Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor

Abstract

The host restriction factor TRIM5alpha mediates species-specific, early blocks to retrovirus infection; susceptibility to these blocks is determined by viral capsid sequences. Here we demonstrate that TRIM5alpha variants from Old World monkeys specifically associate with the HIV type 1 (HIV-1) capsid and that this interaction depends on the TRIM5alpha B30.2 domain. Human and New World monkey TRIM5alpha proteins associated less efficiently with the HIV-1 capsid, accounting for the lack of restriction in cells of these species. After infection, the expression of a restricting TRIM5alpha in the target cells correlated with a decrease in the amount of particulate capsid in the cytosol. In some cases, this loss of particulate capsid was accompanied by a detectable increase in soluble capsid protein. Inhibiting the proteasome did not abrogate restriction. Thus, TRIM5alpha restricts retroviral infection by specifically recognizing the capsid and promoting its rapid, premature disassembly.

Fig. 1.

Specific association of TRIM5α variants with HIV-1-CA–NC complexes. (A) The diagram shows the domain structure of TRIM5α_rh. The sequences included in each of the mutant proteins are indicated by the solid lines. All of the proteins contain a C-terminal HA epitope tag. (B) In vitro-assembled CA–NC complexes were mixed with 293T lysates containing WT TRIM5α_rh-HA or the indicated TRIM5α_rh-HA deletion mutants and layered onto 70% sucrose before centrifugation. Immediately before mixing, an aliquot of the cell lysate was removed and blotted with α-HA antibodies to determine the steady-state expression levels of the different TRIM5 variants (input). After centrifugation, the pellet was resuspended in SDS sample buffer and analyzed by Western blotting with an anti-HA antibody (to detect TRIM5) or an anti-p24 antibody (to detect CA-NC). (C) An experiment similar to that described in B was carried out, except that the 293T cell lysates contained the indicated TRIM proteins. (D) An experiment similar to that described in B was carried out, except that the 293T cell lysates contained TRIMCyp-HA and were mixed with CA–NC complexes in the presence of 0.1% DMSO (−) or 25 μM cyclosporine (Cs) (+).

Fig. 2.

Assay to follow the fate of the retroviral capsid in infected cells. (A) Lysates from HeLa cells incubated with HIV-1(VSV-G) or HIV-1(Env⁻) viruses were cleared and analyzed on sucrose gradients, as described in Materials and Methods. A 100-μl sample (the “supernatant”) was collected from the very top of the gradient, avoiding the layer of endosomes/lysosomes at the interface of lysate and cushion. The pellet was also collected and resuspended directly in SDS sample buffer. (B) HeLa cells were incubated with HIV-1(VSV-G) or HIV-1(Env⁻) viruses at 4°C for 30 min and then at 37°C for 4 h. Cell lysates were prepared and analyzed on gradients consisting of the indicated concentrations of sucrose. The pellet was resuspended in sample buffer and Western blotted by using an anti-p24 antibody.

Fig. 3.

Effect of TRIM5α expression on the fate of the retroviral capsid in infected cells. (A) HeLa cells expressing an empty control vector (LPCX) or TRIM5α_rh-HA were incubated with HIV-1(VSV-G) or HIV-1(Env⁻) at 4°C for 30 min, and then at 37°C for the indicated times. Cell lysates were analyzed as described in Fig. 2A. Supernatants and pellets were Western blotted with an anti-p24 antibody. (B) NIH3T3 cells expressing an empty control vector (LPCX) or TRIM5α_hu-HA were incubated with N-MLV at 4°C for 30 min, and then at 37°C for the indicated times. Cell lysates were analyzed as described in Fig. 2A. Supernatants and pellets were Western blotted with an antibody directed against the p30 capsid protein. (C) HeLa cells expressing an empty control vector (LPCX) or the indicated TRIM5α proteins were incubated with HIV-1(VSV-G) or HIV-1(Env⁻) vectors at 37°C for 4 h, washed, and allowed to incubate for an additional 4 h at 37°C. Cell lysates were analyzed as in Fig. 2A. The supernatant and pellet were probed with anti-p24 antibodies. (D) HeLa cells expressing the indicated TRIM5 variants were incubated with SIV_mac(VSV-G) and SIV_mac(Env⁻) vectors and analyzed as described in C. The supernatant and pellet were probed with an antibody directed against the SIV p27 capsid protein. (E) HeLa cells expressing an empty control vector (LPCX) or TRIM5α_rh-HA were incubated as described in C with either WT HIV-1(VSV-G), WT HIV-1(Env⁻), or HIV-1(VSV-G) viruses with the G89A, H87Q, and Q50Y/T54Q (YQ) capsid proteins. Cell lysates were analyzed as in Fig. 2A, and the pellet and supernatant were probed with an anti-p24 antibody.