TRIM5alpha disrupts the structure of assembled HIV-1 capsid complexes in vitro

Abstract

The host restriction factor TRIM5alpha provides intrinsic defense against retroviral infections in mammalian cells. TRIM5alpha blocks infection by targeting the viral capsid after entry but prior to completion of reverse transcription, but whether this interaction directly alters the structure of the viral capsid is unknown. A previous study reported that rhesus macaque TRIM5alpha protein stably associates with cylindrical complexes formed by assembly of recombinant HIV-1 CA-NC protein in vitro and that restriction leads to accelerated HIV-1 uncoating in target cells. To gain further insight into the mechanism of TRIM5alpha-dependent restriction, we examined the structural effects of TRIM5 proteins on preassembled CA-NC complexes by electron microscopy. Incubation of assembled complexes with lysate of cells expressing the restrictive rhesus TRIM5alpha protein resulted in marked disruption of the normal cylindrical structure of the complexes. In contrast, incubation with lysate of control cells or cells expressing comparable levels of the nonrestrictive human TRIM5alpha protein had little effect on the complexes. Incubation with lysate of cells expressing the TRIMCyp restriction factor also disrupted the cylinders. The effect of TRIMCyp was prevented by the addition of cyclosporine, which inhibits binding of TRIMCyp to the HIV-1 capsid. Thus, disruption of CA-NC cylinders by TRIM5alpha and TRIMCyp was correlated with the specificity of restriction. Collectively, these results suggest that TRIM5alpha-dependent restriction of HIV-1 infection results from structural perturbation of the viral capsid leading to aberrant HIV-1 uncoating in target cells.

FIG. 1.

Structural effects of TRIM5α proteins on tubular CA-NC complexes. Electron microscopy analysis of reactions of preassembled CA-NC complexes incubated with lysate of 293T cells expressing rhesus or human TRIM5α proteins is shown. (A) Each reaction mixture was applied to a carbon mesh grid and negatively stained with 2% uranyl acetate. CA-NC complexes without lysate (mock), 293T lysate plus complexes (Control Lysate), human TRIM5α lysate plus complexes (TRIM5α_hu), and rhesus TRIM5α lysate plus complexes (TRIM5α_rh) are shown. Images shown are one representative of six different experiments. (B) Quantification of cylinders on the grids. An average number of cylinders per unit area was determined for 20 randomly selected images for each reaction. Shown are the mean values of six independent determinations, with error bars representing one standard deviation. (C) Immunoblot of TRIM5α proteins cosedimenting with CA-NC cylinders. TRIM5α proteins were detected with an HA tag-specific monoclonal antibody, and the CA-NC protein was detected with an HIV-1 CA-specific monoclonal antibody. (D) Immunoblot analysis of TRIM5α expression in 293T cells. Equivalent quantities of protein from the indicated cell lysates were subjected to immunoblot analysis with the HA tag-specific antibody. (E) Sedimentation of TRIM5α requires CA-NC tubes. Rhesus or human TRIM5α was incubated with (lanes 1 and 2) or without (lanes 3 to 5) preassembled CA-NC tubes, the reaction mixtures were subjected to centrifugation, and the pellets were analyzed by immunoblotting.

FIG. 2.

Effects of TRIMCyp on tubular CA-NC complexes. Lysate of 293T cells expressing owl monkey TRIMCyp were incubated with HIV-1 CA-NC complexes, and the reactions were analyzed by electron microscopy. (A) Representative EM images of the reactions. Mock, reaction mixture without lysate; +293T lysate, reaction mixture containing lysate of control 293T cells; TRIMCyp, lysate from TRIMCyp-expressing cells; TRIMCyp+CsA, lysate of 293T cells expressing TRIMCyp with CsA (5 μM) added. Images shown are one representative of six different experiments. (B) Quantification of cylinders on the grids. Shown are the mean values of six independent determinations, with error bars representing one standard deviation. (C) Immunoblot analysis of TRIMCyp cosedimenting with CA-NC complexes. Parallel samples without or with added CsA were analyzed.

FIG. 3.

Analysis of the effects of TRIMCyp on G89V mutant CA-NC complexes. Tubular complexes were assembled from a mutant recombinant CA-NC protein containing the G89V substitution in CA and incubated with the indicated cell lysates. Shown are the mean values and standard deviations from four independent experiments.