NMR spectroscopy, structure determination, and analysis.

All of the NMR spectra were recorded at 20°C on Varian INOVA 600, 800, and 900 spectrometers equipped with pulsed-field gradient triple-resonance probes. Sequence-specific resonance assignments were made using the standard triple-resonance techniques. The backbone assignment was achieved by the combined analysis of HNCO, (HCA)CO(CA)NH, HN(CO)CA, HNCACB, and CBCA(CO)NH spectra. The side chain resonances were identified by the combinational use of HBHANH, HBHA(CO)NH, (H)CC(CO)NH, (H)CCH-TOCSY, HCCH-TOCSY, and two-dimensional ¹H-¹⁵N HSQC and ¹H-¹³C HSQC spectra. Nuclear Overhauser effect (NOE) data for structure determination were extracted from three-dimensional ¹⁵N- and ¹³C-edited NOESY spectra, recorded with mixing times of 75 and 65 ms, respectively.

The NMRpipe software package (7) and the program KUJIRA (26), created on the basis of NMRView (21), was used for optimal visualization and spectral analysis. Automated NOE cross-peak assignments (18) and structure calculations with the torsion angle dynamics were performed using the software package CYANA2.0.17 (15).

Dihedral angle restraints were derived using the program TALOS (6). A total of 100 conformers were calculated independently. The 20 conformers with the lowest final CYANA target function values were finally selected. The structures were validated by using PROCHECK-NMR (28). The program MOLMOL (27) was used to analyze the resulting 20 conformers and to prepare drawings of the structures, unless noted otherwise in the legends.

The selected 20 conformers have been deposited in the Protein Data Bank (PDB entry 2YRG).

Dynamic light-scattering experiment.

The data were measured at 4°C by using a DynaPro light scattering system (Protein Solutions, Lakewood, NJ) and a microsampler, with a protein concentration of 2.2 mg/ml in 20 mM Tris-HCl buffer (pH 7.0) containing 100 mM NaCl and 1 mM iminodiacetic acid. Dynamics Version 5.0 software (Protein Solutions) was used in the data collection and analysis.

Analytical ultracentrifugation.

Sedimentation equilibrium experiments were performed on a Beckman Optima XL-I centrifuge, in an eight-position An-50Ti rotor with equilibrium six-channel centerpieces and quartz windows. Experiments were carried out at 4°C with the B-box 2 domain protein in solution at concentrations of 1.2, 0.8, 0.4, and 0.2 mg/ml and at rotor speeds of 20,000, 22,000, and 24,000 rpm for the dimeric molecular weight range and 30,000, 31,000, and 32,000 rpm for the monomeric molecular weight range. The data were corrected after 12, 14, and 16 h at each speed. Protein concentration profiles were monitored by the absorbance at 280 nm. To generate the baseline, the samples were then completely sedimented at 40,000 rpm for 6 h at 4°C. For the calculation of the molecular weight, corrected datasets were analyzed in the self-association model with a partial specific volume of 0.705 ml/g and a solvent density of 1.003 g/ml, using the Beckman XL-A/XL-I data analysis software (version 6.03).

Creation of cells stably expressing TRIM5α variants.

Retroviral vectors encoding wild-type or mutant rhesus monkey TRIM5α_rh proteins were created using the pLPCX vector. The TRIM5α_rh proteins contained an influenza hemagglutinin (HA) epitope tag at the C terminus or FLAG epitope tags at the N terminus. Recombinant viruses were produced in 293T cells by cotransfecting the pLPCX plasmids with the pVPack-GP and pVPack-VSV-G packaging plasmids (Stratagene). The pVPack-VSV-G plasmid encodes the vesicular stomatitis virus G envelope glycoprotein, which allows efficient entry into a wide range of vertebrate cells (59). Cf2Th canine thymocytes were transduced and selected in 5 μg of puromycin (Sigma)/ml.

Infection with viruses expressing GFP.

Recombinant HIV-1 and N-MLV expressing green fluorescent protein (GFP) were prepared as described previously (12, 50). All recombinant viruses were pseudotyped with the vesicular stomatitis virus G glycoprotein. For infections, 3 × 10⁴ Cf2Th cells seeded in 24-well plates were incubated at 37°C with virus for 24 h. Cells were washed and returned to culture for 48 h and then subjected to fluorescence-activated cell sorting (FACS) analysis with a FACScan (Becton Dickinson). HIV-1 and N-MLV viral stocks were titrated by serial dilution on Cf2Th cells to determine the concentration of infectious viruses.

Protein analysis.

Cellular proteins were extracted with radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris [pH 7.4], 100 mM NaCl, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1% NP-40, 2 mg of aprotinin/ml, 2 mg of leupeptin/ml, 1 mg of pepstatin A/ml, 100 mg of phenylmethylsulfonyl fluoride/ml). The cell lysates were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE; 10% acrylamide), followed by blotting onto nitrocellulose membranes (Amersham Pharmacia Biotech). Detection of protein by Western blotting utilized monoclonal antibodies directed against the hemagglutinin (HA) epitope tags (Roche) and FLAG epitope tags (Sigma) and monoclonal antibodies to β-actin (Sigma). All monoclonal antibodies were directly conjugated to horseradish peroxidase. Proteins were detected by enhanced chemiluminescence (NEN Life Sciences Products) using the FluorChem FC2 detection system (Alpha Innotech). Signals were acquired as an image file (TIFF) and quantified by using QuantityOne software (Bio-Rad Laboratories).

TRIM5α dimerization.

Approximately 10⁷ 293T cells transfected with 5 μg of plasmids expressing TRIM5α_rh variants were lysed in NP-40 lysis buffer (0.5% NP-40 in phosphate-buffered saline [PBS]) and cross-linked with different concentrations (0to 10 mM) of ethylene glycolbis-succinimidylsuccinate (EGS) for 30 min as previously described (10). The cross-linking reaction was stopped by adding 10 μl of 1 M Tris-HCl. After cross-linking, the samples were resuspended in 2× sample buffer, followed by incubation at 37°C for 30 min. Samples were analyzed by SDS-PAGE and Western blotting with an anti-HA antibody (Roche).

Higher-order self-association of TRIM5α.

Human 293T cells were independently transfected with plasmids encoding FLAG-tagged and HA-tagged mutant and wild-type TRIM5α_rh proteins. After 48 h, the cells expressing each TRIM5α_rh variant were lysed in 1 ml of whole-cell extract buffer (50 mM Tris [pH 8.0], 280 mM NaCl, 0.5% IGEPAL-10% glycerol, 1 mM DTT, protease inhibitor cocktail [Roche]). Lysates were centrifuged at 14,000 rpm for 1 h at 4°C. Post-spin lysates were then precleared using protein A-agarose (Sigma) for 1 h at 4°C; a small aliquot of each of these lysates was stored as an INPUT sample. Precleared lysates containing the differently tagged proteins were mixed in a 1/1 ratio and incubated with anti-FLAG-agarose beads (Sigma) for 2 h at 4°C to precipitate the FLAG-tagged proteins. Beads containing the immunoprecipitate were washed four times in whole-cell extract buffer. Subsequently, immune complexes were eluted using 200 μg of FLAG tripeptide/ml in whole-cell extract buffer. The eluted samples were separated by SDS-PAGE and analyzed by Western blotting using anti-HA or anti-FLAG antibodies conjugated to horseradish peroxidase.

TRIM5α turnover.

The turnover rates of wild-type and mutant TRIM5α proteins were estimated by pulse-chase experiments. Cf2Th cell lines stably expressing TRIM5 proteins were metabolically labeled for 30 min with 0.1 mCi of [³⁵S]methionine-cysteine (³⁵S protein-labeling mix; Perkin-Elmer)/ml in Dulbecco modified Eagle medium lacking methionine and cysteine and supplemented with 5% dialyzed fetal bovine serum. The cells were then chased for different time intervals in Dulbecco modified Eagle medium containing an excess of unlabeled methionine and cysteine. Cells were subsequently lysed in a modified RIPA buffer (140 mM NaCl, 8 mM Na₂HPO₄, 2 mM NaH₂PO₄, 1% NP-40, 0.25% SDS). Samples were subsequently immunoprecipitated with anti-HA antibody-Sepharose beads for 16 h at 4°C. After two washes with modified RIPA buffer, samples were separated by SDS-PAGE (Invitrogen) and analyzed by autoradiography and PhosphorImager (Molecular Dynamics).

Localization of wild-type and mutant TRIM5α proteins.

The localization of TRIM5α_rh and variants was investigated as previously described (8). Briefly, cells were grown overnight on 12-mm-diameter coverslips and fixed in 3.9% paraformaldehyde (Sigma) in PBS (Cellgro) for 30 min. Cells were washed in PBS, incubated in 0.1 M glycine (Sigma) for 10 min, washed in PBS, and permeabilized with 0.05% saponin (Sigma) for 30 min. Samples were blocked with 10% donkey serum (Dako, Carpinteria, CA) for 30 min and incubated for 1 h with antibodies. The anti-HA fluorescein isothiocyanate-conjugated 3F10 antibody (Roche) was used to stain HA-tagged proteins. Subsequently, samples were mounted for fluorescence microscopy by using the ProLong Antifade kit (Molecular Probes, Eugene, OR). Images were obtained with a Bio-Rad Radiance 2000 laser scanning confocal microscope with Nikon 60× N.A.1.4 optics.

HIV-1 CA-NC expression and purification.

The HIV-1 CA-NC protein was expressed, purified, and assembled as previously described (13, 14). The pET11a expression vector (Novagen) expressing the CA-NC protein of HIV-1 was used to transform BL-21(DE3) E. coli. CA-NC expression was induced with 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) when the culture reached an optical density of 0.6 at 600 nm. After 4 h of induction, the cells were harvested and resuspended in 20 mM Tris-HCl (pH 7.5), 1 μM ZnCl₂, 10 mM 2-mercaptoethanol, and protease inhibitors (Roche). Lysis was performed by sonication, and debris was pelleted for 30 min at 35,000 × g. Nucleic acids were stripped from the solution by using 0.11 equivalents of 2 M (NH₄)₂SO₄ and the same volume of 10% polyethylenimine. Nucleic acids were removed by stirring and centrifugation at 29,500 × g for 15 min. The protein was recovered by addition of 0.35 equivalents of saturated (NH₄)₂SO₄. The protein was centrifuged at 9,820 × g for 15 min and resuspended in 100 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 μM ZnCl₂, and 10 mM 2-mercaptoethanol. The CA-NC protein was dialyzed against 50 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 μM ZnCl₂, and 10 mM 2-mercaptoethanol, and stored at −80°C.

In vitro assembly of CA-NC complexes.

HIV-1 CA-NC particles were assembled in vitro by diluting the CA-NC protein to a concentration of 0.3 mM in 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, and 2 mg of DNA oligo-(TG)₅₀/ml. The mixture was incubated at 4°C overnight and centrifuged at 8,600 × g for 5 min. The pellet was resuspended in assembly buffer (50 mM Tris-HCl [pH 8.0], 0.5 M NaCl) at a final protein concentration of 0.15 mM (13, 14) and stored at 4°C until needed.

Binding of TRIM5α_rh variants to HIV-1 capsid complexes.

293T cells were transfected with plasmids expressing wild-type or mutant TRIM5α_rh proteins. At 48 h after transfection, cell lysates were prepared as follows: previously washed cells were resuspended in hypotonic lysis buffer (10 mM Tris [pH 7.4], 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT). The cell suspension was frozen, thawed, and incubated on ice for 10 min. Afterward, the lysate was centrifuged at full speed in a refrigerated Eppendorf microcentrifuge (~14,000 × g) for 5 min. The supernatant was supplemented with 1/10 volume of 10× PBS and then used in the binding assay. In some cases, samples containing the TRIM5α_rh variants were diluted with extracts prepared in parallel from untransfected cells. To test binding, 5 μl of CA-NC particles assembled in vitro were incubated with 200 μl of cell lysate at room temperature for 1 h. A fraction of this mixture was stored (input). The mixture was spun through a 70% sucrose cushion (70% sucrose, 1× PBS, and 0.5 mM DTT) at 100,000 × g in an SW55 rotor (Beckman) for 1 h at 4°C. After centrifugation, the supernatant was carefully removed and the pellet was resuspended in 1× SDS-PAGE loading buffer (pellet). The level of TRIM5α_rh proteins was determined by Western blotting with an anti-HA antibody, as described above. The level of HIV-1 CA-NC protein in the pellet was assessed by Western blotting with an anti-p24 CA antibody.

Concentration dependence of NMR signals.

The residues on the human TRIM5 B-box 2 protein exhibited broadened signals in the ¹H-¹⁵N HSQC spectrum; this contrasts with previous observations with other B-box 2 domains, which usually give sharp signals because of their compact size and stable fold (34). To investigate the basis of the observed broadening, the concentration dependence of the 1H-1D and ¹H-¹⁵N HSQC spectra was measured (Fig. ​(Fig.3A).3A). As the protein concentration decreased, the peaks of the amide protons from K101, L116, and W115 and side chain CH protons from L116 were shifted; the broadened peaks of the amide protons from L104 and E118, and the imino proton from the W115 indole ring, were sharpened or appeared. These results indicate that the B-box 2 domains associated via the cluster 1 region in the millimolar range of concentration and dissociated at lower concentrations (Fig. ​(Fig.3B).3B). We also observed additional chemical shift perturbations (CSPs) in the very low concentration range <100 μM) involving the very narrow region near E108 and H127; this phenomenon may result from the zinc-mediated B-box 2 association at H127 (see below).

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FIG. 3.

HSQC perturbation analysis in concentration-dependent experiments. (A) Eleven ¹H-¹⁵N HSQC spectra were measured at protein concentrations of 660, 560, 490, 400, 300, 250, 170, 110, 40, 20, and 10 μM and superimposed, with colors ranging from purple (highest concentration) to green (lowest concentration). Signal appearances were observed for the backbone amide proton of E118 and imino proton of the W115 indole ring at the lower concentrations. Chemical shift changes and sharpening were observed for other signals. (B) Mapping of perturbed residues on the B-box 2 structure. In the figure on the left, the size of the CSP is indicated by color. The red color means more than 0.03 ppm of CSP were observed, and yellow indicates more than 0.01 ppm of CSP. The magenta color indicates the appearance of the signal at a lower concentration. Most of the implicated residues are proximal to cluster 1 on one face of the B-box 2 domain (dotted circle on the electrostatic surface on the right). (C) Equilibrium analytical ultracentrifugation of the TRIM5 B-box 2 domain. The radial distribution of the absorbance in the centrifuge cell at equilibrium at 22,000 rpm, with a protein concentration of 1.2 mg/ml (~180 μM), is shown in the lower panel. The red line through the data represents the fit to a single species with a molecular weight corresponding to 10,502; the theoretical molecular weight for the single polypeptide chain is 6,682. The upper panel shows the residuals for the fit. (D) The concentration dependence of chemical shift perturbation for the W115 backbone amide proton in ¹H-¹⁵N HSQC spectra. Total chemical shift change was calculated by using the relation: Δδ_tot = [(δ_HN)² + (δ_N/6.5)²]^1/2 (1). Curve fitting was carried out by using the program gnuplot. The calculated equilibrium dissociation constant K_D was 9.33 ± 5.40 mM (see the text).

Size and affinity of the associated state of the B-box 2 domain.

To estimate the oligomeric state of the associated B-box 2 domain in solution, we performed a high-speed sedimentation equilibrium study by analytical ultracentrifugation (Fig. ​(Fig.3C).3C). The radial distributions at 20,000, 22,000, and 24,000 rpm with a B-box 2 domain concentration of 1.2 mg/ml (~180 μM) fitted to a molecular mass of 1.05 × 10⁴ Da, which corresponds to 1.57 molecules of the B-box 2 domain. This implies that the B-box 2 domain exists between monomeric and dimeric forms in solution at this concentration. In addition, dynamic light scattering experiments revealed that the B-box 2 domain in solution had a monomodal profile with a narrow peak corresponding to particles with an average hydrodynamic diameter of 3.3 nm and an estimated molecular mass of 11 kDa (data not shown). Although it is difficult to calculate an exact molecular mass by dynamic light scattering, this result is consistent with the TRIM5 B-box 2 domain existing in solution at an average size between those of monomers and dimers. The partial dimer content of TRIM5 B-box 2 solutions may reflect a zinc-mediated self-association of the B-box 2 domain, because we detected a chemical shift change specifically in H127 in the low protein concentration range (<100 μM); in contrast, we did not observe any additional chemical shift changes in other residues in the concentration range from 10 to 660 μM (data not shown). We also found that reducing the amount of the weak chelator iminodiacetic acid in the buffer resulted in a much higher average molecular mass in the sedimentation equilibrium measurement and reduced solubility of the B-box 2 protein (data not shown). These results support the interpretation that most of the dimer estimated from the sedimentation equilibrium data resulted from zinc-mediated association. Collectively, the results suggest two types of B-box association, a zinc-mediated association and a direct association involving the region from W115 to E118. The zinc-mediated association appears to exhibit a higher affinity but affects the HSQC spectra less. The direct association has a lower affinity but causes major concentration-dependent shifts of NMR peaks that reflected the dynamic equilibrium between zinc-mediated associates.

Since the zinc-mediated association did not cause CSPs except for H127, the CSP data in the other areas of the domain could be fitted independently. The equilibrium dissociation constant (K_D) was calculated to be 9.33 ± 5.40 mM using gnuplot in the dimer model by the analysis of chemical shift changes of the W115 amide proton in the concentration-dependent two-dimensional ¹H-¹⁵N HSQC spectra (Fig. ​(Fig.3D).3D). The fitting error was very large because of the low affinity and limited solubility of the protein. We estimated that only ca. 10% of the protein contributed to dimerization even at the highest concentration (0.66 mM). This explains the small observed chemical shift changes, even though the indole ring of W115 is apparently included in the binding interface. The small chemical shift changes of the backbone amide protons also suggest that the association does not involve a change of the protein fold and is stabilized by inter-side chain interactions.

Mutagenic analysis of the TRIM5α_rh B-box 2 surface.

To investigate the functional implications of the TRIM5 B-box structure, we modified specific amino acid residues of TRIM5α_rh. We chose the TRIM5α_rh protein because it potently restricts HIV-1 infection (50), allowing quantitation of phenotypes over a wide dynamic range. Moreover, most of the assays to evaluate TRIM5α phenotypes have been developed using the rhesus monkey protein as a prototype (11, 32, 36, 50, 51). Because the TRIM5α_rh and TRIM5α_hu B-box 2 primary amino acid sequences differ by only one residue (see the alignment in Fig. ​Fig.2A),2A), structure-function relationships are expected to be similar between these two domains. Amino acid residues predicted to be surface accessible on the TRIM5α_rh B-box 2 domain, including those predicted to reside in clusters 1 and 2, were altered (Fig. ​(Fig.2B).2B). Particular attention was paid to the charged residues surrounding cluster 1, with various combinations of charge substitutions being introduced into this region. The wild-type and mutant TRIM5α_rh proteins were expressed stably in Cf2Th canine thymocytes, which do not express a TRIM5 ortholog (45). All of the TRIM5α_rh mutants studied were expressed at least as well as the wild-type TRIM5α_rh protein (see Fig. S1 in the supplemental material). The steady-state levels of expression of several of the B-box 2 mutants were greater than that of wild-type TRIM5α_rh, which has a half-life of ~48 min (see Fig. S2 in the supplemental material). Indeed, prolonged half-lives, relative to that of the wild-type TRIM5α_rh, were associated with many of the TRIM5α mutants (Table ​(Table2).2). This result is consistent with previous studies that implicated the TRIM5α B-box 2 domain in the regulation of TRIM5α turnover (10, 11). All of the TRIM5α mutants tested formed dimers detected by EGS cross-linking (see Fig. S3 in the supplemental material and Table ​Table2).2). This result is consistent with previous studies indicating that the TRIM5α B-box 2 domain can be deleted without disrupting dimerization (10).

Retroviral restriction by TRIM5α_rh B-box 2 mutants.

To examine the ability of the TRIM5α_rh B-box 2 mutants to inhibit retroviral infection, Cf2Th cells expressing these mutants were challenged with recombinant HIV-1 expressing GFP. The TRIM5α_rh mutants exhibited a range of HIV-1-restricting abilities (Fig. ​(Fig.4),4), and are rank-ordered in Table ​Table22 according to restriction potency. Substitution of an acidic residue for arginine 121 (R121E and R121D) completely eliminated the ability of TRIM5α_rh to inhibit HIV-1 infection, as has been previously seen (10, 32). Of note, some changes in the glutamic acid residues at 102 and 120, which flank the hydrophobic patch in the cluster 1 region, restored various degrees of HIV-1-restricting activity to the R121D or R121E mutants (Table ​(Table2).2). For TRIM5α_rh mutants with an acidic residue at position 121, an arginine substitution for glutamic acid 120 resulted in the best improvement in HIV-1 restriction; lysine and aspartic acid substitutions at residue 120 were less effective. An arginine substitution for E102 was as effective as an arginine substitution for E120 in restoring HIV-1-restricting activity to TRIM5α_rh variants with the R121E change. Thus, while the presence of three negatively charged residues at positions 102, 120, and 121 was associated with only a low level of TRIM5α_rh function, arginine substitutions at position 102 or 120 could partially compensate for the presence of an acidic residue at position 121.

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FIG. 4.

Restriction of HIV-1 infection by TRIM5α_rh mutants. Cf2Th cells were transduced with the LPCX vector expressing HA-tagged wild-type and mutant TRIM5α_rh proteins. Control cells were transduced with the empty LPCX vector. Stably expressing cell lines were selected with 5 μg of puromycin/ml. The cells were incubated with HIV-1-GFP, and the percentage of GFP-positive cells was measured 48 h later by FACS. The results of two independent experiments were similar; the results of a single experiment are shown.

The TRIM5α_rh mutants that retained the wild-type arginine or a lysine residue at position 121 exhibited HIV-1-restricting ability, regardless of the particular amino acid at residue 120. However, the wild-type glutamic acid and aspartic acid residues at position 120 generally allowed the most potent HIV-1 restriction in this case. The alteration of glutamic acid 102 to arginine resulted in only a slight decrease in the potency of HIV-1 restriction when the wild-type arginine 121 was retained on TRIM5α_rh. These results indicate that the nature of the charged residues that rim the hydrophobic center of cluster 1 influence the efficiency of restriction by TRIM5α_rh.

Of the hydrophobic B-box 2 residues that were altered, changes in W117 and L118, in the cluster 1 hydrophobic patch, exerted the greatest impact on HIV-1-restricting activity. Thus, the TRIM5α_rh B-box 2 residues important for HIV-1 restriction (arginine 121, leucine 118, and tryptophan 117) occupy a localized area on the domain surface.

The TRIM5α_rh B-box 2 mutants exhibited a range of HIV-1-restricting activities. The ability of the mutants to restrict N-MLV infection was examined. The wild-type TRIM5α_rh protein exhibited a modest level of restriction against N-MLV (Table ​(Table2).2). Whereas a few of the B-box 2 mutants that exhibited wild-type anti-HIV-1 activity retained the ability to inhibit N-MLV weakly, most of the mutants were devoid of detectable N-MLV-restricting ability. Thus, several of the mutants, including R121K, exhibited a conditional phenotype, restricting HIV-1 potently but exhibiting no inhibitory activity against the more weakly restricted N-MLV.

Inhibition of HIV-1 reverse transcription by TRIM5α_rh mutants.

Potent HIV-1 restriction by TRIM5α_rh occurs prior to the initiation of reverse transcription (22, 50). To examine the ability of the TRIM5α_rh B-box 2 mutants to block HIV-1 reverse transcription, we assayed the level of late reverse transcripts in mutant-expressing cells challenged with HIV-1-GFP. The percentage of GFP-positive cells was assessed in parallel to provide an indicator of successful infection (Fig. ​(Fig.5).5). HIV-1 reverse transcripts were not detected in any of the cells expressing potently restricting TRIM5α_rh B-box 2 mutants (Table ​(Table2).2). In contrast, in cells expressing B-box 2 mutants that did not restrict HIV-1 infection, the levels of HIV-1 reverse transcripts were similar to those in control cells not expressing TRIM5α_rh (Table ​(Table2).2). Intermediate levels of reverse transcripts were found in most cells expressing TRIM5α_rh mutants with modest HIV-1-restricting ability (Table ​(Table2).2). Thus, for this panel of TRIM5α_rh B-box 2 mutants, the levels of HIV-1 late reverse transcripts were lowest when the degree of restriction was most potent.

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FIG. 5.

Blockade of HIV-1 reverse transcription by the TRIM5α_rh B-box 2 mutants. Cf2Th cells expressing the indicated wild-type and mutant TRIM5α_rh proteins or containing the empty LPCX vector were challenged at a multiplicity of infection of 0.4 with DNase-pretreated HIV-1-GFP. After 6 h, the cells were lysed, and the total DNA was extracted. (A) The levels of viral DNA were measured by quantitative real-time PCR, using a probe against GFP, as described in Materials and Methods. The means and standard deviations of the results derived from three independent experiments performed in triplicate are shown. (B) In some cases, infections were left to proceed for 48 h, and infectivity was measured as the percentage of GFP-positive cells by FACS. The values shown represent the means and standard deviations of the results obtained in three independent experiments.

Effect of TRIM5α B-box 2 changes on HIV-1 capsid binding.

Based on early, qualitative assays measuring the binding of TRIM5α mutants to HIV-1 capsid complexes, an effector function was proposed for the TRIM5α B-box 2 domain (10). Subsequently, when more quantitative capsid-binding assays were utilized, the effects of B-box 2 changes on capsid binding were recognized (32, 33). Because the expression of some TRIM5α B-box 2 mutants is elevated compared to that of the wild-type protein (9), the input levels of TRIM5α variants must be adjusted to compare capsid-binding abilities accurately. We tested the hypothesis that the major function of the TRIM5α B-box 2 domain in retrovirus restriction is to increase the avidity of capsid binding. To this end, the ability of the panel of TRIM5α_rh B-box 2 mutants to bind HIV-1 CA-NC complexes assembled in vitro was assessed. The level of each TRIM5α_rh variant in the cell lysate was first measured, and each lysate was diluted with control cell lysates so that comparable levels of TRIM5α proteins were present in each input sample. The adjusted cell lysates containing the TRIM5α_rh variants were then serially diluted and incubated with assembled HIV-1 CA-NC complexes. The mixtures were layered on a 70% sucrose cushion and centrifuged. The amount of TRIM5α_rh protein that cosedimented with the HIV-1 CA-NC complexes was measured. Comparison of the input and bound TRIM5α_rh levels for each mutant with those of the wild-type TRIM5α_rh protein allowed an estimation of the relative capsid-binding affinity of the mutants (Fig. ​(Fig.6).6). The relative capsid-binding ability of each mutant was calculated at the lowest input concentration of the mutant protein that yielded a detectable amount of capsid binding, thus allowing a comparison among the mutants (Fig. ​(Fig.7A7A and Table ​Table2).2). A strong correlation (r_s = 0.8421, P < 0.001) between binding to HIV-1 capsid complexes and HIV-1 restriction was observed for the panel of TRIM5α_rh variants (Fig. ​(Fig.7B).7B). These results support the hypothesis that the enhancement of capsid-binding avidity represents a major contribution of the TRIM5α B-box 2 domain to retroviral restriction.

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FIG. 6.

Binding of TRIM5α_rh B-box 2 mutants to assembled HIV-1 capsids. (A) 293T cells were transfected with plasmids expressing the indicated wild-type and mutant TRIM5α_rh proteins tagged with HA epitopes. At 36 h after transfection, the cells were lysed. Serial dilutions of the cleared lysates were made by mixing with cleared lysates from untransfected 293T cells. The lysates were incubated at room temperature for 1 h with HIV-1 CA-NC complexes that had been assembled in vitro. The mixtures were applied to a 70% sucrose cushion and centrifuged. Input represents the mixtures analyzed by Western blotting before being applied to the 70% cushion. The input mixtures were Western blotted for the HA tag. The pellet from the 70% cushion (pellet) was analyzed by Western blotting with antibodies against the HA tag and HIV-1 CA-NC protein. (B) The Western blots shown in panel A were quantitated as described in Materials and Methods. The amounts of TRIM5α_rh protein in the input and pellet (bound) fractions are shown in arbitrary units.

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FIG. 7.

Binding of additional TRIM5α_rh mutants to assembled HIV-1 capsid complexes. (A) The binding of the indicated TRIM5α_rh variants to HIV-1 CA-NC complexes assembled in vitro were assessed as described in the Fig. ​Fig.66 legend and Materials and Methods. (B) The relative capsid-binding ability and HIV-1-restricting activity of each TRIM5α_rh B-box 2 variant were assessed as described in the Table ​Table22 footnotes and in Materials and Methods. The means for the relative capsid-binding abilities associated with each HIV-1 restriction group are indicated by horizontal bars. The Spearman rank correlation coefficient, r_s, is 0.8421, with a 95% confidence interval of 0.779 to 0.905 (two-sided P value of <0.001).

Some of the B-box 2 mutants were expressed at higher steady-state levels than the wild-type TRIM5α_rh protein (see Fig. S1 in the supplemental material). Although this variable is compensated for by adjusting the input levels of TRIM5α_rh in the capsid-binding assay, varying expression levels could influence the assessment of HIV-1 restriction potency. Thus, we reanalyzed the data in Fig. ​Fig.7B,7B, separating the TRIM5α_rh mutants into those that were highly expressed and those expressed at wild-type TRIM5α_rh levels. The strength of the correlation was not improved by taking expression level into consideration (data not shown). Thus, TRIM5α_rh overexpression only minimally affects HIV-1 restriction potency in this context.

Higher-order association of TRIM5α_rh B-box 2 mutants.

The B-box 2 domain has been shown to be important for the ability of TRIM5α dimers to associate in higher-order complexes (32). To test the ability of the B-box 2 mutants to form higher-order complexes with the wild-type TRIM5α_rh proteins, cell lysates containing FLAG-tagged wild-type TRIM5α_rh and HA-tagged mutant TRIM5α_rh proteins were mixed. After precipitation with an anti-FLAG antibody, the precipitates were Western blotted with antibodies directed against the FLAG and HA epitope tags. The wild-type TRIM5α_rh-HA protein was efficiently coprecipitated by the anti-FLAG antibody (Fig. ​(Fig.8),8), which is consistent with the ability of wild-type TRIM5α_rh to self-associate in higher-order complexes. Of the TRIM5α_rh mutants tested, only E120D was able to associate efficiently with wild-type TRIM5α_rh (Fig. ​(Fig.88 and Table ​Table2).2). Apparently, the ability of TRIM5α_rh to form higher-order hetero-oligomers with the wild-type TRIM5α_rh protein is very sensitive to changes in different surface residues of the B-box 2 domain.

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FIG. 8.

Higher-order self-association of TRIM5α_rh B-box 2 mutants. 293T cells were transfected with plasmids expressing the indicated wild-type or mutant TRIM5α_rh proteins with a FLAG or an HA epitope tag. Cells expressing wild-type and mutant TRIM5α_rh proteins were lysed 48 h after transfection. (A to D) The cell lysates were mixed, and the indicated mixtures were used for immunoprecipitation by an antibody directed against the FLAG epitope, as described in Materials and Methods. Elution of the immunocomplexes was performed with a FLAG tripeptide and analyzed by Western blotting with anti-HA and anti-FLAG antibodies. WB, Western blot; IP, immunoprecipitation. (E) The higher-order self-association and HIV-1-restricting ability, determined as described in the Table ​Table22 footnotes and in Materials and Methods, of each TRIM5α_rh variant were compared. The means of the values for higher-order self-association within each restriction group are indicated by horizontal bars. The Spearman rank correlation coefficient, r_s, is 0.862, with a 95% confidence interval of 0.665 to 1.0 (two-sided P value of <0.001).

We examined the ability of a subset of TRIM5α_rh mutants to self-associate into higher-order homo- oligomers (Fig. ​(Fig.8D8D and see Fig. S4 in the supplemental material). TRIM5α_rh mutants that exhibited efficient ability to form higher-order homo- oligomers restricted retrovirus infection with modest or high efficiency (Table ​(Table2).2). Conversely, most of the weakly restricting TRIM5α mutants either did not self-associate into higher-order homo-oligomers or did so weakly. Thus, there is a significant correlation between homologous higher-order self-association of the TRIM5α_rh variants and HIV-1-restricting ability (r_s = 0.862, P < 0.001) (Fig. ​(Fig.8E).8E). This observation is consistent with a model in which homologous higher-order self-association contributes to the HIV-1-restricting ability of TRIM5α.

We thank Takashi Umehara for analytical ultracentrifugation and dynamic light scattering measurements and useful discussions and Toshio Nagashima for molecular dynamics calculation of the association model and useful discussions. We also thank Satoru Watanabe, Takushi Harada, Takeshi Nagir, Yasuko Tomo, Masaomi Ikari, Kazuharu Hanada, Yukiko Fujikura, and Akiko Tanaka for sample preparation and showing the screening data of B-box 2 domains. We thank Yvette McLaughlin and Elizabeth Carpelan for manuscript preparation.

We acknowledge the National Institutes of Health (grants AI063987 and AI076094 and a Center for AIDS Research Award AI60354), the International AIDS Vaccine Initiative, the Bristol-Myers Squibb Foundation, and the late William F. McCarty-Cooper for research funding. F.D.-G. is a recipient of a K99/R00 Pathway to Independence Award from the National Institutes of Health (1K99MH086162-01), an American Foundation for AIDS Research Mathilde Krim fellowship in basic biomedical research (106987-43-RFHF), and a Claudia Adams Barr award from the Dana-Farber Cancer Institute. The B-box 2 structure determination was supported by the Riken Structural Genomics/Proteomics Initiative of the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science, and Technology of Japan.