Molecular Mechanism Of PABPC4 Mediated Selective Autophagy To Inhibit CoVs Replication By Degrading CoV N Proteins

Porcine epidemic diarrhea virus(PEDV),which causes high morbidity and mortality in piglets,is an enteropathogenic coronavirus with economic importance.PABPC4(cytoplasmic poly(A)binding protein 4)is an mRNA binding protein,it is known to play important roles in the expression of erythroid differentiation,and regulation of telomerase activity and cell growth.The N protein is an RNA-binding protein that is involved in transcription,translation,and virus assembly.In the early stage of the laboratory,mass spectrometry analysis revealed that PABPC4 interacted with PEDV N protein.To further investigate the effect of PABPC4 on coronavirus replication,we conducted the following studies:1.SP1 regulates the expression of PABPC4.Through the amplification of PABPC4 promoter,dual luciferase test and fluorescence quantitative PCR,the results showed that SP1 is a transcription factor of PABPC4.2.PABPC4 inhibits PEDV replication and degrades PEDV N protein through the PABPC4-MARCH8-NDP52 autophagy pathway.First,overexpress and interfere with PABPC4 and infect PEDV in Vero cells and LLC-PK1 cells.The results showed that PEDV proliferation was not only inhibited by the overexpression of PABPC4 in Vero cells but also in LLC-PK15.Second,the Co-IP,Laser confocal and GST pull down tests.The test results showed that PABPC4 interacts with PEDV N protein.Third,PABPC4 was co-transferred to 293 T cells with PEDV N protein.The results showed that the levels of N protein decreased in a dose-dependent manner in response to PABPC4.These data demonstrate that PABPC4 is involved in regulating the level of PEDV N protein.Fourth,the plasmid of N protein and PABPC4 were co-transfected into HEK293 T cells,and the cells were treated with proteasome inhibitor and autophagy inhibitors.The results showed that the degradation of N protein may be mediated by the autophagy-lysosome pathway.Fifth,we co-transfected plasmids encoding FLAG-PABPC4 and HA-N into HEK 293 T cells.This result indicates that co-expression of PABPC4 and N protein could increase the levels of autophagy in HEK 293 T cells.Sixth,To examine whether the PABPC4 mediates the ubiquitination of N protein by recruiting MARCH8 and NDP52 onto the N protein,and the relationship between PABPC4 and autophagy pathway proteins was detected by Co-IP,GST Pull Down and Laser confocal test respectively.Our data suggest that PABPC4 targets the PEDV N protein and MARCH8 toward autophagic degradation by allowing their binding to the cargo receptor NDP52.Seventh,we individually transfected HEK 293 T cells with plasmids encoding HA-tagged N protein of PEDV,and FLAG-PABPC4,together with small interfering RNAs(NDP52,MARCH8,ATG5,and negative-control siRNA).These results suggest that the N protein of PEDV is degraded by autophagy through the PABPC4-MARCH8-NDP52-autophagosome pathway.3.PABPC4 inhibits coronavirus replication and extensively degrades CoV N protein through the PABPC4-MARCH8-NDP52 autophage pathway.First,Co-IP test was used to verify whether PABPC4 interacts with N proteins of different types of coronavirus.The results showed that PABPC4 interacts with N protein of different types of coronavirus.Second,PABPC4 was co-transferred to 293 T cells withCo V N protein.These data demonstrate that PABPC4 is involved in regulating the level of PEDV N protein.Third,the plasmid of SARS-CoV-2 N protein was co-transfected into HEK 293 T cells with PABPC4,and the cells were treated with the proteasome inhibitor and the autophagy inhibitors.Western blotting showed that the degradation of SARS-CoV-2 N protein might be mediated by the autophagy-lysosome pathway.Fourth,we individually transfected HEK 293 T cells with plasmids encoding HA-tagged N protein of CoV,and FLAG-PABPC4,together with small interfering RNAs(NDP52,MARCH8,ATG5,or NC siRNA).At 24 h post-transfection,western blotting was performed.The results showed that the PABPC4-MARCH8-NDP52-autophagosome pathway was broadly involved in Co V degradation.Fifth,In order to investigate whether PABPC4 regulates the levels of CoV N proteins during viral infection,cells were transfected with PABPC4-FLAG for 24 h and infected with Co Vs from different viral genera.These results suggest that PABPC4 plays an anti-viral role by reducing the level of N protein.In summary,this study proves that SP1 is a transcription factor of PABPC4 and directly in the regulation of PABPC4 expression.It has been confirmed that PABPC4 inhibits Co V replication through the PABPC4-MARCH8-NDP52 autophagosome pathway,which indicates that PABPC4 has a broad inhibitory effect on different coronavirus,and PABPC4-MARCH8-NDP52 autophagosome pathway is widely involved in the degradation of CoV.This not only provides a theoretical basis for the development of drugs for the prevention and control of coronavirus,but also further elucidates the new mechanism of host antagonism against coronavirus replication.