Preliminary Study On The Interaction Between Major Structural Proteins Of Serotype 4 Fowl Adenovirus And Leghorn Male Hepatocellular Cells

Fowl adenoviruses(FAdVs)are members of Avian Adenovirus(Ad V)in Adenoviridae,and the serotype 4 fowl adenovirus(FAdV-4)caused serious Hydropericardium hepatitis syndrome(HHS)in poultry,resulting in a mortality rate of 80% in broilers in the 3-6 weeks age,which greatly threatened the poultry industry in China.At present,researches about FAdV-4 mainly focused on the pathogen detection and evolutionary analysis of viral genome.However,the interactions between FAdV-4 and host proteins,and the specific molecular mechanism are still unclear.In this study,we explored the interactions between host proteins in Leghorn male hepatocellular(LMH)cells and detected the effects of different host proteins on FAdV-4.Meanwhile,FAdV-4 Fiber1 polyclonal antibody was prepared to detect the expression of viral protein.This study is able to lay a theoretical foundation for the mechanism of FAdV-4 invasion into host cells and the in-depth study of FAdV-4 pathogenesis.Results are as follows:1.Preparation of polyclonal antibody against viral Fiber 1 protein of serotype 4 Fowl adenovirusIn this research,the Fiber 1 gene was amplified from FAdV-4 infected dead chicken tissues and constructed into the p Cold I prokaryotic expression vector.Then p Cold I-Fiber1 plasmid was transferred into Rosetta(DE3)expression strain and induced by 1m M IPTG at16 ℃ or 37 ℃.Fiber1 protein was successfully expressed in inclusion bodies detecting by SDS-PAGE with a clear band at 53 k Da.The fusion protein was immunized New Zealand white rabbits.After three immunizations,the positive rabbit sera were collected.Western blot analysis confirmed that the Fiber 1 antibody could interact with both purified Fiber 1protein and viral Fiber 1 protein when diluted at 1: 1 000 times.IFA analysis showed obvious green fluorescence when the Fiber 1 polyclonal antibody was diluted at 1: 3 000 times.2.Identification of the chicken host factors interacted with major structural proteins of FAdV-4Through liquid chromatography tandem-mass spectrometry(LC-MS/MS),we identified 141 host factors that could interact with these four viral proteins.The main interaction network between host proteins were obtained by the STRING software.KEGG and GO analysis were employed to enrich host proteins in the cell components,biological processes,participating pathways to deeply understand the regulations of host proteins in virus infection.After screening 34 high-scored host proteins,we verified that host proteins Hsp70 and DnaJC7 could significantly inhibit FAdV-4 replication.And the two host proteins affect each other’s expression,suggesting that they play a role in the form of molecular chaperone complex in FAdV-4 infection.Subsequently,both nucleotide binding domain(NBD)of Hsp70 and J domain of DnaJC7 were identified as the main functional domains for inhibiting FAdV-4 infection,and we further confirmed that the 383-450 aa site of DnaJC7 was essential for the inhibitory of J domain.3.Study on the interaction of FAdV-4 Hexon protein with Hsp70 and DnaJC7The Co-IP experiments verified that Hsp70,DnaJC7 and Hexon could interact with each other.And the laser confocal microscope experiments results showed the co-localization of Hsp70,DnaJC7 and Hexon in LMH cells.Further,GST pull down assay proved that DnaJC7 could directly interact with Hexon and Hsp70,while Hsp70 could only interact with Hexon in the presence of DnaJC7.Therefore,DnaJC7 played an important role for recruiting the Hexon protein and delivering it to Hsp70 as a bridge.Subsequently,the main interaction domains among Hsp70,DnaJC7 and Hexon were identified by Co-IP assay.Results showed that both NBD and SBD of Hsp70 could interact with DnaJC7 and Hexon.Both TPR and J domain of DnaJC7 interacted with Hexon and Hsp70.In addition,the infection of FAdV-4 induced the autophagy and enhanced the endogenous Hsp70 in a dose-dependent manner.Through using protein synthesis inhibitor cycloheximide(CHX)and autophagy inhibitor chloroquine(CQ)respectively,Hsp70 could promote the degradation of Hexon in cells,and the degradation was reduced when the autophagy pathway was blocked,which indicated that Hsp70 promoted the degradation of Hexon via autophagy pathway.In conclusion,the FAdV-4 Fiber 1 polyclonal antibody with a good reactivity,specificity and sensitivity was prepared successfully.Two host factors,Hsp70 and DnaJC7 significantly inhibited the FAdV-4 replication.Furthermore,Hsp70 interacted with viral Hexon through the assistant of DnaJC7 and promoted the degradation of the Hexon protein through autophagy pathway to inhibit viral replication.Meanwhile,the main inhibitory functional domains of Hsp70 and DnaJC7 as well as the major interaction domain between the host proteins and Hexon were explored.This data provided a novel way for antiviral drug targets and in-depth study of FAdV-4 pathogenic mechanism.