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. 2011 Aug;85(16):8116–8132. doi: 10.1128/JVI.00341-11

Fig. 1.

Fig. 1.

A single RING domain residue determines the potency of SIVmac restriction by TRIM5αAGM variants. (A) The domains of TRIM5α are depicted above, with the locations of the major variable regions (v1, v2, and v3) of the B30.2(SPRY) domain represented. The amino acid residues that differ between TRIM5αAGM(Tan) (Tan) and TRIM5αAGM(Pyg) (Pyg) are shown in single-letter code below. The B30.2(SPRY) region chimeras are depicted. B, B-box2 domain. (B) The steady-state expression levels of the TRIM5α variants were determined by Western blotting with an antibody directed against the C-terminal HA epitope tag. As a reference control, the Western blots were probed with an antibody against β-actin. (C and D) HeLa cells expressing the TRIM5αAGM variants or transduced with the empty LPCX vector were incubated with the indicated volume of recombinant VSV G-pseudotyped SIVmac-GFP or HIV-1-GFP virus stocks. After 72 h, GFP-positive cells were analyzed by FACS. The results shown are representative of those obtained in three independent experiments. (E) The TRIM5α proteins in the lysates of stably expressing Cf2Th cells were incubated with sulfo-EGS at the indicated concentrations. The cross-linked TRIM5α proteins were analyzed by SDS-PAGE and Western blotting with an antibody directed against the C-terminal HA epitope tag. The positions of monomeric and dimeric TRIM5α proteins are indicated. (F) The abilities of the TRIM5α variants to bind HIV-1 capsid complexes were tested by incubating the input amounts of TRIM5α proteins with HIV-1 CA-NC complexes assembled in vitro. After centrifugation through a 70% sucrose cushion, the pellet was analyzed by Western blotting for the bound TRIM5α protein. The CA-NC protein was detected by Coomassie brilliant blue staining of the gel. (G) The half-lives of the indicated TRIM5α variants were investigated by incubating Cf2Th cells expressing the TRIM5α proteins at 37°C in cycloheximide (CHX)-containing medium for the indicated times. The cells were then placed on ice and lysed. Equal amounts of protein from the cell lysates were Western blotted with an antibody directed against the HA epitope tag.