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. 2011 Jan 11;108(2):534-9.
doi: 10.1073/pnas.1013426108. Epub 2010 Dec 27.

Hexagonal assembly of a restricting TRIM5alpha protein

Barbie K Ganser-Pornillos  1 , Viswanathan Chandrasekaran, Owen Pornillos, Joseph G Sodroski, Wesley I Sundquist, Mark Yeager
Affiliations

Hexagonal assembly of a restricting TRIM5alpha protein

Barbie K Ganser-Pornillos et al. Proc Natl Acad Sci U S A. .

Abstract

TRIM5α proteins are restriction factors that protect mammalian cells from retroviral infections by binding incoming viral capsids, accelerating their dissociation, and preventing reverse transcription of the viral genome. Individual TRIM5 isoforms can often protect cells against a broad range of retroviruses, as exemplified by rhesus monkey TRIM5α and its variant, TRIM5-21R, which recognize HIV-1 as well as several distantly related retroviruses. Although capsid recognition is not yet fully understood, previous work has shown that the C-terminal SPRY/B30.2 domain of dimeric TRIM5α binds directly to viral capsids, and that higher-order TRIM5α oligomerization appears to contribute to the efficiency of capsid recognition. Here, we report that recombinant TRIM5-21R spontaneously assembled into two-dimensional paracrystalline hexagonal lattices comprising open, six-sided rings. TRIM5-21R assembly did not require the C-terminal SPRY domain, but did require both protein dimerization and a B-box 2 residue (Arg121) previously implicated in TRIM5α restriction and higher-order assembly. Furthermore, TRIM5-21R assembly was promoted by binding to hexagonal arrays of the HIV-1 CA protein that mimic the surface of the viral capsid. We therefore propose that TRIM5α proteins have evolved to restrict a range of different retroviruses by assembling a deformable hexagonal scaffold that positions the capsid-binding domains to match the symmetry and spacing of the capsid surface lattice. Capsid recognition therefore involves a synergistic combination of direct binding interactions, avidity effects, templated assembly, and lattice complementarity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Schematic representation of the rhesus monkey TRIM5α protein. The four principal domains are illustrated as colored boxes: R, RING domain, yellow; B, B-box 2 domain, red; COIL, predicted coiled-coil domain, blue; SPRY/B30.2 domain, orange. The L1 and L2 linker regions are also labeled. The position of residue Arg121 within the B-box 2 domain is indicated by the arrow. In the TRIM5-21R construct used in this study, the RING domain of rhesus TRIM5α was replaced with the RING domain from human TRIM21.
Fig. 2.
Fig. 2.
Hexagonal assemblies of TRIM5-21R. (A) Representative negatively-stained EM image of a hexagonal array formed spontaneously by dimeric TRIM5-21R upon incubation on a carbon-coated grid. (Scale bar,100 nm). The red circle demarcates a region with clear hexagonal order, as shown in the computed Fourier transform (inset). (B) Fourier-filtered image of a TRIM5-21R array preserved in vitreous ice. (C) Projection density map of (B) shown in gray scale (with no imposed symmetry), illustrating that the hexagonal lattice is composed of six-sided rings. The unit cell parameters for the crystals are: (A) a = 347 Å, b = 335 Å, γ = 119.3°; (B) and (C) a = 348 Å, b = 346 Å, and γ = 120.5°.
Fig. 3.
Fig. 3.
TRIM5-21R binding to helical tubes of cysteine-crosslinked HIV-1 CA hexamers requires the SPRY domain and is enhanced by B-box 2 domain interactions. TRIM5-21R proteins were incubated in the absence of CA (lanes 1–3 and 7–9) or in the presence of a 6-fold molar excess of CA subunits assembled into helical tubes that mimic the hexagonal capsid lattice (lanes 4–6 and 10–12). Binding experiments were performed at two different TRIM5-21R protein concentrations (0.5 μM, lanes 1–6 or 1 μM, lanes 7–12) using either wild-type, full-length TRIM5-21R proteins (WT, lanes 1, 4, 7, and 10), TRIM5-21R proteins with the R121E mutation (R121E, lanes 2, 5, 8, and 11), or truncated TRIM5-21R proteins that lacked the SPRY domain (ΔSPRY, lanes 3, 6, 9, and 12). Pelletable CA and associated TRIM5-21R proteins (Pellet, 4% of total), were separated from unassembled and soluble CA proteins and unbound TRIM5-21R (Supernatant, 1% of total) by centrifugation, and analyzed by Western blotting, with the input levels of both proteins shown for reference (Input, 1% of total).
Fig. 4.
Fig. 4.
TRIM5-21R assembly is promoted by preformed, stabilized two-dimensional CA-NCA14C/E45C/W184A crystals that mimic the surface of the HIV-1 capsid. (A) Representative EM image of a CA-NC/TRIM5-21R cocrystal (1∶1 molar protein ratio, 24 h incubation). (B) Expanded view of the region boxed in red in (A). The contrast was stretched using Adobe Photoshop to enhance the clarity of the TRIM5-21R hexagons. (C) EM image of CA-NC alone at the same magnification as (B), shown for comparison. Scale bars,100 nm. (D) and (E) Computed Fourier transforms of the crystals shown in (B) and (C), respectively. In (D), diffraction from the TRIM5-21R lattice is evident (circled in green). Note that in this case, there are at least two stacked TRIM lattices (the first-order reflections of each correspond to a hexagonal lattice with ∼350 Å spacing), and at least three stacked CA lattices (a = 93.1 Å, b = 93.1 Å, and γ = 119.0°; a = 92.5 Å, b = 94.2 Å, and γ = 118.9°; a = 93.1 Å, b = 92.6 Å, and γ = 118.8°). The TRIM and CA diffraction patterns do not overlap in spatial frequency owing to the size of the TRIM lattice and its lack of long-range order. As expected, TRIM diffraction is absent in the transform of CA-NC alone, which shows two CA lattices (E).
Fig. 5.
Fig. 5.
Possible modes of interaction between the TRIM5-21R and CA lattices. (A) Fourier-filtered images of CA-NCA14C/E45C/W184A crystals alone (top) and TRIM5-21R crystals alone (bottom) illustrate the dramatic difference in unit cell size of the two lattices. (Scale bars,100 nm). (B) Assuming rigid, planar lattices, we can envision at least three different binding modes between the larger TRIM5-21R hexagonal lattice (colored) and the smaller CA lattice (gray). In these different binding modes, the TRIM5-21R lattice vectors (colored arrows) are rotated by either 0° (top), 30° (center), or 46.1° (bottom) relative to the CA lattice vectors (black arrows), and the interactions require TRIM5-21R lattice spacings of 360–400 Å, 312–346 Å, or 324–361 Å, respectively (given CA lattice spacings of 90–100 Å). The experimentally observed range of lattice spacings in our TRIM5-21R arrays is shown in Table 1. (C) Schematic model of an HIV-1 fullerene cone illustrating how TRIM5α could recognize the capsid lattice. Notice that only a small number of TRIM5α hexameric rings are required to create high avidity and that the lattice-matching geometry must be able to accommodate a greater radius of curvature in the outer TRIM5α lattice as compared to the inner CA lattice. Although the reconstructions do not allow us to position the different domains unambiguously, we modeled the coiled-coil domains within the narrowest density along each edge of the TRIM5α hexagon because the coiled-coil is expected to be the thinnest of the TRIM5α domains. The length of each hexagon edge (∼17 nm), together with the apparent twofold symmetry, suggests that each edge may be composed of two TRIM5α dimers, with coiled-coil elements linking L2/SPRY and RING/B-box 2 domains that occupy the regions of high local density at the midpoint and ends of each edge. We tentatively positioned six RING/B-box 2 domains at each threefold symmetry axis and four SPRY domains about the midpoint of each ring edge. Other models are possible, however, and higher resolution reconstructions will be required to define the TRIM lattice architecture unambiguously.
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