofilaments in CA helical assemblies. Our results are consistent with previous studies that TRIM5a rh binding to CA-NC assemblies did not increase soluble CA-NC monomers and dimers [32,37], and further suggest that binding of TRIM5a rh disrupts the hexamer-hexamer interfaces, thereby releasing protofilaments along one of the three principal helical directions. A model based on the above findings is depicted schematically in Figure 7. CA assembles into helical tubes in vitro with a hexagonal surface unit formed by CA NTDs that is connected by CTD-CTD dimer and trimer interfaces on the inner surfaces of the three-dimensional tube or cone [38,40,41]. In these helical tubes, three slightly different inter-hexamer interactions were observed (see Fig. 3 in [38]). Binding of TRIM5a rh may disrupt these interactions differentially, weakening the CTD-CTD interfaces between hexamers. In turn, this causes release of CA protofilament fragments, such as those illustrated in Figure 7, and, indeed, similar types of fragments were observed in the cryoEM images (Fig. 4C). For TRIM5-21R interacting with CA-NC, shortening of tubes was observed in vitro, in addition to fragmentation [32]. Examples of this type of fragmentation of helical tubes have also been observed in other biological systems, including microtubules in vivo and in vitro [42,43], actin filaments [44] and dynamin spirals and tubes [45]. Thus, the disassembly of the CA tubes into helical-type fragments is not unprecedented. Importantly, the use of two mutants, A14C/E45C and P207C/ T216C, containing engineered disulfide bonds, allowed us to assign the site of TRIM5a rh action to the inter-hexamer interface (vs. the intra-hexamer interface), both, for in vitro assemblies and isolated HIV-1 cores, providing compelling evidence for specific structural disruption of the trimer interface of the HIV-1 capsid upon TRIM5a binding. In this manner, key insights into the mechanistic aspects of TRIM5a rh -capsid interaction were obtained.
Retroviral uncoating is a poorly characterized process, generally defined as viral capsid disassembly following release of the viral core into the target cell. Studies using HIV-1 CA mutants indicate that the stability of the viral core is optimally balanced for efficient viral replication [46]. Therefore, a plausible mechanism for restriction by TRIM5a involves binding to the viral capsid, capsid destabilization, and perturbation of uncoating. Here, we show by cryoEM that TRIM5a rh CC-SPRY binding to CA assemblies causes massive destruction of assembled HIV-1 CA complexes. A similar effect was observed with purified HIV-1 cores. Intriguingly, this effect was seen with the TRIM5a rh CC-SPRY domain construct lacking the RING and B-box domains, albeit at high concentrations, even though TRIM5a protein devoid of RING and B-box domains was reported to lack restriction activity when expressed in cells [23,24]. These seemingly inconsistent results could be due to several factors, including: (1) reduced binding to the viral capsid in the cell due to lack of self-association mediated by the B-box that can be overcome at high protein concentration in vitro; (2) improper intracellular localization of the deletion protein; or (3) altered association with host cell factors. We favor the first explanation, since the CC-CypA protein has been shown to restrict HIV-1 and FIV when expressed in target cells [47], and oligomerization of CypA appears sufficient to induce HIV-1 restriction [48]. Given the ability of the B-box domain to promote higher-order TRIM5a association [21], it seems plausible that this domain in intact TRIM5a rh may potentiate the effects observed here for TRIM5a rh CC-SPRY. Most importantly, while the CC-SPRY from rhesus TRIM5a was active on our in vitro assemblies and isolated cores, the corresponding human TRIM5a fragment was inactive. Thus, binding of the CC-SPRY domain to CA is essential for TRIM5a retroviral restriction and for structural disruption of the capsid. However, our current results do not exclude the possibility of additional structural consequences induced by higher-ordered oligomerization of TRIM5a on the viral capsid.
Although the molecular mechanism of TRIM5a restriction is not fully understood, current models hypothesize that after capsid release into the target cell, TRIM5a binds and triggers premature capsid disassembly. Our results suggest that direct binding of TRIM5a to the capsid is sufficient to inflict direct structural damage. Yet, cellular proteasome activity is clearly involved in the block to reverse transcription induced by TRIM5a [27]. Recruitment of proteasomes, most likely via the TRIM5a RING domain, may further disaggregate capsid fragments and also degrade TRIM5a [28], thereby mediating the irreversible block to infection. In contrast to TRIM5a-mediated restriction, Fv1 restriction of MLV does not result in inhibition of reverse transcription, yet both TRIM5a and Fv1 target the retroviral capsid. We speculate that the common feature in TRIM5a and Fv1 restriction is the structural damage to the capsid, with the major mechanistic difference involving recruitment of the proteasome in the case of TRIM5a-dependent restriction.
The findings presented here represent the first detailed structural analysis of TRIM5a disruption of the CA lattice to date. Additional structural studies of TRIM5a effects, especially with regard to the CTD-CTD interfaces in CA assemblies and HIV-1 cores, as well as the involvement of the RING and B-box domains, will further aid to elucidate the molecular mechanisms of TRIM5-mediated HIV-1 restriction and may offer insights into the HIV-1 virus-cellular interplay as well as lead to novel approaches in antiviral therapy.

Protein expression and purification
cDNAs encoding the coiled-coil and SPRY domains of human and rhesus TRIM5a (TRIM5a CC-SPRY; residues 132-493 and 134-497, respectively) were amplified and cloned into the pENT-TOPO vectors (Invitrogen), modified to encode a Strep-tag at the N-terminus and a His 6 -tag at the C-terminus of the proteins. The cDNAs encoding HIV-1 capsid (CA) and capsid-nucleocapsid (CA-NC) were amplified from pNL4-3 and cloned into the pET21 vector (Invitrogen). All clones were verified by sequencing of the entire coding region.
Baculoviruses expressing human and rhesus TRIM5a CC-SPRY were prepared using the Baculdirect C-term (Invitrogen) according to the manufacturer's protocols. Proteins were expressed in SF21 insect cells by infecting cells with recombinant baculoviruses at a MOI of 2 for 40 h. Cells were lysed by sonication in a buffer containing 25 mM sodium phosphate, pH 7.5, 250 mM NaCl, 10 mM beta-mercaptoethanol, and 0.02% sodium azide. Soluble proteins were purified over a 5 mL Ni-NTA column followed by passage over a Hi-Load Superdex 200 16/60 column (GE Healthcare) in a buffer containing 25 mM sodium phosphate, pH 7.5, 150 mM NaCl, 2 mM DTT, 10% glycerol, and 0.02% sodium azide. The fraction containing TRIM5a CC-SPRY was further purified over a 5 mL Hi-Trap QP column (GE Healthcare) using a gradient of 0-1 M NaCl or a 5 mL StrepTrap-HP column (GE-Healthcare) using 2.5 mM desbiotin for elution. CA-NC proteins were expressed in E. coli Rosetta 2 (DE3), cultured in Luria-Bertani medium, using 0.4 mM IPTG for induction and growth at 18uC for 23 h. The proteins were purified as described in Ganser et al [49]. Briefly, soluble proteins were precipitated with 40% (w/v) ammonium sulfate after DNA was removed by precipitation with polyethylenimine. The precipitates were dialyzed against a buffer containing 25 mM TrisHCl, pH 7.5, 50 mM NaCl, 1 mM ZnSO 4 , 10 mM betamercaptoethanol, and 0.02% azide. Proteins were separated by column chromatography over a 5 mL Hi-Trap SP (GE Healthcare) with a 0-1 M NaCl gradient and Hi-Load Superdex75 26/ 60 columns, equilibrated with a buffer containing 25 mM TrisHCl, pH 7.5, 150 mM NaCl, 1 mM ZnSO 4 , 10 mM betamercaptoethanol, and 0.02% azide. CA proteins were prepared as described in Byeon et al [38].

Isolation of HIV-1 core structures
HIV-1 cores were isolated from virions by a modification of the ''spin-thru'' method previously described [50]. HIV-1 viruses were derived from the R9 molecular clone [51] and mutants thereof. CA mutations were created by overlap PCR. SpeI-ApaI fragments were transferred into R9, and the transferred region was verified by PCR. HIV-1 viruses were produced by transient transfection of sixty dishes of 6610 6 293T cells with 10 mg plasmid DNA (using 10 mg of HIV-1 construct R9, R9.Env-, or R9.A14C/E45C) using polyethylenimine (3.6 mg/ml, Polysciences) [52] in each 10 cm dish. Two days after transfection, virus-containing supernatants were collected and clarified by filtration (0.45 mm pore-size). Particles in clarified supernatants (600 ml) from 293T cells were pelleted through 3ml cushions of 20% sucrose (120,000 6g, 2.5 h) in a Beckman SW32Ti rotor then gently suspended in a total of 1.2 ml STE buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA) for 2 h at 4uC. The concentrated virus suspension was subjected to equilibrium ultracentrifugation (120,000 6 g, 16 h, 4uC, Beckman SW-32Ti rotor) through a layer of 1% Triton X-100 into a linear gradient of 30%-70% sucrose in STE buffer. Twelve 1-ml fractions were collected. CA concentrations were determined by p24 ELISA [53]. The peak p24 fractions near the bottom of the gradient were pooled and concentrated to ,100 ml by diafiltration with an Ultracel-10K protein concentrator (Amicon). The sample was diluted with STE buffer and reconcentrated to reduce the final sucrose concentration in the sample to less than 0.5%. The concentrated samples of cores were then assayed for p24 by ELISA. Multi-angle light scattering Light-scattering data were obtained using an analytical Super-dex200 column (1 cm 630 cm, GE Healthcare) with in-line multiangle light scattering (HELEOS, Wyatt Technology), variable wavelength UV (Agilent 1100 Series, Agilent Technology) and refractive index (Optilab rEX, Wyatt Technology.) detection. Approximately 100 mL of 2 mg/mL protein solutions were injected into the pre-equilibrated column using 25 mM sodium phosphate buffer (pH 7.5), 250 mM NaCl, 10% glycerol, and 0.02% (w/v) sodium azide at a flow-rate of 0.5 ml/minute for equilibration and elution. Molecular masses were determined from the scattering data using the ASTRA program (Wyatt Technology).

Circular Dichroism (CD)
CD spectra of TRIM5a CC-SPRY (5.4 mg/mL) were collected in a buffer containing 1 mM sodium phosphate, pH 7.5, 14 mM NaCl with a Jasco-810 CD spectrophotometer (Easton, MD). Data were collected with a scan rate of 1 nm/sec from 260 to 200 nm at a constant temperature of 12uC and averaged over 40 scans.

Binding assays
CA and CA-NC tubes were assembled containing 80 mM (2 mg/ml) CA, 1 M NaCl and 50 mM Tris-HCl (pH 8.0) at 37uC for one hour or 300 mM CA-NC, 60 mM TG50 oligonucleotide in 250 mM NaCl, 50 mM Tris-HCl (pH 8.0) buffer at 4uC for 19 hr, respectively. For the TRIM5a CC-SPRY binding assays, the binding buffer, 10 mM Tris pH 7.5, 330 mM NaCl, 1 mM TCEP, 0.02% Azide, 5% Glycerol, is also the stock buffer for TRIM5a CC-SPRY proteins. Briefly, binding buffer containing different concentrations of TRIM5a CC-SPRY was added to preassembled CA and CA-NC tubes. CA concentration was slightly reduced to 64 mM in the binding assays. The CA-NC assemblies were diluted to final concentrations of 80 mM (comparable to the amount of total protein used with CA) or 10 mM (comparable to the number of tubes seen with CA) with assembly buffer prior to the binding assays. TRIM5a hu CC-SPRY or TRIM5a rh CC-SPRY aliquots from 4 mg/ml stock solutions were added to preassembled CA and CA-NC tubes. The reaction mixture was incubated on a rocking platform at room temperature for 1 hr with gentle mixing at 10 min intervals. At the end of incubation, 5 ml samples were withdrawn from the reaction mixtures and immediately used for cryoEM analysis. 6 ml samples from the same reaction mixtures were mixed with 4X LDS loading buffer (Invitrogen) supplemented with 10 mM DTT for SDS-PAGE analysis (t). The remaining sample was pelleted at 20,000 g with an Eppendorf centrifuge 5417R for 15 min and supernatants (s) and pellets (p, resuspended in 1/3 of volume) were mixed with 4X LDS loading buffer for gel analysis. Total, supernatant, and pellet samples, without boiling, were loaded on 10% SDS-PAGE and stained with Coomassie Blue. Each experiment was carried out at least three times.

Gold labeling of TRIM5a CC-SPRY
His-tagged TRIM5a proteins at the C-terminus, TRIM5ahu CC-SPRY and TRIM5a rh CC-SPRY, were labeled using 5 nm Ni-NTA-Nanogold gold beads from Nanoprobes (Yaphank, NY). For gold labeling, wild type CA protein was assembled into tubes using 80 mM (2 mg/ml) CA in the assembly buffer (1 M NaCl and 50 mM Tris-HCl (pH 8.0)) at 37uC for one hour. TRIM5a hu CC-SPRY or TRIM5a rh CC-SPRY (2 ml) was added to the assembly mix (20 ml) to a final concentration of 18 mM and incubated on a rocking platform at room temperature for 1 hr with gentle mixing at 10 min intervals. 2.7 ml of 5 nm Ni-NTA-Nanogold gold beads (stock concentration, 0.5 mM) in 100 mM imidazole (pH 8.0) was added to the assemblies and allowed to incubate at room temperature for 20 minutes. The mixture was then centrifuged at 3,000 g and the pellet was resuspended in assembly buffer. Samples were immediately applied to glowdischarged EM grids for negative staining with 1% uranyl acetate solution after resuspension. Images were acquired on an FEI Tecnai TF20 electron microscope at a nominal magnification of 50,000 and with underfocus values about 2 mm, using a Gatan ultrascan 4KX4K CCD camera (Gatan Inc., Pleasanton, CA, U.S.A.).

CA double-cysteine mutant cross-linking reactions
The cross-linking experiment was set up as previously described [54]. Briefly, 30 ml P207C/T216C or A14C/E45C CA were preassembled in the presence of 50 mM DTT under the conditions described above. The assembled material was then subjected to centrifugation at 20,000 g at room temperature in an Eppendorf centrifuge 5417R for 15 minutes. The pellet was resuspended in 30 ml assembling buffer and oxidized with 1 ml of 30x oxidizer mix (60 mM CuSO 4 , (Sigma) dissolved in water, and 267 mM 1,10-Phenanthroline (Sigma) dissolved in 100% ethanol in a 1:1 ratio) for 5 seconds, immediately followed by quenching with 20 mM iodoacetamide (Sigma) and 3.7 mM Neocuproine (Sigma).

SDS-PAGE gel densitometry analysis
For the dose-dependent TRIM5a rh CC-SPRY binding assay, the SDS-PAGE gels were scanned (Epson 4990 scanner) and the integrated intensities of CA, CA-NC, and TRIM5a rh protein bands in pellet fractions were measured using Image J 1.40 g program (NIH). The molar ratios were calculated according to the formula (TRIM5a rh band intensity/TRIM5a rh molecular weight)/(CA band intensity/CA molecular weight).

Cryo-EM analysis
Aliquots from the binding assays (above) were subjected to cryoEM analysis. 2 ml were applied to the carbon side of a glow discharged perforated Quantifoil grids (Quantifoil Micro Tools, Jena, Germany), and 2.5 ml binding buffer was added to the back side of the grids. Grids were blotted and plunge-frozen in liquid ethane using a manual gravity plunger. Low dose (10,15 e 2 /Å 2 ) projection images were collected on an FEI Tecnai TF20 electron microscope at a nominal magnification of 50,000 and with underfocus values ranging from 1.0 to 2.5 mm, using a Gatan ultrascan 4KX4K CCD camera (Gatan Inc., Pleasanton, CA, U.S.A.).

Quantification of A14C/E45C and P207C/T216C cores in the presence of human and rhesus TRIM5a CC-SPRY
The effect of Rhesus TRIM5a CC-SPRY on HIV-1 cores was examined and quantified using cryoEM. 18 mM rhesus or human TRIM5a CC-SPRY proteins were added to a solution of isolated HIV-1 A14C/E45C or P207C/T216C cores (,11 mg/ml). After one hour incubation at room temperature with gentle agitation, the samples were subjected to cryoEM analysis. For each sample, about 80 low dose projection images were collected at 19,000x magnification. Each field of view covers about 5 mm 2 . The image areas were chosen randomly, owing to the nature of cryoEM imaging. The number of cores in each sample was quantified using average number of cores per image frame. Mean values from four totally independent experiments are plotted in Fig. 6 with the standard deviation indicated. Figure S1 SDS-PAGE analysis of binding of TRIM5a CC-SPRY to pre-assembled wild-type CA tubes. Samples of the reaction mix before centrifugation (t), of supernatant (s), and of pellet (p) are shown. Controls for TRIM5a without CA, CA without TRIM5a and CA with human TRIM5a are shown as indicated.

Supporting Information
(TIF)  Figure S6 SDS-PAGE analysis of TRIM5a rh CC-SPRY binding to A14C/E45C CA tubes. A14C/E45C CA assemblies were incubated with either TRIM5a hu CC-SPRY, TRIM5a rh CC-SPRY or reaction buffer followed by oxidization. Samples of the reaction mixture before high speed centrifugation (t), and pellets of non-reduced (p) and reduced (pDTT) samples were analyzed by non-reducing SDS-PAGE and stained with Coomassie Blue. A CA dimer is observed (lane 1, 3,5,7,9) in non-oxidized samples, whereas a dimer of CA hexamers is only seen in oxidized A14C/E45C CA assemblies without TRIM5a rh CC-SPRY treatment (lane 2 and 8).