Lenacapavir (LEN) is a highly potent, long-acting HIV-1 capsid inhibitor that holds exceptional promise for pre-exposure prophylaxis. LEN causes the mature viral capsid to rupture and lose integrity, but the underlying mechanism has been unclear. Here, we show that LEN is an allosteric modulator of HIV-1 capsid structure that breaks the capsid's fullerene cone architecture in two steps: loss of high-curvature declinations occurs early, followed by failure of the capsid body. At the molecular level, LEN alters the non-covalent bonding interactions between capsid subunits and reduces local lattice curvature. LEN also alters the material properties of the capsid, by increasing brittleness. These results provide molecular rationales for how LEN remodels HIV-1 capsid structure and impairs the replication capacity of the virus.

Retroviruses can be detected by the innate immune sensor cyclic GMP-AMP synthase (cGAS), which recognizes reverse-transcribed DNA and activates an antiviral response. However, the extent to which HIV-1 shields its genome from cGAS recognition remains unclear. To study this process in mechanistic detail, we reconstituted reverse transcription, genome release, and innate immune sensing of HIV-1 in a cell-free system. We found that wild-type HIV-1 capsids protect viral genomes from cGAS even after completing reverse transcription. Viral DNA could be "deprotected" by thermal stress, capsid mutations, or reduced concentrations of inositol hexakisphosphate (IP6) that destabilize the capsid. Strikingly, the capsid inhibitor lenacapavir also disrupted viral cores and dramatically potentiated cGAS activity, both in vitro and in cellular infections. Our results provide biochemical evidence that the HIV-1 capsid lattice conceals the genome from cGAS and that chemical or physical disruption of the viral core can expose HIV-1 DNA and activate innate immune signaling.

HIV-1 DNA is preferentially integrated into chromosomal hot spots by the preintegration complex (PIC). To understand the mechanism, we measured the DNA integration activity of PICs-extracted from infected cells-and intasomes, biochemically assembled PIC substructures using a number of relevant target substrates. We observed that PIC-mediated integration into human chromatin is preferred compared to genomic DNA. Surprisingly, nucleosomes lacking histone modifications were not preferred integration compared to the analogous naked DNA. Nucleosomes containing the trimethylated histone 3 lysine 36 (H3K36me3), an epigenetic mark linked to active transcription, significantly stimulated integration, but the levels remained lower than the naked DNA. Notably, H3K36me3-modified nucleosomes with linker DNA optimally supported integration mediated by the PIC but not by the intasome. Interestingly, optimal intasome-mediated integration required the cellular cofactor LEDGF. Unexpectedly, LEDGF minimally affected PIC-mediated integration into naked DNA but blocked integration into nucleosomes. The block for the PIC-mediated integration was significantly relieved by H3K36me3 modification. Mapping the integration sites in the preferred substrates revealed that specific features of the nucleosome-bound DNA are preferred for integration, whereas integration into naked DNA was random. Finally, biochemical and genetic studies demonstrate that DNA condensation by the H1 protein dramatically reduces integration, providing further evidence that features inherent to the open chromatin are preferred for HIV-1 integration. Collectively, these results identify the optimal target substrate for HIV-1 integration, report a mechanistic link between H3K36me3 and integration preference, and importantly, reveal distinct mechanisms utilized by the PIC for integration compared to the intasomes. IMPORTANCE HIV-1 infection is dependent on integration of the viral DNA into the host chromosomes. The preintegration complex (PIC) containing the viral DNA, the virally encoded integrase (IN) enzyme, and other viral/host factors carries out HIV-1 integration. HIV-1 integration is not dependent on the target DNA sequence, and yet the viral DNA is selectively inserted into specific "hot spots" of human chromosomes. A growing body of literature indicates that structural features of the human chromatin are important for integration targeting. However, the mechanisms that guide the PIC and enable insertion of the PIC-associated viral DNA into specific hot spots of the human chromosomes are not fully understood. In this study, we describe a biochemical mechanism for the preference of the HIV-1 DNA integration into open chromatin. Furthermore, our study defines a direct role for the histone epigenetic mark H3K36me3 in HIV-1 integration preference and identify an optimal substrate for HIV-1 PIC-mediated viral DNA integration.

During the first half of the viral life cycle, HIV-1 reverse transcribes its RNA genome and integrates the double-stranded DNA copy into a host cell chromosome. Despite progress in characterizing and inhibiting these processes, in situ mechanistic and structural studies remain challenging. This is because these operations are executed by individual viral preintegration complexes deep within cells. We therefore reconstituted and imaged the early stages of HIV-1 replication in a cell-free system. HIV-1 cores released from permeabilized virions supported efficient, capsid-dependent endogenous reverse transcription to produce double-stranded DNA genomes, which sometimes looped out from ruptured capsid walls. Concerted integration of both viral DNA ends into a target plasmid then proceeded in a cell extract-dependent reaction. This reconstituted system uncovers the role of the capsid in templating replication.

TRIM5α combines distinct modes of binding into successively higher-order structures to recognize HIV-1 and retroviral capsids.

ABSTRACT Restriction factors are intrinsic cellular defense proteins that have evolved to block microbial infections. Retroviruses such as HIV-1 are restricted by TRIM5 proteins, which recognize the viral capsid shell that surrounds, organizes, and protects the viral genome. TRIM5α uses a SPRY domain to bind capsids with low intrinsic affinity (KD of >1 mM) and therefore requires higher-order assembly into a hexagonal lattice to generate sufficient avidity for productive capsid recognition. TRIMCyp, on the other hand, binds HIV-1 capsids through a cyclophilin A domain, which has a well-defined binding site and higher affinity (KD of ∼10 μM) for isolated capsid subunits. Therefore, it has been argued that TRIMCyp proteins have dispensed with the need for higher-order assembly to function as antiviral factors. Here, we show that, consistent with its high degree of sequence similarity with TRIM5α, the TRIMCyp B-box 2 domain shares the same ability to self-associate and facilitate assembly of a TRIMCyp hexagonal lattice that can wrap about the HIV-1 capsid. We also show that under stringent experimental conditions, TRIMCyp-mediated restriction of HIV-1 is indeed dependent on higher-order assembly. Both forms of TRIM5 therefore use the same mechanism of avidity-driven capsid pattern recognition. IMPORTANCE Rhesus macaques and owl monkeys are highly resistant to HIV-1 infection due to the activity of TRIM5 restriction factors. The rhesus macaque TRIM5α protein blocks HIV-1 through a mechanism that requires self-assembly of a hexagonal TRIM5α lattice around the invading viral core. Lattice assembly amplifies very weak interactions between the TRIM5α SPRY domain and the HIV-1 capsid. Assembly also promotes dimerization of the TRIM5α RING E3 ligase domain, resulting in synthesis of polyubiquitin chains that mediate downstream steps of restriction. In contrast to rhesus TRIM5α, the owl monkey TRIM5 homolog, TRIMCyp, binds isolated HIV-1 CA subunits much more tightly through its cyclophilin A domain and therefore was thought to act independently of higher-order assembly. Here, we show that TRIMCyp shares the assembly properties of TRIM5α and that both forms of TRIM5 use the same mechanism of hexagonal lattice formation to promote viral recognition and restriction.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract TRIM5 proteins are restriction factors that block retroviral infections by binding viral capsids and preventing reverse transcription. Capsid recognition is mediated by C-terminal domains on TRIM5α (SPRY) or TRIMCyp (cyclophilin A), which interact weakly with capsids. Efficient capsid recognition also requires the conserved N-terminal tripartite motifs (TRIM), which mediate oligomerization and create avidity effects. To characterize how TRIM5 proteins recognize viral capsids, we developed methods for isolating native recombinant TRIM5 proteins and purifying stable HIV-1 capsids. Biochemical and EM analyses revealed that TRIM5 proteins assembled into hexagonal nets, both alone and on capsid surfaces. These nets comprised open hexameric rings, with the SPRY domains centered on the edges and the B-box and RING domains at the vertices. Thus, the principles of hexagonal TRIM5 assembly and capsid pattern recognition are conserved across primates, allowing TRIM5 assemblies to maintain the conformational plasticity necessary to recognize divergent and pleomorphic retroviral capsids. https://doi.org/10.7554/eLife.16269.001 eLife digest After infecting a cell, a virus reproduces by forcing the cell to produce new copies of the virus, which then spread to other cells. However, cells have evolved ways to fight back against these infections. For example, many mammalian cells contain proteins called restriction factors that prevent the virus from multiplying. The TRIM5 proteins form one common set of restriction factors that act against a class of viruses called retroviruses. HIV-1 and related retroviruses have a protein shell known as a capsid that surrounds the genetic material of the virus. The capsid contains several hundred repeating units, each of which consists of a hexagonal ring of six CA proteins. Although this basic pattern is maintained across different retroviruses, the overall shape of the capsids can vary considerably. For instance, HIV-1 capsids are shaped like a cone, but other retroviruses can form cylinders or spheres. Soon after the retrovirus enters a mammalian cell, TRIM5 proteins bind to the capsid. This causes the capsid to be destroyed, which prevents viral replication. Previous research has shown that several TRIM5 proteins must link up with each other via a region of their structure called the B-box 2 domain in order to efficiently recognize capsids. How this assembly process occurs, and why it enables the TRIM5 proteins to recognize different capsids was not fully understood. Now, Li, Chandrasekaran et al. (and independently Wagner et al.) have investigated these questions. Using biochemical analyses and electron microscopy, Li, Chandrasekaran et al. found that TRIM5 proteins can bind directly to the surface of HIV-1 capsids. Several TRIM5 proteins link together to form large hexagonal nets, in which the B-box domains of the proteins are found at the points where three TRIM5 proteins meet. This arrangement mimics the pattern present in the HIV-1 capsid, and just a few TRIM5 rings can cover most of the capsid. Li, Chandrasekaran et al. then analysed TRIM5 proteins from several primates, including rhesus macaques, African green monkeys and chimpanzees. In all cases analyzed, the TRIM5 proteins assembled into hexagonal nets, although the individual units within the net did not have strictly regular shapes. These results suggest that TRIM5 proteins assemble a scaffold that can deform to match the pattern of the proteins in the capsid. Further work is now needed to understand how capsid recognition is linked to the processes that disable the virus. https://doi.org/10.7554/eLife.16269.002 Introduction Mammalian hosts have evolved a series of different innate immune strategies to combat retroviruses (reviewed in [Altfeld and Gale, 2015; Bieniasz, 2003, 2004; Fitzgerald et al., 2014; Harris et al., 2012; Neil and Bieniasz, 2009; Rustagi and Gale, 2014; Sparrer and Gack, 2015; van Montfoort et al., 2014; Yoo et al., 2014]). TRIM5α and the related TRIMCyp protein (collectively TRIM5) are restriction factors that recognize the capsid surfaces of incoming retroviral core particles, induce their dissociation, and inhibit reverse transcription (Sayah et al., 2004; Stremlau et al., 2004; 2006). The mechanistic basis for core inactivation is not yet well established, but current models invoke the involvement of proteasomes (Anderson et al., 2006; Campbell et al., 2008; Diaz-Griffero et al., 2007; Kutluay et al., 2013; Lukic et al., 2011; Rold and Aiken, 2008; Wu et al., 2006), autophagosomes (Mandell et al., 2014a; 2014b), and/or the establishment of a general antiviral state (Pertel et al., 2011). Like other members of the tripartite motif (TRIM) family (Reymond et al., 2001), TRIM5 proteins comprise a RING E3 ubiquitin (Ub) ligase domain (Meroni and Diez-Roux, 2005; Yamauchi et al., 2008), an L1 linker, a B-box 2 self-assembly domain (Diaz-Griffero et al., 2009; Javanbakht et al., 2005; Li and Sodroski, 2008), an antiparallel dimeric coiled-coil, and an L2 linker that folds back on the coiled-coil (Goldstone et al., 2014; Li et al., 2014; Sanchez et al., 2014; Weinert et al., 2015) (Figure 1A). TRIM5 proteins also contain one of two different C-terminal viral core recognition domains, a B30.2/SPRY domain in TRIM5α (hereafter termed SPRY) or a cyclophilin A (CypA) domain in TRIMCyp (Brennan et al., 2008; Newman et al., 2008; Nisole et al., 2004; Sayah et al., 2004; Stremlau et al., 2005; 2006; Virgen et al., 2008). Figure 1 Download asset Open asset ECT analysis of TRIM5-21R 2D crystals. (A) Schematic of the TRIM5 dimer. The two RING (yellow) and B-box 2 (red) domains are separated by a ~17 nm, antiparallel dimeric coiled-coil (blue). The two L2 linkers (green) fold back towards the 2-fold axis of the coiled-coil to orient two capsid-binding SPRY domains (orange). (B) Schematic of the TRIM5 hexagonal lattice model. (C and D) Tomographic slice (top) of (C) full-length TRIM5-21R, and (D) TRIM5-21RΔSPRY lattices. Scale bars are 100 nm. In both cases, the computed Fourier transform (bottom, left) and subtomogram average without imposed rotational symmetry (bottom, middle) exhibit six-fold symmetry. Iso-surface representations of the densities are also shown (bottom, right). (E) A density difference map of the subtomogram averages of full-length TRIM5-21R and TRIM5-21RΔSPRY lattices reveals positive density (red) at the center of each hexagon edge, corresponding to the SPRY domain position, supporting the TRIM5α dimer and hexamer models shown in (A and B). (F) Heat maps (bottom) of lattice arm lengths and angles measured from refined lattice points (top) selected from the TRIM5-21R tomogram in (C). (G) Histograms showing the distributions of measured arm lengths (n = 155) and angles (n = 111). The most abundant arm length (18.5–19 nm) and arm angle (120°) are consistent with the structure models in (A and B) and the p6 plane group symmetry of TRIM5-21R 2D crystals (Ganser-Pornillos et al., 2011). Note that this analysis probably underestimates the extent of hexamer variability owing to the initial selection of well-ordered lattice points. https://doi.org/10.7554/eLife.16269.003 TRIM5 proteins act by binding the outer capsid shell of the viral core (Biris et al., 2013; Black and Aiken, 2010; Diaz-Griffero et al., 2006b; Kar et al., 2008; Kovalskyy and Ivanov, 2014; Langelier et al., 2008; Sayah et al., 2004; Sebastian and Luban, 2005; Stremlau et al., 2006; Zhao et al., 2011). The capsid protects and organizes the internal ribonucleocapsid, which comprises the viral NC protein, the RNA genome, and associated replicative enzymes. Closed retroviral capsids are constructed from several hundred CA protein hexamers and exactly 12 CA pentamers ([Ganser et al., 1999; Gres et al., 2015; Li et al., 2000; Obal et al., 2015; Pornillos et al., 2011; Zhao et al., 2013] and reviewed in [Ganser-Pornillos et al., 2012; Zhang et al., 2015]). Although all retroviral capsids are organized following these principles, individual capsids are unique, asymmetric objects that can differ in hexamer numbers and pentamer distributions (Benjamin et al., 2005; Briggs et al., 2006; Butan et al., 2008; Ganser-Pornillos et al., 2004; Heymann et al., 2008). For example, HIV-1 capsids are typically conical, but their sizes and cone angles can vary, and cylindrical and spherical capsids also form (Benjamin et al., 2005; Briggs et al., 2006; Briggs et al., 2003; Ganser-Pornillos et al., 2004; Heymann et al., 2008; Welker et al., 2000). Indeed, spherical and cylindrical capsids predominate in other retroviral genera, and capsid surface properties can vary considerably because CA proteins from different genera share low sequence identity (Berthet-Colominas et al., 1999; Campos-Olivas et al., 2000; Cornilescu et al., 2001; Ganser et al., 1999; Jin et al., 1999; Khorasanizadeh et al., 1999; Kingston et al., 2000; Momany et al., 1996; Mortuza et al., 2008; Mortuza et al., 2004; von Schwedler et al., 1998; Zlotnick et al., 1998). To function effectively, individual TRIM5 proteins must overcome these variations in retroviral capsid shape and sequence (Hatziioannou et al., 2003; Wilson et al., 2008). We, and others, have proposed that TRIM5 proteins recognize pleomorphic capsids by recognizing repeating patterns on the capsid surface (Biris et al., 2012; Ganser-Pornillos et al., 2011; Goldstone et al., 2014; Yang et al., 2012). This model supposes that flexible loops on the SPRY and CypA domains can adopt multiple different conformations and can bind weakly to conserved elements on the capsid surfaces (Biris et al., 2012; Caines et al., 2012; Kovalskyy and Ivanov, 2014; Price et al., 2009; Song et al., 2005a; Stremlau et al., 2005; Ylinen et al., 2010). These weak interactions are then amplified by TRIM5 assembly into a higher-order hexagonal lattice, which positions arrays of SPRY/CypA domains to interact with repeating epitopes on the capsid surfaces (Ganser-Pornillos et al., 2011; Li and Sodroski, 2008). This 'pattern recognition' model has been supported by biochemical and structural analyses of a TRIM5 protein construct called TRIM5-21R, which is an artificial chimera in which the RING domain from human TRIM21 replaces the RING domain of rhesus TRIM5α (Diaz-Griffero et al., 2006a; Kar et al., 2008; Langelier et al., 2008). The TRIM5-21R construct retains HIV-1 restriction activity, and has been used in several studies owing to its unusually favorable stability, solubility and assembly properties. Consistent with the pattern recognition model, TRIM5-21R was shown to assemble into open hexagonal lattices, both alone and on the surface of 2D CA crystals that mimic the surface of the HIV-1 capsid (Ganser-Pornillos et al., 2011). TRIM5-21R assemblies could be microns in size but lacked strict long-range order, and 2D projections could therefore only be reconstructed to a resolution of ~7.5 nm. Domain positions therefore had to be inferred, and were interpreted in the absence of high-resolution information on the structure of the TRIM5 protein core. Technical challenges in purifying authentic HIV-1 cores have also been a significant experimental limitation, and all published biochemical and structural studies of TRIM5α-capsid interactions have therefore either employed crude viral core preparations or artificial mimics of the capsid surface (Black and Aiken, 2010; Ganser-Pornillos et al., 2011; Langelier et al., 2008; Sebastian and Luban, 2005; Stremlau et al., 2006; Zhao et al., 2011). Thus, the interactions between authentic viral capsids and TRIM5 proteins have yet to be investigated biochemically or structurally. To address these different shortcomings, we have developed methods for preparing authentic recombinant TRIM5 proteins, co-assemblies of TRIM5 and CA proteins, and stable HIV-1 cores. These reagents were then used to demonstrate that TRIM5 proteins form hexagonal arrays on HIV-1 capsids. Results Structure-based models for TRIM5-21R assembly A composite model for the domain organization within the TRIM5α dimer is shown in Figure 1A. We (Sanchez et al., 2014), and others (Goldstone et al., 2014; Weinert et al., 2015), have suggested that these dimers form the edges of the hexameric rings observed within the hexagonal TRIM5-21R lattice (modeled in Figure 1B). This model is attractive because the edges of each hexagon are ~19 nm long, which would accommodate the antiparallel TRIM5α coiled-coil (~17 nm), and the RING and B-box 2 domains would associate at the three-fold vertices, which is consistent with the trimeric B-box 2 domain structure presented in the accompanying paper (Wagner et al., 2016). Finally, the L2 linkers fold back and form a four-helix bundle at the center of the coiled-coil, and this platform could, in principle, buttress and orient the TRIM5α SPRY domains. None of the domain positions have been determined experimentally, however, and we therefore began our studies by defining the location of the SPRY domains within the hexagonal TRIM5-21R lattice. To visualize TRIM5-21R assemblies in three dimensions and define the SPRY domain positions, we generated electron cryotomograms (ECT) from tilt series of vitrified 2D crystals of both full length TRIM5-21R (Figure 1C) and a construct that lacked the SPRY domain (TRIM5-21RΔSPRY, residues 1–300, Figure 1D). The 3D reconstructions were refined and improved by subtomogram averaging of densities centered at equivalent lattice vertices. As expected, TRIM5-21R and TRIM5-21RΔSPRY both assembled into similar planar lattices of hexagonal rings, with inter-ring spacings and protein densities matching those of the previous 2D projection structures (Ganser-Pornillos et al., 2011). Difference density maps clearly revealed that the SPRY domains are located at the center of each hexagon edge (Figure 1E), thereby supporting the model shown in Figure 1B. The TRIM5-21R lattice is a hexagonal net with variable arm lengths and angles Although the paracrystalline arrays of TRIM5-21R exhibited long-range order, they diffracted poorly, suggesting variability within the lattice. To quantify this variability, we performed a nearest neighbor analysis of the refined positions of the lattice vertices used for subtomogram averaging. The relative positions of 153 vertices in the TRIM5-21R 2D crystals were used to define individual hexamer edge lengths and angles (see Figure 1F). The length distribution of hexamer edges was centered about a mean of 19 nm, but individual lengths varied by up to ±5 nm (Figure 1G, upper panel). Similarly, the distribution of hexamer vertex angles was centered about 120°, but varied by up to ±20° (Figure 1G, lower panel). Thus, individual rings within the TRIM5 lattice exhibited considerable conformational variability, explaining the poor crystalline order and modest diffraction resolution. Expression and purification of authentic primate TRIM5α and TRIMCyp proteins Recombinant owl monkey TRIMCyp has been purified (Pertel et al., 2011), but TRIM5α proteins are more challenging to purify because these proteins tend to self-assemble, both in cells and in vitro. Hence, previous biochemical and structural studies of TRIM5α proteins have been performed with impure proteins, protein fragments, or non-native chimeric constructs (Biris et al., 2012; Goldstone et al., 2014; Kar et al., 2008; Langelier et al., 2008; Sanchez et al., 2014; Yang et al., 2012; Ganser-Pornillos et al., 2011). To overcome this limitation, we tested a variety of different expression and purification conditions, with the goal of developing a general method for preparing milligram quantities of authentic, full-length primate TRIM5α and TRIMCyp proteins. The strategy that was ultimately successful entailed expressing TRIM5 proteins in insect cells using a baculoviral expression system. As described in greater detail in the Materials and methods, expressed TRIM5 proteins formed cytoplasmic bodies that could be solubilized by lysing the cells in a low ionic strength, alkali buffer that contained the non-ionic detergent Triton X-100, as well as a non-detergent small molecule, sulfobetaine-256 (NDSB-256) that has previously been shown to inhibit protein aggregation (Sainsbury et al., 2008; Vuillard et al., 1995). Once solubilized, primate TRIM5 proteins typically remained dimeric and soluble under low salt, alkaline conditions in the absence of Triton X-100 and NDSB-256, even at concentrations greater than 1 mg/ml. The proteins could therefore be purified, provided they were maintained at high pH, low salt and/or low protein concentrations. Our stepwise protein purification protocol is illustrated for rhesus TRIM5α in Figure 2A. Briefly, various N-terminal OneSTrEP-FLAG- (OSF-) or C-terminal FLAG-OneSTrEP- (FOS-) tagged TRIM5 proteins were initially purified using Strep-Tactin affinity chromatography, the affinity tag was removed by HRV14-3C protease treatment, and the proteins were then purified to homogeneity by anion exchange and gel filtration chromatography. Analogous approaches were used to express and purify wild type and mutant TRIM5α proteins from rhesus macaques (Macaca mulatta, here abbreviated TRIM5αrh), African green monkeys (Chlorocebus pygerythrus, TRIM5αAGMpyg), chimpanzees (Pan troglodytes, TRIM5αcpz), humans (Homo sapiens, TRIM5αhu), and the TRIMCyp protein from owl monkeys (Aotus trivirgatus, TRIMCyp). Yields ranged between 1.3 and 9.6 mg/L of insect cell cultures, and all of the proteins eluted with similar retention times during the final gel filtration chromatography step, indicating that they were all dimers of similar shape. All of the proteins could be purified to >95% purity (Figure 2B) with the exception of TRIMCyp, where our preparations also contained breakdown contaminants that mapped to proteolytic cleavage at residues Lys283 and Gln287 (not shown). These breakdown contaminants were eliminated by creating a mutant construct that expressed TRIMCypK283D,Q287D (Figure 2B, lane 4). These mutations are not expected to affect functionally relevant properties of the protein because TRIMCypK283D,Q287D retains potent HIV-1 restriction activity (Figure 2—figure supplement 1). Figure 2 with 1 supplement see all Download asset Open asset Purification and characterization of recombinant TRIM5 proteins. (A) Coomassie-stained SDS-PAGE showing the stepwise purification of rhesus TRIM5α (TRIM5αrh). Samples correspond to: soluble lysate from control SF9 cells (Uninfected, lane 1); soluble lysate from SF9 cells expressing OSF-TRIM5αrh (Infected, lane 2); Strep-Tactin affinity-purified OSF-TRIM5αrh (Affinity, lane 3); TRIM5αrh after removal of the OSF tag by HRV14-3C protease treatment (Tag Cleavage, lane 4); dimeric TRIM5αrh purified by Q anion exchange chromatography (Anion Exchange, lane 5); dimeric TRIM5αrh purified by Superdex 200 gel filtration chromatography (Gel Filtration, lane 6). (B) Coomassie-stained SDS-PAGE showing 1.5 µg of purified rhesus, African green monkey pygerythrus (AGMpyg), chimpanzee TRIM5α, proteolysis-resistant owl monkey TRIMCypK283D,Q287D, human TRIM5α, and HIV-1-restricting human TRIM5αR332P. (C,D) TRIM5 hexagonal assembly is a conserved property of primate TRIM5 proteins. Negatively stained EM image of hexagonal arrays formed by (C) TRIM5αAGMpyg and (D) TRIMCyp. Computed Fourier transforms (top right insets) show clear hexagonal order and filtered projection density maps of 2-dimensional crystals (bottom left insets) also reveal hexagonal rings and density distributions reminiscent of TRIM5-21R lattices (Ganser-Pornillos et al., 2011). The unit cell parameters are a = 345 Å, b = 345 Å, γ = 120° (TRIM5αAGMpyg); and a = 345 Å, b = 344 Å, γ = 119° (TRIMCyp). Note that the TRIMCyp samples contained a mixture of full-length TRIMCyp and fragments that were proteolyzed to the C-terminus of residues K283 or Q287 (see Results and Materials and methods for details). The relatively thinner two fold density in the TRIMCyp projection map could either reflect low crystal occupancy of the CypA domain (due to proteolysis) or inherently flexible CypA domains in TRIMCyp as has been proposed by (Goldstone et al., 2014). https://doi.org/10.7554/eLife.16269.004 Conservation of hexagonal TRIM5 protein assembly To determine whether the ability to assemble into hexagonal nets is a conserved property of authentic TRIM5 proteins, we screened for conditions that promoted assembly of different primate TRIM5 proteins, using negative stain EM imaging to assay assembly states. These screens identified conditions under which two of the TRIM5 proteins, TRIM5αAGMpyg (Figure 2C) and TRIMCyp (Figure 2D) spontaneously formed 2D hexagonal assemblies that were similar in appearance to those formed by TRIM5-21R (Ganser-Pornillos et al., 2011). Assembly efficiencies varied, however, as TRIM5αAGMpyg assembled very efficiently under the same conditions as TRIM5-21R, whereas TRIMCyp assembled inefficiently and required additional precipitants (see Materials and methods). Negatively stained 2D crystals of TRIM5αAGMpyg and TRIMCyp were imaged and processed to generate Fourier-filtered 2D projection reconstructions (Figure 2C,D; bottom). Both TRIM5 proteins formed lattices comprising open hexameric rings that were similar in appearance and size to the TRIM5-21R rings, demonstrating that diverse TRIM5 proteins from different primates that share 73% pairwise identity across their TRIM domains share the ability to assemble into analogous hexagonal nets. Templated hexagonal TRIM5 protein assembly on HIV-1 CA surfaces The pattern recognition model for TRIM5 restriction predicts that binding to the surface of the viral capsid promotes hexagonal TRIM5 assembly. We therefore tested whether hexagonal 2D crystals of HIV-1 CA, which mimic the capsid surface, could promote the assembly of three different restricting TRIM5 proteins; TRIM5αrh, TRIMCypK283D,Q287D, and TRIM5αhu,R332P, and two different non-restricting TRIM5 proteins; wild type TRIM5αhu and TRIM5αcpz (Hatziioannou et al., 2004; Nisole et al., 2004; Sayah et al., 2004; Song et al., 2005b; Stremlau et al., 2004; 2005; Yap et al., 2005). To test for templated assembly of TRIM5 proteins, soluble dimeric proteins were incubated together with preassembled 2D CA crystals under solution conditions that were sufficiently stringent to prevent untemplated assembly. TRIM5αAGMpyg was not used in these studies because it assembled very robustly even in the absence of a template. As shown in Figure 3, the three restricting TRIM5 proteins assembled into visible hexagonal nets on the surfaces of preformed HIV-1 CA crystals, whereas templated assembly was not observed for either one of the non-restricting TRIM5 proteins (Figure 3C and data not shown). Templated assembly therefore correlated well with restriction activity, emphasizing the coupling of CA binding and TRIM5 lattice assembly. Computed Fourier transforms of the images of decorated crystals (Figure 3, insets) revealed well-defined first- and second-order reflections from the smaller underlying CA lattice (red and blue), as well as more diffuse peaks (TRIM5αrh and TRIMCypK283D,Q287D, green) or a powder diffraction ring (TRIM5αhu,R332P) corresponding to the first-order reflections from the hexagonal TRIM5 lattices. Thus, all three restricting TRIM5 proteins bound the CA surfaces and assembled into hexagonal nets that were clearly visible, but lacked extensive crystalline order. Figure 3 Download asset Open asset Assembly of restricting TRIM5 proteins on 2D crystals of HIV-1 CA. Negative stain EM images of CA 2D crystals decorated with (A) TRIM5αrh, (B) TRIMCypK283D,Q287D, (C) TRIM5αhu (non-restricting allele), (D) TRIM5αhu,R332P (restricting mutant). Scale bars are 100 nm. Computed Fourier transforms (insets) and indexing (second insets) show the first and second order reflections of two CA lattices and their unit cell parameters (red and blue) as well as diffraction spots (A, B) or rings (D) corresponding to the first order reflections of the TRIM5 lattices (green). https://doi.org/10.7554/eLife.16269.006 TRIM5 binding to helical HIV-1 CA tubes The helical tubes formed by pure recombinant HIV-1 CA provide another regularized model for the curved, symmetric arrays of CA hexagons on conical viral capsid surfaces (Campbell and Vogt, 1995; Li et al., 2000). We employed a sucrose co-sedimentation assay to test whether the restricting TRIM5αAGMpyg and TRIMCypK283D,Q287D proteins bound disulfide-crosslinked helical tubes formed by a mutant HIV-1 CA protein that assembled into discrete helical tubes stabilized by intrahexamer disulfide crosslinks (CAA14C,E45C,A92E) (Byeon et al., 2009; Ganser-Pornillos et al., 2011; Langelier et al., 2008; Li et al., 2000; Pornillos et al., 2009; 2010; Zhao et al., 2011). Both TRIM5αAGMpyg and TRIMCypK283D,Q287D bound the CA tubes, as judged by their co-sedimentation through the sucrose cushion when the CA tubes were present, but not when they were absent (compare the 'pellet' fractions in lanes 1 vs. 2 and lanes 5 vs. 6 in Figure 4—figure supplement 1A). Binding was specific because TRIM5αAGMpyg did not co-sediment with CA tubes when the SPRY domain was removed (compare lanes 2 vs. 4) and because TRIMCypK283D,Q287D did not co-sediment with CA tubes in the presence of cyclosporine A, which competitively inhibits the CypA-CA interaction (compare lanes 6 vs. 7). Thus, pure recombinant TRIM5 proteins bind the hexagonal lattices of both helical CA tubes and 2D CA crystals. Negative stain and deep-etch electron microscopy were used to image the TRIM5-decorated HIV-1 CA tubes. Four restricting TRIM5 proteins, TRIM5αAGMpyg, TRIMCypK283D,Q287D, TRIM5αrh, and TRIM5αhu,R332P, formed thin ring-like decorations on the surfaces of CA tubes (Figure 4—figure supplement 1B). The TRIM5 decorations typically appeared as light, string-like nets against the darker underlying CA tubes when the assemblies were stained with either uranyl acetate (UA) or phosphotungstate (PTA). Equivalent decorations were not observed for control CA tubes alone or for CA tubes plus TRIM5αhu, which does not restrict HIV-1. TRIM5 decoration of CA tubes therefore correlated with restriction activity. Deep-etch EM images often exhibit even greater contrast than negative stain transmission EM images, and this effect was evident in deep-etch images of undecorated HIV-1 CA tubes, where rows of individual CA hexamers were readily visible (Figure 4A, upper panel). Strings and rings of TRIM5αAGMpyg were often readily visible on the decorated CA tube surfaces, and networks of rings were sometimes observable (Figure 4A, lower panel). Hence, TRIM5 proteins can form hexagonal nets on the surfaces of helical HIV-1 CA tubes. Figure 4 with 1 supplement see all Download asset Open asset Assembly of TRIM5α proteins on HIV-1 CA tubes. (A) Deep-etch electron micrographs of control hyperstable CA tubes (top) and TRIM5α decorated CA tubes (bottom) with blow-up views of boxed regions to the right. Scale bar is 50 nm. (B) Negative stain electron micrographs of co-assembled TRIM5αAGMpyg-coated CA tubes (top). Scale bar is 100 nm. Expanded views of negatively stained CA assembly in the absence (bottom left) or presence of TRIM5αAGMpyg proteins (bottom right). TRIM5α formed ring-like decorations (yellow arrows) on the tube surface and displayed protrusions along the edge of the tube (red arrows). Similar decorations were not observed in the control case. The protrusions were regularly spaced and we speculate that they are either well-ordered coiled-coil arms from adjacent hexagons wrapping around the CA tubes or possibly ordered RING domains projecting outward from the lattice. Scale bars are 50 nm. (C) Histograms showing the distributions of measured inter-ring spacings in the TRIM5αAGMpyg–decorated tubes (blue bars, n = 170) and in co-assemblies (red bars, n = 164), and of inter-protrusion spacings in co-assemblies (green bars, n = 166). The most abundant inter-ring spacing (30–35 nm) is consistent with the spacing of TRIM5-21R 2D crystals, indicating similar structures. (D) Electron cryotomography (ECT) reveals that TRIM5α forms hexagonal nets on the surface of CA tube. Hexagon-like rings are marked by magenta arrows. Scale bar is 80 nm. (E) Histograms showing the distributions of arm lengths (n = 35) and angles (n = 42) measured from three-fold vertices in ECT. https://doi.org/10.7554/eLife.16269.007 The efficiency of TRIM5 assembly on disulfide-crosslinked CA tubes was relatively low, which may explain why the decorations were not noted in previous studies. We therefore screened additional assembly conditions to determine the source of this variability. These experiments revealed that co-incubation of native CA protein with TRIM5α under low ionic strength conditions produced CA tubes that were extensively decorated with TRIM5α. Co-assembly was reproducible, and both tagged and untagged TRIM5-21R, TRIM5AGMpyg, and TRIM5rh proteins formed ring-like decorations on CA tube surfaces at a variety of different CA/TRIM5 molar ratios (Figure 4B, data not shown, and Wagner et al., 2016). The TRIM5αAGMpyg inter-ring spacing distribution on the tubes centered at 30–35 nm, as was also the case for TRIM5αAGMpyg assemblies on pre-formed disulfide-crosslinked CA tubes (Figure 4C). In all cases, the rings were irregular and their spac

TRIM5 proteins are restriction factors that block retroviral infections by binding viral capsids and preventing reverse transcription. Capsid recognition is mediated by C-terminal domains on TRIM5α (SPRY) or TRIMCyp (cyclophilin A), which interact weakly with capsids. Efficient capsid recognition also requires the conserved N-terminal tripartite motifs (TRIM), which mediate oligomerization and create avidity effects. To characterize how TRIM5 proteins recognize viral capsids, we developed methods for isolating native recombinant TRIM5 proteins and purifying stable HIV-1 capsids. Biochemical and EM analyses revealed that TRIM5 proteins assembled into hexagonal nets, both alone and on capsid surfaces. These nets comprised open hexameric rings, with the SPRY domains centered on the edges and the B-box and RING domains at the vertices. Thus, the principles of hexagonal TRIM5 assembly and capsid pattern recognition are conserved across primates, allowing TRIM5 assemblies to maintain the conformational plasticity necessary to recognize divergent and pleomorphic retroviral capsids.

Restriction factors and pattern recognition receptors are important components of intrinsic cellular defenses against viral infection. Mammalian TRIM5α proteins are restriction factors and receptors that target the capsid cores of retroviruses and activate ubiquitin-dependent antiviral responses upon capsid recognition. Here, we report crystallographic and functional studies of the TRIM5α B-box 2 domain, which mediates higher-order assembly of TRIM5 proteins. The B-box can form both dimers and trimers, and the trimers can link multiple TRIM5α proteins into a hexagonal net that matches the lattice arrangement of capsid subunits and enables avid capsid binding. Two modes of conformational flexibility allow TRIM5α to accommodate the variable curvature of retroviral capsids. B-box mediated interactions also modulate TRIM5α's E3 ubiquitin ligase activity, by stereochemically restricting how the N-terminal RING domain can dimerize. Overall, these studies define important molecular details of cellular recognition of retroviruses, and how recognition links to downstream processes to disable the virus.

TRIM5α is an antiviral, cytoplasmic, E3 ubiquitin (Ub) ligase that assembles on incoming retroviral capsids and induces their premature dissociation. It inhibits reverse transcription of the viral genome and can also synthesize unanchored polyubiquitin (polyUb) chains to stimulate innate immune responses. Here, we show that TRIM5α employs the E2 Ub‐conjugating enzyme Ube2W to anchor the Lys63‐linked polyUb chains in a process of TRIM5α auto‐ubiquitination. Chain anchoring is initiated, in cells and in vitro, through Ube2W‐catalyzed monoubiquitination of TRIM5α. This modification serves as a substrate for the elongation of anchored Lys63‐linked polyUb chains, catalyzed by the heterodimeric E2 enzyme Ube2N/Ube2V2. Ube2W targets multiple TRIM5α internal lysines with Ub especially lysines 45 and 50, rather than modifying the N‐terminal amino group, which is instead αN‐acetylated in cells. E2 depletion or Ub mutation inhibits TRIM5α ubiquitination in cells and restores restricted viral reverse transcription, but not infection. Our data indicate that the stepwise formation of anchored Lys63‐linked polyUb is a critical early step in the TRIM5α restriction mechanism and identify the E2 Ub‐conjugating cofactors involved.

Transient transfection of small interfering RNA (siRNA) provides a powerful approach for studying cellular protein functions, particularly when the target protein can be re-expressed from an exogenous siRNA-resistant construct in order to rescue the knockdown phenotype, confirm siRNA target specificity, and support mutational analyses. Rescue experiments often fail, however, when siRNA-resistant constructs are expressed at suboptimal levels. Here, we describe an ensemble of mammalian protein expression vectors with CMV promoters of differing strengths. Using CHMP2A rescue of HIV-1 budding, we show that these vectors can combine high-transfection efficiencies with tunable protein expression levels to optimize the rescue of cellular phenotypes induced by siRNA transfection.

A ubiquitin ligase (E3) functions at the crossroad between ubiquitin activation and the attachment of ubiquitin to protein substrates. During this process, the E3 interacts with both a substrate and a ubiquitin-conjugating enzyme (E2). Although a major goal when investigating an E3 is to identify its substrates, recent evidence indicates that the E2 dictates the type of ubiquitin modification that will occur on the substrate. There are similar to 30 E2s identified in the human genome, many of which remain to be characterized. We found that the RING E3 BRCA1/BARD1 can interact with 10 different E2s. The ability of BRCA1 to interact with multiple E2s is likely to be a common feature among other RING and U-box E3s. We and others have also found that certain E2s show a preference for attaching either the first ubiquitin to a substrate lysine or ubiquitin to itself (chain building), suggesting that E2s may play a role in dictating product formation. Therefore, when investigating the functions of an E3 it is advisable to identify all E2s that interact with the E3 so that these can be used in E3-dependent substrate-ubiquitination assays. We describe a method used to identify all the E2s that interact with BRCA1. Defining the set of E2s that interact with other RING and U-box E3s will open the door for predictive models and lead to a better understand of substrate ubiquitination.

The rhesus monkey intrinsic immunity factor TRIM5alpha(rh) recognizes incoming capsids from a variety of retroviruses, including human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV), and inhibits the accumulation of viral reverse transcripts. However, direct interactions between restricting TRIM5alpha proteins and retroviral capsids have not previously been demonstrated using pure recombinant proteins. To facilitate structural and mechanistic studies of retroviral restriction, we have developed methods for expressing and purifying an active chimeric TRIM5alpha(rh) protein containing the RING domain from the related human TRIM21 protein. This recombinant TRIM5-21R protein was expressed in SF-21 insect cells and purified through three chromatographic steps. Two distinct TRIM5-21R species were purified and shown to correspond to monomers and dimers, as analyzed by analytical ultracentrifugation. Chemically cross-linked recombinant TRIM5-21R dimers and mammalian-expressed TRIM5-21R and TRIM5alpha proteins exhibited similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities, indicating that mammalian TRIM5alpha proteins are predominantly dimeric. Purified TRIM5-21R had ubiquitin ligase activity and could autoubquitylate with different E2 ubiquitin conjugating enzymes in vitro. TRIM5-21R bound directly to synthetic capsids composed of recombinant HIV-1 CA-NC proteins and to authentic EIAV core particles. HIV-1 CA-NC assemblies bound dimeric TRIM5-21R better than either monomeric TRIM5-21R or TRIM5-21R constructs that lacked the SPRY domain or its V1 loop. Thus, our studies indicate that TRIM5alpha proteins are dimeric ubiquitin E3 ligases that recognize retroviral capsids through direct interactions mediated by the SPRY domain and demonstrate that these activities can be recapitulated in vitro using pure recombinant proteins.

An E3 ubiquitin ligase mediates the transfer of activated ubiquitin from an E2 ubiquitin-conjugating enzyme to its substrate lysine residues. Using a structure-based, yeast two-hybrid strategy, we discovered six previously unidentified interactions between the human heterodimeric RING E3 BRCA1-BARD1 and the human E2s UbcH6, Ube2e2, UbcM2, Ubc13, Ube2k and Ube2w. All six E2s bind directly to the BRCA1 RING motif and are active with BRCA1-BARD1 for autoubiquitination in vitro. Four of the E2s direct monoubiquitination of BRCA1. Ubc13-Mms2 and Ube2k direct the synthesis of Lys63- or Lys48-linked ubiquitin chains on BRCA1 and require an acceptor ubiquitin attached to BRCA1. Differences between the mono- and polyubiquitination activities of the BRCA1-interacting E2s correlate with their ability to bind ubiquitin noncovalently at a site distal to the active site. Thus, BRCA1 has the ability to direct the synthesis of specific polyubiquitin chain linkages, depending on the E2 bound to its RING.

Protein ubiquitination is a powerful regulatory modification that influences nearly every aspect of eukaryotic cell biology. The general pathway for ubiquitin (Ub) modification requires the sequential activities of a Ub-activating enzyme (E1), a Ub transfer enzyme (E2), and a Ub ligase (E3). The E2 must recognize both the E1 and a cognate E3 in addition to carrying activated Ub. These central functions are performed by a topologically conserved α/β-fold core domain of ∼150 residues shared by all E2s. However, as presented herein, the UbcH5 family of E2s can also bind Ub noncovalently on a surface well removed from the E2 active site. We present the solution structure of the UbcH5c/Ub noncovalent complex and demonstrate that this noncovalent interaction permits self-assembly of activated UbcH5c∼Ub molecules. Self-assembly has profound consequences for the processive formation of polyubiquitin (poly-Ub) chains in ubiquitination reactions directed by the breast and ovarian cancer tumor susceptibility protein BRCA1.