The Interaction Between FHC And CSFV NS4B And The Effects Of It On The Propagation Of CSFV

Classical swine fever virus(CSFV)is the etiology of classical swine fever(CSF),which causes enormous economic losses to the pig industry in various countries and regions.CSF is a contagious disease of animals that must be reported ruled by the World Organization for Animal Health(OIE),and it is also a Class A animal disease in China.Although a few countries have eradicated CSF,but it still spreads in most swine-raised countries and regions.The preventive policy of CSF of China is a combination between vaccine inoculation and cul ing.In spite of the effects on prevention it has achieved,it appears a non-classical and mild CSFV,seriously affecting and constraining the development of the pig farming industry.Therefore,there is still an intense need for our country to explore an effective policy to prevent the spread of CSFV.Virus is intracellular parasitic microorganism which must rely on host cell to replicat e.Furthermore,it will provide new perspective to demonstrate the mechanisms of CSFV infect io n.Apoptosis is an essential and effective mechanism for host cell to resist viral infect io n.Many viruses have evolved efficient strategies to limit or delay the apoptosis process of host cell for persistent infection.Ferritin heavy chain(FHC)is a well-known anti-apoptotic protein,however,the role of FHC in CSFV infection has not been reported.In this study,in order to explore the interaction between FHC and CSFV NS4 B and the effects of FHC on CSFV propagation,we conducted a series assays and obtained the following results:(1)FHC interacts with CSFV NS4 B.The related plasmids were constructed,interact io n between FHC protein and CSFV NS4 B was confirmed by glutathione S-transferase(GST)-pull down(GST pull-down),co-immunoprecipitation(co-IP)assays.In addition,the co-localiza t io n between FHC and CSFV NS4 B was confirmed by confocal imaging assays.(2)FHC promotes the propagation of CSFV in PK-15 cells.Real time quantitive PCR(RT-qPCR),Western blot and Indirect immuno-fluorescence assays(IFA)were conducted to determine CSFV propagation in FHC-knockdown or FHC-overexpression PK-15 cells.The results showed that recombinant lentivirus-mediated knockdown or overexpression of FHC inhibited or enhanced CSFV propagation,respectively;Furthermore,both NS4 B expression and CSFV infection increased the expression of FHC.(3)FHC plays an anti-apoptotic role in CSFV infection.Apoptotic cell rates and ROS levels were determined by flow cytometry and Dihydroethidium(DHC)staining.The results showed that FHC plays an anti-apoptotic role in CSFV infection and FHC limits apoptosis by stabilizing cel ular ROS level.In conclusion,we found that FHC promotes CSFV propagation and interacts with the CSFV NS4 B protein.In addition,both NS4 B expression and CSFV infection inhibited ROSmediated apoptosis via upregulating FHC levels.