Molecular Cloning, Expression, And Functional Analysis Of IFIT5 Gene In Gallus Gallus Domesticus

The IFIT family is among hundreds of IFN-stimulated genes which is predominantly induced by interferons, virus and lipopolysaccharide. It has become clear that these encoded proteins inhibits viral infections through multiple mechanisms, such as by binding and sequestering specific viral RNA or proteins, suppressing translation initiation, and also regulating cell-intrinsic and cell-extrinsic immune responses. However, relatively few studies have investigated the molecular characterization, expression pattern, and potential function of IFIT5 in chickens. In present study, we cloned and characterized the full-length coding sequences of chicken IFIT5 and detected its tissue expression profile in chicken by qRT-PCR. We also detected the differential expression of chicken IFIT5 with or without different virulence of NDV strains or virus analog challenges, both in vitro and in vivo. To verify whether the chicken MDA-5, LGP-2, MAVS and IRF-3 are upstream regulatory genes of IFIT5, we determined the induction endogenous of mRNA expression of IFIT5 by qRT-PCR, after stimulating cells by NDV-Mukteswar infection or poly I:C transfection in the presence of siRNA or eukaryotic expression vector of chicken MDA-5, LGP-2, MAVS and IRF-3. Furthermore, to explore effect of chicken IFIT5 on IRF-3 and NF-κB pathway, we determined the induction of endogenous mRNA expression of IRF-3-and NF-KB-responsive genes by qRT-PCR, after stimulating cells by NDV-Mukteswar infection, poly dA:dT or poly I:C transfection in the presence of eukaryotic expression vector of chicken IFIT5. The main results are as follows:(1) The full-length coding sequence of chicken IFIT5 is 1440bp encoding a putative protein of 479 amino acids and is composed of two exons. The deduced amino acids sequence identity between chicken and Columba livia IFIT5 orthologues is highest by 66.5%, but there is not more than 50% similarity between chicken and mammalia. It suggests that IFIT family genes may had been affected by strong natural selection in the process of evolution.(2) The chicken IFIT5 mRNA expression was highest in the spleen, followed by the heart, lung, thymus, kidney and small intestine; a relatively weak signal was detected in bursa of fabricius, brain, pectoral and leg muscles; and the lowest in the liver.(3) The thymus and spleen tissues infected with NDV-mukteawar or F48E9 strains showed that the chicken IFIT5 mRNA expression was significantly increased at 1 dpi, reached a peak at 2 dpi and decreased thereafter. The bursa of fabricius tissue infected with NDV-mukteawar strain showed that the chicken IFIT5 mRNA expression was significantly increased and reached a peak at 1 dpi, whereas infected with NDV-F48E9 strain, it was steadily increased among 4 dpi.(4) The expression of chicken IFIT5 was significantly increased in CEF cells following challenged with poly I:C or poly dA:dT. It suggests that chicken IFIT5 might play an important role in RNA virus or DNA virus infection.(5) The expression of chicken IFIT5 and IFN-β were significantly co-increased in CEF cells following infected with NDV-Mukteswar strain. More importantly, the chicken IFIT5 was lated obviously to respond the secondary infection that compared with IFN-β. It suggests that chicken IFN-β had regulatory effects on the activation of chicken IFIT5.(6) The expression of chicken IFIT5 was significantly increased in CEF cells following infection with NDV-Mukteswar strain in the presence of eukaryotic expression vector of chicken MDA-5, MAVS and 1RF-3. Consistently, the expression of chicken IFIT5 was significantly decreased in CEF cells following challenged with NDV-Mukteswar strain or poly I:C in the presence of siRNA of chicken MDA-5, LGP-2, MAVS and IRF-3. Taken together, these data suggests that chicken MDA-5, LGP-2, MAVS and IRF-3 had positive regulatory effects on the activation of chicken IFIT5.(7) The expression of chicken IRF-3-and NF-κB-responsive genes were significantly increased in CEF cells following challenge with poly I:C in the presence of eukaryotic expression vector of chicken IFIT5. It suggests that chicken IFIT5 had positive regulatory effects on the activation of chicken IRF-3 and NF-κB pathway.