Fig 2: RSV NS1 inhibits IFNβ response by inducing TUFM-dependent autophagy in vivo. (A–E) Wide-type and TUFM knockdown Balb/c mice were generated and then infected with RSV (3 × 106 PFU per mouse) . Relative expression of indicated proteins (A) and IFNβ, TUFM (B), and RSV N (D) mRNA, viral titers (C), pathology (E), fluorescence intensity of GFP and TUFM protein (F) of mouse lungs were analyzed via WB, qRT-PCR, viral plaque assay, H&E staining, and IHF, respectively. Scale bar = 40 µm (E)/50 µm (F). (G–J) ATG5WT or ATG5KO C57BL/6 mice were infected with AAV-NC or AAV-flag-NS1 (1 × 1011 AAV virions), followed by infection with RSV (3 × 106 PFU per mouse). Relative expression of indicated proteins (G) and IFNβ mRNA (H), viral titers (I), and pathology (J) of mouse lungs were analyzed via WB, qRT-PCR, viral plaque assay, and H&E staining, respectively. Scale bar = 40 µm (J). Results are means ± SD for five mice per group. *: P＜0.05; **: P＜0.01, ***: P＜0.001.

Fig 3: RSV NS1 could interact with mitochondria protein TUFM. (A) Cytosolic or mitochondrial localization of NS1 in stable NS1-expressing HEp-2 cells (HEp-2-NS1-OE) was analyzed via WB. (B, C, I, and J) HEK 293 cells were co-transfected with indicated plasmids for 48 h. Lysates were subjected to IP and analyzed via WB. (D) His or His-NS1 protein was incubated with lysates of HEK 293 cells. The mixture was subjected to Ni-NTA pulldown and analyzed via WB. (E) His or His-TUFM protein was incubated with lysates of HEK 293 cells, which was transfected with indicated plasmids for 48 h. The mixtures were subjected to Ni-NTA pulldown and analyzed via WB. (F and G) Transient NS1-expressing HEp-2 cells (F) or HEp-2-NS1-OE cells (G) were transfected with siNC or siTUFM for 16 h, mitochondrial and cytoplasmic proteins were analyzed via WB. Domain structures of wild-type TUFM and truncation mutants.