a, Control or ∆FHL1 HAP1 cells were transfected with CHIKV-M-Gluc capped genomic RNA expressing Gaussia luciferase (Gluc). Gluc activity was monitored at the indicated time points. RLU, relative light units. Data are mean ± s.e.m. n = 3 independent experiments performed in quadruplicate. Two-tailed multiple t-tests with Holm-Sidak correction. b, Confocal microscopy of the colocalization of CHIKV nsP3 with FHL1 in fibroblasts inoculated with CHIKV nsP3–mCherry (MOI of 2), fixed 48 h after infection and stained with anti-FHL1 antibody. Images are representative of three experiments. c, Immunoassay of the interaction between CHIKV nsP3 and FHL1 isoforms in HEK293T cells transfected with Flag-tagged CHIKV nsP3 and either an empty vector or plasmids encoding the three HA-tagged FHL1 isoforms. Proteins from cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblot analysis with anti-Flag and anti-HA antibodies. d, Immunoassay of the interaction between CHIKV nsP3 and FHL2 in HEK293T cells transfected with Flag-tagged CHIKV nsP3 and either an empty vector or plasmids encoding HA-tagged FHL1 and FHL2. Proteins from cell lysates were immunoprecipitated with anti-Flag followed by immunoblot analysis with anti-Flag and anti-HA. e, Endogenous FHL1, G3BP1 or G3BP2 immunoprecipitation from control and ∆FHL1 HEK293T cells transfected with plasmids encoding Flag-tagged CHIKV, Sindbis (SINV) or Semliki forest virus (SFV) nsP3. Proteins from cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblot analysis with anti-Flag, anti-FHL1, anti-G3BP1 and anti-G3BP2 antibodies. f, Endogenous FHL1 immunoprecipitation from HEK293T cells transfected with plasmids encoding Flag-tagged full length CHIKV nsP3, CHIKV nsP3 carrying the SINV HVD (CHIKV/HVD-SINV) or Sindbis nsP3 carrying CHIKV HVD (SINV/HVD-CHIKV). Proteins from cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblot analysis with anti-Flag and anti-FHL1 antibodies. g, Purified GST-tagged nsP3 constructs and HA-tagged FHL1A detected by Coomassie blue staining. c–i, One representative experiment of three experiments is shown.