General model for retroviral capsid pattern recognition by TRIM5 proteins

Permission to archive final published versionRestriction 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 recognitionYe

Mechanism of TRIM25 Catalytic Activation in the Antiviral RIG-I Pathway

SUMMARY Antiviral response pathways induce interferon by higher-order assembly of signaling complexes called signalosomes. Assembly of the RIG-I signalosome is regulated by K63-linked polyubiquitin chains, which are synthesized by the E3 ubiquitin ligase, TRIM25. We have previously shown that the TRIM25 coiled-coil domain is a stable, antiparallel dimer that positions two catalytic RING domains on opposite ends of an elongated rod. We now show that the RING domain is a separate self-association motif that engages ubiquitin-conjugated E2 enzymes as a dimer. RING dimerization is required for catalysis, TRIM25-mediated RIG-I ubiquitination, interferon induction, and antiviral activity. We also provide evidence that RING dimerization and E3 ligase activity are promoted by binding of the TRIM25 SPRY domain to the RIG-I effector domain. These results indicate that TRIM25 actively participates in higher-order assembly of the RIG-I signalosome and helps to fine-tune the efficiency of the RIG-I-mediated antiviral response

TRIM5α SPRY/coiled-coil interactions optimize avid retroviral capsid recognition

<div><p>Restriction factors are important components of intrinsic cellular defense mechanisms against viral pathogens. TRIM5α is a restriction factor that intercepts the incoming capsid cores of retroviruses such as HIV and provides an effective species-specific barrier to retroviral infection. The TRIM5α SPRY domain directly binds the capsid with only very weak, millimolar-level affinity, and productive capsid recognition therefore requires both TRIM5α dimerization and assembly of the dimers into a multivalent hexagonal lattice to promote avid binding. Here, we explore the important unresolved question of whether the SPRY domains are flexibly linked to the TRIM lattice or more precisely positioned to maximize avidity. Biochemical and biophysical experiments indicate that the linker segment connecting the SPRY domain to the coiled-coil domain adopts an α-helical fold, and that this helical portion mediates interactions between the two domains. Targeted mutations were generated to disrupt the putative packing interface without affecting dimerization or higher-order assembly, and we identified mutant proteins that were nevertheless deficient in capsid binding <i>in vitro</i> and restriction activity in cells. Our studies therefore support a model wherein substantial avidity gains during assembly-mediated capsid recognition by TRIM5α come in part from tailored spacing of tethered recognition domains.</p></div

Dimerization of CC-L2 mutants.

<p>Purified mutant proteins were analyzed by using SEC-MALS (size exclusion chromatography coupled with multi-angle light scattering). The solid curves represent the normalized UV absorbance trace (arbitrary units) of eluting components. The dotted curves show the population averaged molecular mass calculated from the measured protein concentration and light scattering data. Dashed gray lines indicate the expected masses of the monomer and dimer species. (A) Wildtype control. (B) D186A. (C) I193A. (D) E197A. The major peak had a substantial trailing edge indicating dissociation into monomers. (E) E201A.</p

Capsid binding activities of TRIM5-21R proteins.

<p>Representative results of pull-down assays. Purified TRIM5-21R (5 μM) was incubated with disulfide-stabilized HIV-1 CA tubes, fractionated by centrifugation, and visualized by SDS-PAGE with Coommassie staining. L, load; S, soluble fraction; P, pellet fraction. Band intensities were quantified by densitometry. Experiments were repeated at least 2 times for each mutant using independent protein preparations, with similar results.</p

Restriction activities rhesus TRIM5α proteins.

<p>HeLa cells that stably expressed the indicated HA-tagged TRIM5α proteins were infected with GFP-labeled HIV and the extent of viral replication was quantified. (A) Class I mutants. (B) Class II mutants. (C) Expression levels were quantified by immunoblotting. Experiments were repeated 6 times independently with similar results.</p

<i>In vitro</i> assembly activities of class II TRIM5-21R mutants.

<p>Purified TRIM5-21R proteins were incubated in assembly buffer overnight and the resulting precipitates were examined by negative stain electron microscopy. (A) I193A. (B) E201A. Insets: Fourier transforms of the associated images. Scale bars = 200 nm.</p

Differential scanning fluorimetry thermal stability profiles of purified TRIM5α proteins.

<p>(A) Comparison of CC-L2, CC-L2-SPRY, and SPRY constructs. (B) Effect of model-based mutations in context CC-L2. (C) Effect of model-based mutations in context of the restriction-competent TRIM5-21R protein.</p

Modeling of SPRY/coiled-coil packing in TRIM5α.

<p>(A) Ribbon model of the TRIM5α dimer, which was obtained by combining the crystal structures of the B-box/coiled-coil dimer [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006686#ppat.1006686.ref012" target="_blank">12</a>] and isolated SPRY domain [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006686#ppat.1006686.ref030" target="_blank">30</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006686#ppat.1006686.ref031" target="_blank">31</a>]. Domains and structural elements are colored as follows: RING, not included in model; B-box 2, orange; coiled-coil, green; L2 linker helix, gray; SPRY, blue. (B) Expanded view of the central region of the antiparallel dimer, in the same orientation as A. Residues selected for mutagenesis in this study are represented by sticks and transluscent spheres. Orange, class I residues; pink, class II residues. (C) Orthogonal view rotated as indicated, with individual residues labeled. The L2 helices are omitted in this panel for clarity. The two subunits are distinguished by an apostrophe. The coiled-coil dimer’s two-fold symmetry axis is indicated by the black oval.</p

Restriction activities of TRIMCyp proteins.

<p>(A) HeLa cells that stably expressed the indicated HA-tagged owl monkey TRIM5α proteins were infected with GFP-labeled HIV and the extent of viral replication was quantified. (B) Expression levels were quantified by immunoblotting. Experiments were repeated 2 times independently with similar results.</p