Fig 1.
Conformationally heterogeneous packing of the terminal L2 helix against the coiled-coil scaffold in context of the CC-L2 fragment of rhesus TRIM5α.
(A-D) Distance distribution curves show a single well-defined peak when R1 is attached to coiled-coil helix (W196R1). Multiple peaks are obtained when labels are appended to the indicated L2 helix residues (D288R1, B; E292R1, C; W300R1, D). Note that since the protein is a dimer, a single cysteine substitution can be used to attach two labels for distance measurements. The shaded regions represent fits to the DEER data that are within 15% root mean square deviation of the best fit (dark trace). Corresponding time-traces and subtracted dipolar evolutions are shown in S1 Fig. Expected distances calculated from the static structural model are indicated by the histograms in magenta. These are based upon all the available rotamers given the steric constraints in the model.
Fig 2.
Differential scanning fluorimetry thermal stability profiles of purified TRIM5α proteins.
(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.
Fig 3.
NMR analysis of N-terminal extensions of the isolated TRIM5α SPRY domain.
(A) Normalized backbone amide chemical shift differences in comparing rhesus TRIM5α constructs spanning residues 292–497 and 281–497. Chemical shift changes were also mapped onto the SPRY domain structure (PDB 2LM3 [30]). Dashed circles highlight the loop that undergoes the greatest changes, apart from the N-terminal residues. Black arrow indicates the trajectory of the putative extended helix. (B) Normalized chemical shift indices calculated from assigned Cα, Cβ, C (carbonyl), and Hα resonances by using the program PREDITOR [64], shown for each of the indicated residues in the putative L2/SPRY helix. Values indicate predicted secondary structure: 1 = β-strand, 0 = random coil, -1 = α-helix. (C) Sections of an 15N-edited 3-dimensional NOESY spectrum with well-resolved sequential Hα-HN cross-peaks. Resonance overlap precluded identification of cross-peaks for the entire helical sequence.
Fig 4.
Modeling of SPRY/coiled-coil packing in TRIM5α.
(A) Ribbon model of the TRIM5α dimer, which was obtained by combining the crystal structures of the B-box/coiled-coil dimer [12] and isolated SPRY domain [30, 31]. 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.
Table 1.
Functional phenotypes of TRIM5α mutants.
Fig 5.
Dimerization of CC-L2 mutants.
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.
Fig 6.
Capsid binding activities of TRIM5-21R proteins.
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.
Fig 7.
Cytoplasmic body assembly activities of YFP-TRIM5α proteins.
(A) The number of cytoplasmic bodies was counted in each cell and normalized to the intracellular YFP concentration as described [24]. (B) Representative image of HeLa cells stably expressing the wildtype control. (C) D186A. (D) I193A. (E) E201A. Cytoplasmic bodies appear as green puncta. DAPI was used to stain nuclei blue. Scale bars = 10 μ.
Fig 8.
In vitro assembly activities of class II TRIM5-21R mutants.
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.
Fig 9.
Restriction activities rhesus TRIM5α proteins.
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.
Fig 10.
Restriction activities of TRIMCyp proteins.
(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.