| Foot and mouth disease(FMD) is an acute and highly infectious diseases in cloven-hoofed animals infected with Foot and mouth disease virus(FMDV). Innate immunity is the first line to defense the virus infection. PRRs recognize viral nucleic acids or viral products to activate series of cascade downstream, and induce the production of I-IFN and cytokines to play an antiviral effect.Autophagy is an important life activity to maintain homeostasis in eukaryotic cells. It is a degradation pathway mainly rely on lysosomes. By engulfing the biological macromolecules the body does not required, autophgosome is formed, and it fuses with lysosomes to degrade the contents. The new study found that autophagy is closely related to the occurrence of many diseases, such as cancer,infectious diseases and liver dysfunction.Autophagy as having the function of pathogen clearance, is linked together with innate immunity to help the body to resist pathogenic microorganism infection.ATG5-ATG12 conjugate is an essential complex in the process of autophagy, which plays an important role in the extending autophagic membranes. Studies have shown that downregulation of ATG5-ATG12 can block autophagy effectively, and many of the functions of autophagy may related with it, for example, ATG5 involved in regulating the expression of type I interferons. Studying the mechanism of cellular ATG5-ATG12 anti-FMDV response can provide new ideas and theoretical basis for the research of antiviral drugs and the development of vaccines.Previous studies have shown that FMDV activate autophagy in human cancer cell lines. In the present study, we choosed porcine kidney passage cells(PK-15) who was FMDV classic host cell line exposure to FMDV, then ATG5-ATG12 and autophagy marker proteins LC3-II / LC3-I were examined by Western blot, as well as confocal laser microscopy were used to observe the gathering LC3 green fluorescence, to verify that FMDV activated autophagy in host cells. Further study found that FMDV replication was significantly inhibited at different levels detection by Western blot, Real-time RT-PCR and TCID50 in PK-15 cells over-expressed ATG5-ATG12. Then we analyzed expression of cytokines and inflammatory cytokine by Western blot and Real-time RT-PCR, and found that IFN-β, IL-6,CXCL10, PKR and MX1 were significantly increased compared with the control group, confirming that the ATG5-ATG12 promote cellular anti-virus innate immunity. We detected the crucial adapter protein in I-IFN pathway by Western blot, found that ATG5-ATG12 increased IκBα phosphorylation levels as well as ubiquitination degradation, and the expression of NF-κB / p65 was significantly up-regulated.We observed the increase of the distribution of p65 in nucleus by confocal laser fluorescence microscope, thus confirming the ATG5-ATG12 promotes type I interferons transcriptional by enhancing NF-κB signaling. And interference test further verified the above test results. At the same time, we do further test on baby hamster kidney cells(BHK-21) and another porcine kidney cells(IBRS-2). We found in the type I interferon deficient BHK-21 cells, ATG5-ATG12 did not inhibit FMDV replication which confirmed ATG5-ATG12 inhibit the FMDV replication by promoting the expression of type I interferons. As expected, ATG5-ATG12 significantly inhibits FMDV replication in IBRS-2 cells.In summary, this study with various methods and different techniques studied ATG5-ATG12 antiviral effect from different levels. And it reveals the mechanism of ATG5-ATG12 playing an antiviral role by positive regulation I-IFN pathway. The study provides a new horizon for further study of infection mechanism of FMDV and anti-viral drugs. |
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