Fig. 6
TRIM5α is SUMOylated on residue K84 within a predicted pSUM consensus motif. a Schematic representation of the human TRIM5α protein domains (to scale). The 4 lysines within putative consensus SUMO modification sequences are indicated in red. The corresponding sequences in rhesus TRIM5α are indicated on the right for comparison purposes. b HEK 293T (original blots in Supplementary Figure 5a) and c MDTF cells (original blots in Supplementary Figure 5b) were transfected with the indicated pcDNA human TRIM5α expression constructs. Inputs were monitored by Western blotting using anti-Flag and -GAPDH antibodies. TRIM5α was immunoprecipitated using anti-Flag beads as in Fig. 1a. TRIM5α SUMOylation was assessed by Western blotting on immunoprecipitated TRIM5α using anti-Flag and -SUMO1 antibodies. The TRIM5α*S1 band was readily detectable for all TRIM5α mutants but was noticeably reduced for the K84R mutant, indicating that SUMO is conjugated at this residue. Western blots are representative of 2 independent experiments. d SUMOylation on K84 is required for the antiviral function of TRIM5α. MDTF cells were transfected with the wild-type and mutant human TRIM5α constructs then infected with N-MLV at MOI 2. Top panels show representative fields of wide-field microscopy. Equal transfection efficiency was monitored by flow cytometry (plots below). Mean fluorescence intensity (MFI) was quantified by Fiji software. The graph shows the values from 3 independent fields ± SD and is representative of 2 independent experiments. Human TRIM5α and the K10R, K111R, and K167R mutants all restricted N-MLV, whereas the K84R mutant lost the ability to do so. Scale bar = 50 μm
