Fig 2: The establishment of a TRIM5-dependent antiviral state requires autophagy factors.Control or knockout THP-1 macrophages were primed with P90A (CrFK MOI 3) for 24 hours prior to infection with WT HIV-GFP (CrFK MOI 1) for two days. (A) Representative images from high content imager showing HIV-GFP exposed primed or unprimed THP-1 cell lines. Scale bar, 50 μm. (B) The percentage of cells showing GFP-positivity. Each data point represents one well of a 96 well plate (>2000 cells analyzed). Data shown is from one representative experiment. (C) The relative restriction imposed by P90A priming in control THP-1 macrophages or in THP-1 cell lines knocked out for the indicated genes as shown in (A,B). N = 4 independent experiments. (D) The impact of NF-κB, AP1, and autophagy inhibition on P90A-induced restriction. THP-1 cells were treated with BAY 117085 (NF-κB inhibitor; 10 μM), SP600125 (JNK/AP1 inhibitor; 10 μM) or 3-methyladenine (PI3 kinase/autophagy inhibitor; 1 mM) or DMSO (vehicle control) for 1 h prior to infection with P90A or mock infection. See also S4G Fig. Data, mean + SEM. N = 4 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; †, not significant by ANOVA.

Fig 3: Autophagy factors are required for TRIM5-driven inflammatory gene expression in macrophages.(A) Differentiated THP-1 cell lines stably expressing APEX2-V5 alone or APEX2-V5 tagged TRIM5 (human or rhesus) were infected with HIV-GFP and the percentage of infected (GFP-positive) cells was determined by high content imaging. White mask, automatically identified cell boundaries. Scale bar, 50 μm. Plot, the mean above-background GFP intensity per cell (a measure of transduction efficiency) following infection of increasing amounts of HIV-GFP. N = 5 independent biological replicates, >500 cells analyzed per replicate. (B-D) Quantitative PCR analysis of THP-1 macrophage cells stably transduced with lentiviruses expressing rhesus or human TRIM5 fused to APEX2 or APEX2 alone and subjected to the indicated siRNA-mediated knockdowns. The abundance of IFNB1 (B), NLRP1 (C), and IL6 (D) were determined relative to the abundance of 18S ribosomal RNA. Quantitative PCR analysis of knockdown efficiency is shown in S2C and S2D Fig. Data, each data point represents an independent biological replicate; *, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001; †, not significant by ANOVA.

Fig 4: Stimulation of TRIM5 signaling induces an antiviral state.(A) Schematic overview of experimental model for sequential viral “priming and challenge” experiments. PMA-differentiated THP-1 macrophages were cultured in the presence of the “priming” virus for 24 hours prior to subsequent “challenge” with GFP-expressing WT HIV-1 for next 48h. The percentage of cells showing GFP-positivity was determined by high content imaging. (B,C) THP-1 cells were primed with HIV-1 CA P90A (CrFK MOI = 3) prior to challenge with different CrFK MOI of WT HIV-1. Representative images (B) of mock infected, infected, or primed and infected cells captured by high content imager. Scale bar, 50 μm. Plot (C) shows the percentage of cells that become GFP-positive following infection with the challenge virus. (D) THP-1 cells were primed with VSV-G pseudotyped N-MLV (CrFK MOI = 0.5) and then challenged with WT HIV-1 at the indicated MOI. For C and D, each data point represents one well of a 96-well plate with >2000 cells analyzed per well. Data shown is from one representative experiment of 3. See also S4A and S4B Fig. (E) Differentiated THP-1 cells were primed or not with HIV-1 CA WT (TRIM5 resistant) or P90A (TRIM5 sensitive) and then challenged with HIV-1 GFP. The data are expressed as the ratio of infected cells before priming to relative to what is seen in unprimed cells (relative restriction). (F) The effect of TRIM5 knockout on the ability of priming with HIV-1 CA P90A to protect THP-1 macrophages from infection with WT HIV-1 GFP. (G) The relative restriction seen in P90A primed WT or TRIM5 KO THP-1 macrophages stably expressing APEX2 or TRIM5-APEX2 (TRIM5 add-back). For panels E-G, priming virus was used at a CrFK MOI of 3 and HIV-GFP infections were performed at a CrFK MOI of 1. (H) Q-RT PCR analysis of the impact of P90A (CrFK MOI = 3) or poly(I:C) priming on the permissivity of WT or TRIM5 KO THP-1 macrophages to Sendai virus infection. **, P < 0.01; ***, P < 0.001, ****, P < 0.0001; †, not significant by t test or ANOVA.

Fig 5: Multiple components of autophagy/lysosome system are involved in TRIM5 signaling.(A-E) The effect of the indicated knockdowns on TRIM5-mediated NF-κB and AP1 activation in a dual luciferase reporter assay system. Immunoblots demonstrating knockdown efficiency are shown in S1C Fig. (F) HEK293T cells were transfected as above and treated or not with the protease inhibitors e64d and pepstatin A (PepA) for 48 h prior to lysis. Both inhibitors were use at a concentration of 10 μM. Luciferase activity determined as above. Each data point represents an independent biological replicate. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; †, not significant by ANOVA.