Supplementary MaterialsS1 Table: Patient data. presence of mutations known to modulate restriction by Mx2 and TRIM5. (a) Bar graph of the contingency table of mutations at the G116 position (Mx2) in individuals bearing B27/B57 or other alleles (Chi-square test; = 0.0169). (b) Bar graphs showing the presence or the absence of mutations previously shown to be associated with TRIM5 sensitivity according to the HLA status (Fishers exact test; = 0.0412).(PDF) ppat.1007398.s009.pdf (2.0M) GUID:?88EFAD25-D996-4E31-993E-DFC02CEECF4B S3 Fig: TRIM5 and Mx2 knockdown validation. (a) mRNA levels were determined by RT-qPCR and normalized on GAPDH mRNA levels. Shown are mean mRNA levels calculated by RT-qPCR performed in duplicates on total RNA extracted from IFN–treated Jurkat cells, and normalized to the shLuc control. (b) Same analysis in THP-1 cells.(PDF) ppat.1007398.s010.pdf (2.0M) GUID:?165FFAF6-48FF-4F59-8DF6-98EB15F0E9E2 S4 Fig: Sensitivity of NRC10GFP and NL43GFP to restriction by Mx2 and TRIM5. Jurkat cells knocked down for Mx2 or TRIM5 or both were infected with increasing amounts of the two HIV-1vectors. Infectivity was measured by FACS as the % of GFP+ cells 48 h post-infection.(PDF) ppat.1007398.s011.pdf (2.0M) GUID:?BD681744-EFC1-4FFD-B50F-F916C4BC762B S5 Fig: CRISPR/Cas9-mediated editing of TRIM5 in human cell lines. (a) Cas9 was targeted to exon 1 of the TRIM5 gene (green) by two selected gRNAs, whose binding sites are schematized with scissors. Arrowheads indicate the positions of the binding sites for SCR7 irreversible inhibition the ODNs used in the PCR-Surveyor assay. (b) Surveyor assay. Briefly, PCR products amplified from 293T cells transfected with pLCv2-hT5g1, pLCv2-hT5g2, or pLCv2-CAG (control) were subjected to denaturation, reannealing, and digestion with the SCR7 irreversible inhibition Surveyor enzyme. Arrows indicate cleavage products of Dicer1 the expected size. (c) Sanger sequencing analysis. THP-1 cells were transduced with lentiviral vectors SCR7 irreversible inhibition produced using pLCv2-hT5g2 or the control vector, pLCv2-CAG. Following puromycin selection, the targeted locus was PCR amplified and the PCR product was Sanger sequenced. The figure shows an alignment of the obtained sequence plots. (d, e) Decomposition of sequencing plots by TIDE assay. The graphs on the left show the percentages of aberrant peaks upstream and downstream of the cut site in the sequencing reactions shown in panel c in THP-1 (d) and in Jurkat (e) cells. The graphs on the right display the frequency of aberrant sequence signals in sequences in corresponding T5KO (test sequences in green) and control (CAG in black) cells. SCR7 irreversible inhibition The percentage of indel-containing alleles was computed by the TIDE assay. (f) T5KO and control THP-1 were infected with N-MLVGFP and B-MLVGFP. Infectivity was assessed by flow cytometry 72 h post-infection. (g) Knockdown validation for Ubc13 and TAK1 in TRIM5 knockout and control cells. mRNA levels were determined by RT-qPCR and normalized on GAPDH mRNA levels. Shown are mean mRNA levels calculated by RT-qPCR performed in duplicates on total RNA extracted from TRIM5-KO and control CAG THP-1 and Jurkat cells as indicated.(PDF) ppat.1007398.s012.pdf (2.0M) GUID:?E9B6D271-EF35-4FCA-A681-ED1F2E052871 S6 Fig: Effect of inhibitors on HIV-1 vector infectivity. THP-1 cells were pre-treated or not with (a) BX795 (iTBK1), (b) BAY11-7085 (iNF-B) or (c) SP600125 (iAP-1) for 1 h, infected with DsRed-expressing chimeric vectors (virus 1), and 48 h later infected with NRC1GFP (virus 2). Infectivity of DsRed-virus 1 was assessed by flow cytometry 48 h later. Data are from the same infections as those shown in Fig 5C, Fig 6D and Fig 6E, respectively.(PDF) ppat.1007398.s013.pdf (2.0M) GUID:?27959534-693A-4A23-956E-B9B4572A1549 S7 Fig: Productively infected cells in microscopy experiments. (a) Microscopy images corresponding to Fig 6A with the GFP field included. (b) Frequency of infected (GFP+) cells quantified by analyzing 100 cells from 10 pictures and plotted according to TRIM5 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) T5KO and control THP-1 cells were treated for 60 min with Raltegravir (RAL) or left untreated then infected with the GFP-expressing EC5-2 vector at a CRFK MOI = 2. Infectivity (% GFP+ cells) was measured by SCR7 irreversible inhibition FACS at 48 h post-infection.(PDF) ppat.1007398.s014.pdf (2.0M) GUID:?D1BA59D6-22AA-415A-84F4-CE32A6358D06 S8 Fig: Hypothetical model. Following entry, viruses from a B27/B57+ subject escape Mx2 restriction but are recognized by TRIM5. TRIM5 disrupts the proper uncoating process and may trigger pro-inflammatory signals through Ubc13- and TAK1-dependent.