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. Author manuscript; available in PMC: 2015 Aug 25.
Published in final edited form as: Dev Cell. 2014 Aug 7;30(4):394–409. doi: 10.1016/j.devcel.2014.06.013

Figure 7. Autophagy degrades protein target of TRIM5α in a manner requiring direct cargo recognition and mAtg8-interacting motifs in TRIM5α.

Figure 7

(A,B) Levels of intracellular p24 were determined by immunoblotting from rhesus cells (FRhK4) that had been subjected to knockdown of autophagy factors (Scr, scrambled siRNA) or TRIM5α and then exposed to pseudotyped HIV-1 for 4 h under full media or starvation conditions. (C,D) Immunoblot-based assessment of HIV-1 p24 in primary rhesus CD4+ T cells subjected to indicated knockdowns, infected with VSVG-pseudotyped HIV-1, and induced for autophagy by starvation for 4 h. (E,F) Luciferase activity of fed or starved FRhK4 cells subjected to indicated knockdowns and infected with luciferase-expressing pseudotyped HIV-1 (panel E) or SIVmac239 (panel F). (G,H) Binding of GST-GABARAP to a TRIM5α peptide array. A 20-mer peptide array corresponding to the entire TRIM5α sequence scanned in 3 amino acid residue shifts was subjected to a dot blot with GST-GABARAP as a probe. (H) Identification of a GABARAP interacting region on TRIM5α. Bars indicate peptide spots on the arrays with black bars indicating strongest binding (corresponding to the charcoal-framed dots in G) and gray bars to weaker binding (dashed-framed dots in G). Highlighted sequences, canonical LIR (LIR-1) and alternative LIR (LIR-2) involved in GST-GABARAP binding. (I) Mutational analysis of RhTRIM5α LIR motifs for effects on TRIM5α binding to GABARAP by GST pull-down. (J) High-content imaging analysis of the punctate distribution (vs. diffuse cytoplasmic) of GFP-tagged WT or double-LIR mutant (LIR-1*&2*) RhTRIM5α. (K) High-content analysis of the abundance of punctate p-ULK1 (Ser-317) in HeLa cells expressing GFP alone or GFP-RhTRIM5α (WT or LIR-1*&2*). (L) High-content analysis of the abundance of endogenous LC3B puncta in cells as in K. (M) Transfected 293T cells transiently expressing GFP alone or GFP-RhTRIM5α (WT or LIR-1*&2*) were infected with VSVG-pseudotyped HIV-1, induced for autophagy by starvation for 4 h, and levels of intracellular p24 were determined by immunoblotting. Data, means ± SE; n ≥ 3 experiments; *, P < 0.05; †, P ≥ 0.05 (t test or ANOVA). See also Figure S7.