Figure 4: Deletion of N-terminally truncated isoforms triggers aggregation and activation of full-length MAVS.

(a) A diagram showing various MAVS mutants with methionine replaced by leucine, including MAVS-(M2L), MAVS-(M2&6L), MAVS-(M2-5L), MAVS-(M2-6L), which abolish the production of corresponding N-terminally truncated forms. (b) Plasmids encoding MAVS, MAVS-(M2L), MAVS-(M2&6L), MAVS-(M2-5L), MAVS-(M2-6L) were transfected into HEK293T Mavs^−/− cells. Cells were collected 36 h post transfection. P5 fractions were isolated and used to analyse various MAVS isoforms by immunoblotting with anti-MAVS-(C), anti-MAVS-(M) and anti-MAVS-(N) antibodies. The original full blot can be found in Supplementary Fig. 12c. (c,d) Transfection was performed as described in b. After cells were collected, RNA was extracted and qPCR was performed to measure IFN induction (c). P5 fractions were isolated to examine MAVS aggregation and activity in inducing IRF3 dimerization in vitro (d). (e,f) Plasmid encoding MAVS-(M2-6L) was transfected into HEK293T Mavs^−/− cells, together with pcDNA3-flag-sumo or increasing amounts of pcDNA3-flag-sumo-MAVS-TM. As described in (c), IFN induction was measured by qPCR (e). P5 fractions were used to examine MAVS aggregation and activity in inducing IRF3 dimerization in vitro (f). Cell lysates were subjected to SDS-PAGE and immunoblotting. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student’s t-test. **P<0.01 and ***P<0.001. N.S. indicates no statistically significant difference.