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. 2012 Nov 6;109(45):18372-7.
doi: 10.1073/pnas.1210903109. Epub 2012 Oct 22.

Structural insight into HIV-1 capsid recognition by rhesus TRIM5α

Haitao Yang  1 , Xiaoyun Ji, Gongpu Zhao, Jiying Ning, Qi Zhao, Christopher Aiken, Angela M Gronenborn, Peijun Zhang, Yong Xiong
Affiliations

Structural insight into HIV-1 capsid recognition by rhesus TRIM5α

Haitao Yang et al. Proc Natl Acad Sci U S A. .

Abstract

Tripartite motif protein isoform 5 alpha (TRIM5α) is a potent antiviral protein that restricts infection by HIV-1 and other retroviruses. TRIM5α recognizes the lattice of the retrovirus capsid through its B30.2 (PRY/SPRY) domain in a species-specific manner. Upon binding, TRIM5α induces premature disassembly of the viral capsid and activates the downstream innate immune response. We have determined the crystal structure of the rhesus TRIM5α PRY/SPRY domain that reveals essential features for capsid binding. Combined cryo-electron microscopy and biochemical data show that the monomeric rhesus TRIM5α PRY/SPRY, but not the human TRIM5α PRY/SPRY, can bind to HIV-1 capsid protein assemblies without causing disruption of the capsid. This suggests that the PRY/SPRY domain alone constitutes an important pattern-sensing component of TRIM5α that is capable of interacting with viral capsids of different curvatures. Our results provide molecular insights into the mechanisms of TRIM5α-mediated retroviral restriction.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
(A) Schematic depiction of dimeric TRIM5α. The four domains are colored differently and their respective molecular masses are indicated. The two linker regions are labeled L1 and L2. (B) Size-exclusion chromatograms of rhesus (solid) and human (dashed) MBP-TRIM5α PRY/SPRY. The molecular masses of protein standards are indicated at the top.
Fig. 2.
Fig. 2.
PRY/SPRYrh is a conserved module with unique structural features. (A) Two views of the crystal structure of PRY/SPRYrh in ribbon and surface representations. Density for V1 is not observed. An example conformation of the V1 loop obtained by homology modeling is shown by the black dashed line in the molecular surface (red). The arrowed line around this part of the structure indicates the conformational space available to V1. The variable regions (V1–V4) are colored in red. (Inset) Dimensions of the triangular-shaped core. (B) Four variable regions are shown separately by superimposing 15 SPRY-containing protein structures. TRIM5α is colored red and the others are grouped into two clusters, colored cyan and green, respectively. (C) Structure-based phylogenetic tree of the 15 SPRY-containing proteins [using the program SHP (26) and PHYLIP (27)]. The following structures with PDB ID in parentheses are included: PRY/SPRY-19q13.4.1 (2FBE), human TRIM21 (2IWG), murine TRIM21 (2VOK), TRIM20 (2WL1), TRIM72 (2KB5), Ash2L (3TOJ), SPRY-containing protein3 (2YYO), GUSTAVUS (2FNJ), GUSTAVUS/VASA (2IHS), SSB-1/VASA (3F2O), SSB-1/hPar-4 (2JK9), SSB-4 (2V24), human SSB-2/VASA (3EMW), and murine SSB-2 (3EK9). Protein names are color-coded according to B. Proteins analyzed in D are underlined. (D) Structure-based amino acid sequence alignment. The secondary structure elements of rhTRIM5α are indicated. Fully conserved residues are marked in the solid red box; similar residues are shown in red type. Residues that do not fit into the structure-based alignment are shaded in semitransparent red. Large protein-specific insertions were omitted in regions where orange dots are shown.
Fig. 3.
Fig. 3.
Interaction of TRIM5α PRY/SPRY domains with CA hexamers and wild-type CA tubular assemblies. (A) Size-exclusion chromatographic profiles of individual CA hexamers (CA concentration 400 μM) (black dashed line), PRY/SPRYrh (200 μM) (black solid line), and their mixture (gray solid line). (B and C) Binding of MBP-TRIM5α PRY/SPYR to preassembled wild-type CA tubes. Binding reactions were analyzed by SDS/PAGE using CA tubular assemblies (69 μM), incubated with MBP-PRY/SPRYrh (24 μM), MBP-PRY/SPRYhu (21 μM), and binding buffer (B) or MBP-PRY/SPRYrh at various concentrations of 5–52 μM (C). Samples of the reaction mixture before centrifugation (t), of supernatant (s), and of pellet (p) are shown. (D–G) Cryo-EM images of the reaction mixture. Low-dose projection image of wild-type CA tubes (72 µM) incubated with MBP-TRIM5α PRY/SPRY at nominal magnifications of 4,700× (D and F) and 59,000× (E and G). CA tubes are well separated (D) and decorated with protein density (E) upon binding of MBP-PRY/SPRYrh (24 µM). Incubation with MBP-PRY/SPRYhu (21 µM) yields bundled CA tubes (F) similar to CA assembly alone, and no additional protein density is observed on the CA tube surface (G). (Scale bars: 1 µm in D and F; 100 nm in E and G.)
Fig. 4.
Fig. 4.
Analysis of the interaction of the PRY/SPRYrh domain and assembled HIV CA tubes. (A) The potential binding surfaces on the PRY/SPRY domain and two neighboring HIV CA hexamers are colored in red and green, respectively, in intrahexamer (Upper) and interhexamer (Lower) binding scenarios. The molecules are drawn to the same scale and are shown in surface representations with the interaction elements marked. The HIV-1 CA model is from the hexamer crystal structure (PDB ID: 3H4E). (B) A conceptual model of two PRY/SPRY molecules (red and yellow surface) with each binding to a neighboring CA hexamer (tan: N-terminal domain; light blue: C-terminal domain) separately. (C) Side view of B. (D) A conceptual model for binding between the PRY/SPRY domain and interhexamer CA interfaces in different directions on an HIV-1 CA tubular assembly. Note that the binding surface in the PRY/SPRY core (red) fits well at the CA hexamer interface (buried in the top view of the CA hexamer assembly) and that the flexible V1 loop adopts various conformations to fit the varying curvature along different directions. The CA tube model was created by docking the crystal structure of HIV-1 CA hexamer (PDB ID: 3H4E) to the EM map of the HIV-CA helical tube (Electron Microscopy Data Bank accession code: EMD-5136). (E) A side view of PRY/SPRY binding to the interhexamer interface in the a-b direction in D. The CA-NTD is shown in ribbon representation. The arrow marks the range of distances that PRY/SPRY needs to span across neighboring CA hexamers in the HIV capsid. (F) A schematic depiction of how the dimeric TRIM5α causes the disruption of the HIV CA tubular assembly.
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