Fig. 3

ACTN4 inhibits SARS-CoV-2 replication and interacts with nsp12. a, d WB assays of ACTN4 and viral protein N protein levels. ACTN4 was knocked down (a) or overexpressed (d) in Huh7 cells infected with WT SARS-CoV-2 or the BA.5 mutant at MOI = 1. GAPDH was used as the loading control. b, c ACTN4 knockdown promotes viral replication. RT-qPCR was used to determine the SARS-CoV-2 WT (b) or BA.5 (c) RNA levels in WTAP knockdown Huh7 cells at 48 hpi. Relative levels of SARS-CoV-2 RNA were quantified using RT-qPCR with specific primers targeting the N gene and S gene. Data are means ± SEMs (n = 3). *P ≤ 0.05, **P ≤ 0.01, ***P < 0.001, one-way ANOVA. e, f Overexpression of ACTN4 inhibits viral replication. RT-qPCR was used to determine SARS-CoV-2 WT (e) or BA.5 (f) RNA levels in WTAP-overexpressing Huh7 cells at 48 hpi. Relative levels of SARS-CoV-2 RNA were quantified using RT-qPCR with specific primers targeting the N gene and S gene. Data are means ± SEMs (n = 3). *P ≤ 0.05, **P ≤ 0.01, ***P < 0.001, one-way ANOVA. g ACTN4 interacts with viral RdRp nsp12. Huh7 cells were transfected with pHA-nsp12 and pFlag-ACTN4. Co-IP was performed using anti-HA/anti-Flag Abs or IgG. The immuno-blots were probed with the anti-Flag or anti-HA Abs. Left panels were input, middle panels were incubated with anti-HA Abs then detected with anti-Flag/anti-HA Abs, right panels were incubated with anti-Flag Abs then detected with anti-HA/anti-Flag Abs. h Confocal microscopy images of Huh7 cells transfected with empty vector or pHA-nsp12. Co-staining was performed using an anti-ACTN4 Abs (green) and anti-HA Abs (red), together with Hoechst to stain the nucleus (blue). The scale on the picture indicates 10 μm