ATP synthase subunit ATP5B interacts with TGEV Nsp2 and acts as a negative regulator of TGEV replication
Description
Figure 1. Expression of TGEV Nsp2. Western blot analysis of IPEC-J2 cell lysates at different time points after overexpression of Nsp2 was performed using anti-HA antibody and anti-β-Actin antibody. Figure 2. Identification of the cellular proteins that interact with TGEV Nsp2 by immunoprecipitation (IP). (A) pCMV-HA-Nsp2 and empty vector pCMV-HA were transfected into IPEC-J2 cells. 24 h later, the cell protein samples were collected, and the immunoprecipitation (IP) test was carried out with Anti-HA Magnetic Beads. The obtained protein samples were dyed with protein glue silver. (B) Venn diagram of the identified protein candidates interacting with TGEV Nsp2 from mock infected. Figure 3. Construction and analysis of the protein-protein interactions (PPIs) network using STRING database. Figure 4. The annotation of proteins interacting with TGEV Nsp2 using Gene Ontology. Figure 5. Classification of the top 20 cellular protein-enriching KEGG pathways interacting with TGEV Nsp2. Figure 6. Confirmation of the interaction of TGEV Nsp2 with ATP5B and Citrin. (A)-(B) Plasmids expressing host cell protein were co-transfected with PCMV-HA- Nsp2 into HEK-293T cells, while plasmids co-transfected with host cell protein and pCMV-HA cells were used as negative controls. 24 h after transfection, Co-IP assay was performed using Anti-c-HA magnetic beads, followed by Western blot assay using HA and Myc monoclonal antibodies. (C)-(D) The plasmid expressing host cell protein and pCMV-HA-Nsp2 were co-transferred into IPEC-J2 cells, and the co-localization was observed by confocal laser microscopy 24 h after transfection. Figure 7. The expression of ATP5B was down-regulated after TGEV infection. The expression level of ATP5B in IPEC-J2 cells at different time points after TGEV infection was detected by QPCR (A) or Western blot (B). Figure 8. ATP5B inhibits TGEV replication. (A) Western blot and IFA (B) were used to detect ATP5B overexpression. pCMV-Myc-ATP5B and pCMV-Myc were transfected into IPEC-J2 cells for Western blot and IFA assay 24 hours later. (C) ATP5B was overexpressed in IPEC-J2 cells and TGEV was inoculated 24h later. Total cell cultures were collected at different time points (12h-72h) after infection, virus titer was detected, and a one-step growth dynamic curve was drawn. (D) Relative protein expression levels of ATP5B in cells transfected with SiRNAs against ATP5B and control SiRNA analyzed by western blot analysis with anti-ATP5B polyclonal antibodies. (E) Assay of cell viability after transfection with SiRNA. (F) SiATP5B was transfected into IPEC-J2 cells and TGEV was inoculated 24h later. Total cell cultures were collected at different time points (12h-72h) after infection, virus titer was detected, and a one-step growth dynamic curve was drawn.