Impairment of TRIM5α_hu anti-HIV-1 activity by a positively charged residue at position 332. (A) The TRIM5α_hu protein is depicted, with the domains shown (B2, B-box 2). Residue numbers are indicated, as are the locations of the v1 to v3 variable regions of the B30.2 domain. The primary amino acid sequence of a segment of v1 is shown, with the changes in residue 332 for each of the mutants indicated. The analogous region of TRIM5α_rh is shown below for comparison. (B) The expression of the TRIM5α variants was measured by Western blotting HeLa cell lysates with an antibody directed against the HA epitope tag. As a control, the lysates were Western blotted with an anti-GAPDH antibody. (C) HeLa cells expressing the wild-type and mutant TRIM5α proteins or control HeLa cells transduced with the empty LPCX vector were incubated with various amounts of HIV-1-GFP or Moloney MLV (MLV-GFP). Infected GFP-positive cells were counted by FACS. The results of a typical experiment are shown. Similar results were obtained in three independent experiments. RT, reverse transcriptase.

Effects of the expression of TRIM5α_hu mutants on SIV_mac infection. (A and B) HeLa cells expressing the wild-type and mutant TRIM5α proteins or control HeLa cells transduced with the empty LPCX vector were incubated with various amounts of SIV-GFP. Infected GFP-positive cells were counted by FACS. The results of a typical experiment are shown. Similar results were obtained in three independent experiments. RT, reverse transcriptase.

Effects of deletion of TRIM5α_hu residue 332 on viral inhibition and capsid binding. (A) The expression of the TRIM5α variants in Cf2Th cells was measured by Western blotting cell lysates with an antibody directed against the HA epitope tag. As a control, the lysates were Western blotted with an anti-GAPDH antibody. (B and C) Cf2Th cells expressing the indicated TRIM5α variants or control Cf2Th cells transduced with the empty LPCX vector were incubated with various amounts of HIV-1-GFP (B) or SIV-GFP (C). Infected GFP-positive cells were counted by FACS. Reverse transcriptase (RT) activity (in counts per minute [in thousands] [Kcpm]) is shown. (D) HIV-1 CA-NC complexes were mixed with 293T cell lysates containing the indicated TRIM5α proteins and layered onto 70% sucrose cushions before centrifugation. The input and pelleted proteins were analyzed by Western blotting as described in the legend to Fig. 4.

Effects of amino acid substitutions adjacent to TRIM5α_hu residue 332 on HIV-1 infection. (A) A diagram of amino acid substitutions in the v1 region of the TRIM5α_hu B30.2 domain is shown. (B) The expression of the TRIM5α variants was measured by Western blotting HeLa cell lysates with an antibody directed against the HA epitope tag. As a control, the lysates were Western blotted with an anti-GAPDH antibody. (C) HeLa cells expressing the wild-type and mutant TRIM5α proteins or control HeLa cells transduced with the empty LPCX vector were incubated with various amounts of HIV-1-GFP or MLV-GFP. Infected GFP-positive cells were counted by FACS. The results of a typical experiment are shown. Similar results were obtained in three independent experiments. Reverse transcriptase (RT) activity (in counts per minute [in thousands] [Kcpm]) is shown.

Contribution of HIV-1 capsid binding to the HIV-1-restricting activity of TRIM5α_hu mutants. (A) Transmission electron microscopic image of negatively stained CA-NC complexes formed by the assembly of the HIV-1 CA-NC protein and DNA oligonucleotides. Conical structures are marked by the red arrows. (B) To determine the abilities of TRIM5α variants to bind the HIV-1 capsid complexes, in vitro-assembled HIV-1 CA-NC complexes were mixed with 293T cell lysates containing the TRIM5α proteins and layered onto 70% sucrose cushions before centrifugation. Immediately prior to mixing, aliquots of the cell lysates were removed and blotted with anti-HA antibodies to determine the steady-state expression levels of the different TRIM5α variants (Input). After centrifugation, the pellets were resuspended in SDS sample buffer and analyzed by Western blotting using an anti-HA antibody (to detect TRIM5α) or an anti-p24 antibody (to detect the HIV-1 CA-NC protein). (C, D, and E) The input cell lysates and pellets from the binding assays were analyzed for the presence of TRIM5α and the HIV-1 CA-NC protein by Western blotting. In panel E, the amount (in micrograms) of plasmid transfected into the 293T cells was varied as indicated to allow comparison of the HIV-1 capsid-binding ability of the wild-type and mutant TRIM5α_hu proteins.

Sensitivity of TRIM5α_hu R332P binding and restriction to a change in the HIV-1 capsid. (A) HeLa cells transduced with the empty LPCX vector or expressing TRIM5α_rh or TRIM5α_hu R332P were incubated with various amounts of wild-type (WT) HIV-1-GFP or HIV-1(P90A/A92E)-GFP. Infected GFP-positive cells were counted by FACS. The results of a typical experiment are shown. Similar results were obtained in three independent experiments. RT, reverse transcriptase. (B) Binding of TRIM5α_rh and TRIM5α_hu R332P to wild-type (WT) or P90A/A92E HIV-1 CA-NC complexes was measured using the assay described in the legend to Fig. 4B and Materials and Methods. Proteins in the input and pellet were detected by Western blotting.