Journal: Journal of Virology

Article Title: SGIV envelope protein VP088 facilitated virus replication via interacting with other viral proteins and promoting p62-dependent autophagic degradation of TBK1

doi: 10.1128/jvi.01193-25

Figure Lengend Snippet: VP088 down-regulated host IFN response. ( A ) VP088 overexpression alone decreased the transcription levels of IFN- related genes. The mRNA levels of ISG15, ISG56, Viperin, and MX1 in pFlag-VP088-transfected GS cells were measured using qPCR. ( B ) The silencing effects of siRNA on VP088 protein synthesis during SGIV infection. GS cells transfected with siRNA-NC, si-VP088-1, si-VP088-2, or si-VP088-3 were infected with SGIV for 24 h and then collected for IB analysis using anti-VP088 antibody. ( C ) The transcription levels of IFN-related genes in VP088 silenced cells upon SGIV infection. GS cells were transfected with si-VP088 and then infected with SGIV for 24 h. Cells were collected for qPCR analysis to evaluate the mRNA levels of ISG15, ISG56, Viperin, and MX1. ( D–F ) VP088 decreased EccGAS-EcSTING-induced promoter activities of IFN1 ( D ), IFN3 ( E ), or ISRE ( F ). ( H–J ) VP088 reduced EcTBK1-induced promoter activities of IFN1 ( H ), IFN3 ( I ), or ISRE ( J ). Cells were co-transfected with the reporter gene plasmids (IFN1-Luc, IFN3-Luc, or ISRE-Luc), internal control plasmid pRL-SV40, pHA-VP088 and pFlag-EccGAS plus pGFP-EcSTING, or pGFP-EcTBK1 for 24 h, and then collected for dual fluorescence reporter gene assay. ( G and K ) VP088 decreased EccGAS-EcSTING ( G ) and EcTBK1 ( K ) induced IFN response. Cells were transfected with pHA-VP088 and pFlag-EccGAS plus pGFP-EcSTING or pGFP-EcTBK1 for 48 h, and then the mRNA levels of IFN-related genes were detected using qPCR. * P < 0.05.

Article Snippet: To evaluate the effect of VP088 knockdown on IFN immune response during SGIV infection, three specific small interfering RNA (siRNA) oligonucleotides targeting VP088 (si-VP088) and stealth RNAi negative control (siRNA-NC) were designed by Sangon Biotech (Shanghai), and the specific sequences were listed in .

Techniques: Over Expression, Transfection, Infection, Control, Plasmid Preparation, Fluorescence, Reporter Gene Assay

Journal: Journal of Virology

Article Title: SGIV envelope protein VP088 facilitated virus replication via interacting with other viral proteins and promoting p62-dependent autophagic degradation of TBK1

doi: 10.1128/jvi.01193-25

Figure Lengend Snippet: Involvement of Ecp62 in VP088-mediated autophagic degradation of EcTBK1. ( A ) VP088 degraded EcTBK1 in a dose-dependent manner. GS cells were co-transfected with pHA-EcTBK1 and pFlag-VP088 under different concentrations, and then the cells were collected for western blotting analysis. ( B ) The schematic diagram of the truncated mutants of EcTBK1. ( C ) The interaction between VP088 and different domains of EcTBK1. Cells were transfected with pGFP-VP088 and pHA-EcTBK, pHA-EcTBK1-∆C, or pHA-EcTBK1-∆N, and then the cells were collected for Co-IP assay and western blotting analysis. ( D ) Co-localization analysis between VP088 and different truncations of EcTBK1 in GS cells. Cells were transfected with pGFP-VP088 and pHA-EcTBK1-∆C or pHA-EcTBK1-∆N and then fixed at 48 h post-transfection for IFA. The samples were visualized under a confocal microscope. Scale bar, 5 µM. ( E–G ) The effects of different inhibitors, including MG132 ( E ), 3-MA ( F ), or NH 4 Cl ( G ), on the degradation effect of VP088 on exogenous EcTBK1. GS cells were co-transfected with pFlag-VP088 and pHA-EcTBK1, then treated without or with MG132, 3-MA, or NH 4 Cl, and collected for western blotting. ( H, I ) The interactions between Ecp62 and VP088 ( H ) or EcTBK1 ( I ) in vitro . GS cells were transfected with Ecp62 and VP088 or EcTBK1 and then collected for Co-IP assay and western blotting assay. ( J ) The silencing effect of siRNA on Ecp62 protein synthesis. GS cells were transfected with siRNA-NC, si-Ecp62-1, si-Ecp62-2, or si-Ecp62-3 and then collected for western blotting analysis. ( K ) Ecp62 was essential for the degradation effect of VP088 on EcTBK1. GS cells were co-transfected with si-Ecp62-1, pFlag-VP088, and pHA-EcTBK1, and then the cells were collected for western blotting analysis.

Article Snippet: To evaluate the effect of VP088 knockdown on IFN immune response during SGIV infection, three specific small interfering RNA (siRNA) oligonucleotides targeting VP088 (si-VP088) and stealth RNAi negative control (siRNA-NC) were designed by Sangon Biotech (Shanghai), and the specific sequences were listed in .

Techniques: Transfection, Western Blot, Co-Immunoprecipitation Assay, Microscopy, In Vitro