Vimentin inhibits α-tubulin acetylation via enhancing α-TAT1 degradation to suppress the replication of human parainfluenza virus type 3
Fig 3
VIM suppresses HPIV3 replication and inhibits formation of HPIV3 IBs.
(A) VIM did not impair the binding and entry of HPIV3. Schematic diagram of HPIV3 binding and entry assay in WT/VIM-KO A549 cells. Results for HPIV3 RNA levels were obtained using RT-qPCR, and the ratios of the relative changes normalized to the control group are shown. Three technical replicates were carried out in each analysis. At least three replicate experiments were performed. (B) VIM suppressed HPIV3 replication. An HPIV3 minigenome (MG) activity assay showed that VIM suppressed HPIV3 MG activity in a dose-dependent manner, but VIMΔH did not. HeLa cells infected with VTF7-3 were transfected with plasmids encoding N, P, L, and viral MG. “-” means that only P plasmid was not transfected; “+” means that N, P, L, and MG plasmids were all transfected. VIM and VIMΔH were both transfected in the “-” and “+” groups, to exclude their regulation of HPIV3 MG activity. Both protein expression and HPIV3 MG relative activity were detected. The activity of the “+” group was shown as 100% activity. (C) VIM suppressed the formation of HPIV3, but VIMΔH did not. Flag–VIM and Flag–VIMΔH were transfected alone or co-transfected with N-Myc and HA–P complex in HeLa cells, and an immunofluorescence assay was conducted. N-Myc was considered a component of IBs and indicated the location and sizes of IBs. Mouse anti-c-Myc antibody and rabbit anti-FLAG antibody were used. Scale bar: 20 μm. (D) The IB sizes were divided into large, medium, and small, and statistical analysis was carried out. IBs in three virus-infected fields were analyzed. Values are means ± SDs. (E) Live cell imaging showed that VIM suppressed the fusion of HPIV3 viral IBs. HeLa-GFP–P cell lines were infected with HPIV3, and mCherry–VIM was transiently expressed. GFP–P-labeled IBs adjacent to VIM are shown in the upper row, and IBs in non-VIM regions are shown in the lower row. The white arrows indicate IBs adjacent to VIM in the upper row and the IB fusion process in the lower row. Scale bar: 5 μm. (F) The IB sizes were divided into large, medium, and small, and statistical analysis was carried out. IBs adjacent to VIM in three areas were counted as shown in the S1 Movie. Values are means ± SDs. For panels A, D and F, Student’s t test was used with * p value<0.05, ** p value<0.01, *** p value<0.001, **** p value<0.0001, and ns = not significant.