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. 2015 Dec 31;14(2):355–369. doi: 10.1016/j.celrep.2015.12.039

Figure 4.

Figure 4

SUMOylation-Dependent Mislocalization of TRIM5α in DCs Accounts for Lack of Restriction

(A) Human DCs were labeled with anti-TRIM5α antibody (above), or transduced with mRFP-TRIM5αhu for 48 hr (below). Two representative confocal images are shown for each condition. Scale bar represents 10 μm.

(B) Human DCs were transduced or not with mRFP-TRIM5αhu for 2 days and infected with EIAV-eGFP vector at MOI 1. Representative flow cytometry charts at 3 dpt are shown left, while the graph shows the mean of three independent experiments ± SEM.

(C) RM TRIM5α was fused in frame at its amino terminus with the first 112 aa of human coilin, comprising the Cajal body targeting domain (CBD) and a NLS. P4-CCR5 cells transduced with TRIP-TRIM5αrh, TRIP-Coilin-TRIM5αrh, or untransduced (NT) were labeled for TRIM5α for immunofluorescence detection and challenged with HIV-1 LAI with either wild-type envelope or VSV-G. β-galactosidase activity at 48 hpi is shown normalized by Bradford as the mean of two independent experiments carried out in triplicate ± SD.

(D) Co-localization of TRIM5α and Coilin was measured as in Figure 3B in human PBLs and DCs ± 10 or 100 μM GA for 24 hr. Graph shows Manders’ coefficients for the fraction of coilin-labeled Cajal bodies overlapping with TRIM5α, mean of three independent experiments ± SEM with a minimum of ∼50 cells analyzed for each condition. Statistical significance was assessed by unpaired t test (∗∗∗p ≤ 0.001 ∗∗p ≤ 0.01 ∗p ≤ 0.05; ns, non-significant). White arrows point to areas of co-localization.

(E) Human DCs were treated or not with 100 μM GA for 24 hr and infected with EIAV-eGFP vector at MOI 1. Representative flow cytometry charts at 3 dpt are shown left, while the graph shows the mean of three independent experiments ± SEM.

(F) Human DCs were lipofected or not with two different siRNAs targeting human TRIM5α (HM1 and HM3) or control siLuc (day 0) ± 100 μM GA (day 1) and EIAV-eGFP at MOI 1 (day 2). TRIM5α mRNA expression on day 3 is normalized for housekeeping transcript RPL13A (60S ribosomal protein L13a) with siLuc arbitrarily set to 1. GFP expression on day 4 is the mean of two experiments ± SD.