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. 2008 Sep 17;82(23):11682–11694. doi: 10.1128/JVI.01562-08

FIG. 5.

FIG. 5.

TRIM5-21R binds CA-NC assemblies. (A) Schematic summary (above) and SDS-PAGE/Coomassie blue analyses (below) of purified full-length dimeric TRIM5-21R (lane 3, denoted WT), a dimeric TRIM5-21R construct missing part of the V1 loop (lane 2, denoted ΔV1), and a monomeric TRIM5-21R construct missing the entire SPRY domain (lane 1, denoted ΔSPRY). (B) TRIM5-21R protein binding to CA-NC/DNA assemblies. Monomeric (M) or dimeric (D) TRIM5-21R (WT, lanes 3 and 4 and lanes 7 and 8), TRIM5-21RΔV1 (ΔV1, lanes 2 and 6), or TRIM5-21RΔSPRY (ΔSPRY, lanes 1 and 5) proteins were incubated alone (lanes 1 to 4) or with CA-NC/DNA assemblies (lanes 5 to 8) and then centrifuged through a 70% sucrose cushion to separate CA-NC assemblies and bound proteins (pellet) from unbound and lower-molecular-weight proteins (supernatant). Western blots show the distribution of TRIM (α-FLAG, first, third, and fifth panels) and CA-NC (α-CA, second, fourth, and sixth panels). Note that CA-NC assemblies bound full-length TRIM5-21R constructs better than either TRIM5-21RΔV1 or TRIM5-21RΔSPRY. Ratios of bound CA-NC and TRIM5-21R proteins were obtained by combining known input protein concentrations, quantification of bound CA-NC levels using a standard curve of known CA-NC protein concentrations, and quantification of Western blot band intensities. Estimated bound ratios were as follows: CA-NC/TRIM5-21R dimer = 1.1, CA-NC/TRIM5-21R monomer = 0.8, and CA-NC/TRIM5-21RΔV = 0.18. (C) CA-NC/DNA assemblies bind better to dimeric TRIM5-21R proteins than to monomeric proteins under stringent conditions. Monomeric (lanes 2, 4, and 6) or dimeric (lanes 1, 3, and 5) proteins were incubated alone (lanes 1 and 2) or with CA-NC/DNA assemblies (lanes 3 to 6) and then centrifuged through a 70% sucrose cushion to separate CA-NC assemblies and bound proteins (pellet) from unbound and lower molecular weight proteins (supernatant). Western blots show the distribution of TRIM5-21R (α-FLAG, TRIM panels) and CA-NC (α-CA, CA-NC panels). The concentrations of TRIM5-21R proteins in lanes 5 and 6 were 3.33-fold higher than in lanes 3 and 4, and all binding assays were performed at lower protein concentrations than those shown in part (B) (see Materials and Methods). Note that under these conditions, CA-NC assemblies bound dimeric TRIM5-21R better than monomeric TRIM5-21R. The estimated ratios of bound proteins were as follows: CA-NC/TRIM5-21R dimer (0.15 μM input) = 0.025; CA-NC/TRIM5-21R monomer (0.15 μM input) = 0.01, CA-NC/TRIM5-21R dimer (0.5 μM input) = 0.068; and CA-NC/TRIM5-21R monomer (0.5 μM input) = 0.028.

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