NF-κB and AP-1 are involved in the induction of the antiviral state in THP-1 cells.

(a) Representative IF microscopy staining of P-p65 and P-cJun in shTRIM5 and control shLuc PMA-differentiated THP-1 cells following infection for 48 h with NL43GFP and NRC10GFP (CRFK MOI = 10), as well as following infection with empty vectors. Where indicated, cells were pre-treated with Raltegravir for 1h. (b) Frequency of nuclear P-cJun and P-p65 quantified by analyzing ≥100 cells from ≥ 10 pictures and plotted according to TRIM5α expression and viral infection. The Kruskal-Wallis test and the Dunn's Multiple Comparison Test were used to assess statistical significance. Shown are means with SEM. noV = No virus, Vir = virus, RAL = Raltegravir, EV = empty vector. (c) Infectivity levels of virus 2 (NRC1GFP) in TRIM5-KO and control PMA-differentiated THP-1 cells pre-infected with NRC10Ds-Red. Differences in the % of GFP+ cells were analyzed using a two-way ANOVA. (d) THP-1 cells were treated with BAY11-7085 (“iNF-κB”) for 1 h and washed prior to infection with the patient-derived chimeric DsRed-expressing HIV-1 vectors (“virus 1”); 48 h later, they were exposed to NRC1GFP (“virus 2”). The fold inhibition of NRC1GFP infectivity was measured with or without inhibitor in T5KO and in CAG cells (p = 0.0085 in absence of inhibitor, n = 24; p = 0.2734 in presence of inhibitor, n = 8). The Wilcoxon's matched pair signed rank test was used. (e) THP-1 cells were pre-treated or not with SP600125 (“iJunB”) and washed before being infected with NRC10DsRed. 48 h later, cells were infected with NRC1GFP and the % GFP+ cells were analyzed by FACS. Bars are means +SEM, and individual values are shown as well. Experiments were performed three times in triplicates. Numbers represent the fold decrease in % GFP+ cells relative to mock-infected cells treated similarly. The Kruskal-Wallis and Dunn’s multiple comparison tests were used (*pp<0.0001).