Fig 5. NiV-M interacts with TRIM6 and inhibits TRIM6-medited IFNβ induction and signaling.

A) NiV-M interacts with TRIM6. HEK293T cells were transfected with NiV-M, empty vector or HA-TRIM6. Cells were harvested and whole cell extracts (WCE) were used for TRIM6 immunoprecipitation using anti-HA-beads. (B-C) the C-terminal SPRY domain of TRIM6 interacts with NiV-M. B) TRIM6 deletion mutants used for coIP (GST-tagged). C) HEK293T cells were transfected with the GST-TRIM6 deletion mutants indicated together with NiV-M. Cells were harvested and WCE were used for TRIM6 immunoprecipitation using GST-beads. D) NiV-M co-localizes with TRIM6 in cytoplasmic bodies. HeLa cells were transfected with HA-TRIM6 and NiV-M-WT or NiV-M mutants K258A or K258R. Cells were fixed and stained with the indicated antibodies followed by confocal microscopy. Colocalization profiles are shown on the right. E) NiV-M inhibits the RIG-I induced IFNβ by targeting TRIM6. HEK293T were transfected with a non-targeting siRNA control (siControl) or aTRIM6-targeting siRNA (siTRIM6). After 24 hours, cells were transfected with IFNβ luciferase reporter and a Renilla control plasmid, in the presence or absence of NiV-M, or reconstitution with HA-TRIM6, followed by luciferase assay. Data were normalized by none stimulated sample to obtain fold induction. Depicted is the mean ± SD (n = 3). (F-G) NiV-M inhibits TRIM6-mediated IFNβ-induced ISG54 but not ISG15 reporter activity. HEK293T cells were transfected with ISG54-ISRE luciferase reporter (F) or ISG15 luciferase reporter (G) and a Renilla control plasmid and HA-TRIM6 in the presence of increasing concentrations of NiV-M-WT or NiV-MK258A, followed by luciferase assay. Data were normalized by none stimulated sample to obtain fold induction. Depicted is the mean ± SD (n = 3). Representatives of at least 2 independent experiments are shown. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001, by Student’s t test.