VIM negatively regulates the acetylation of α-tubulin, inhibiting the formation of HPIV3 IBs.

(A) VIM downregulated α-tubulin acetylation. N-Myc, HA–P, and Flag–VIM plasmids were transfected alone or together, and the acetylation of α-tubulin was detected as well as the overexpressed proteins. (B) VIM downregulated the α-TAT1 protein level and the acetylation of α-tubulin. The expression of VIM protein in HeLa cells was gradually increased. The protein level of α-TAT1 and acetylation of α-tubulin were detected. Two endoplasmic reticulum proteins, calnexin and PDI, were also detected. (C) Acetylation level of α-tubulin in KD and VIM-KO cells. Acetylation of α-tubulin in WT/VIM-KD/VIM-KO cells (HeLa/A549) was detected. (D) Acetylated α-tubulin morphology in WT and VIM-KO cells. WT A549 and KO-VIM A549 cells were mixed (1:1), and the acetylated α-tubulin was detected using specific antibodies in the IF assay. Scale bar: 20 μm. (E) Fluorescence intensity of acetylated α-tubulin in (D) was analyzed using ImageJ, and the results are shown in the histogram with difference analysis. Ten visible fields of cells were observed and analyzed. (F) HPIV3 viral IBs fused to increase in size in the VIM-KO A549 cells, HPIV3HA-P-infected A549 cells, and A549 VIM-KO cells. HPIV3 viral IBs were identified with HA-tagged P protein using the IF assay. Cellular VIM was also observed. Rabbit anti-VIM antibody and mouse anti-HA antibody were used. Scale bar: 20 μm. (G) Inclusion body size statistical chart. HPIV3 IBs were divided into large, medium, and small; the numbers were counted; and statistical analysis carried out. IBs in three virus-infected fields were analyzed. (H) HPIV3 MG activity in VIM WT/KO A549 cells. HPIV3 MG activity assay was applied as previously described, and the relative change is shown. The activity of the “+” group in WT cells was shown as 100% activity. Values are means ± SDs from three experiments. For panels E, G, and H, Student’s t test was used and * p value<0.05, ** p value<0.01, *** p value<0.001, **** p value<0.0001, and ns = not significant.