As an important component of innate immune system, interferon (IFN) system is the first line of defense protecting fish and other vertebrates against virus and other microbes. An ideal cell model to research fish IFN system has been established in our lab. UV-inactivated grass carp hemorrhage virus (GCHV) is capable to induce an antiviral state in crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells as evidenced by production of IFN activity and activation of a number of cellular genes involved in IFN antiviral response. GCHV induced gene 1 (Gig1) and Gig2 are two novel IFN inducible genes cloned and characterized from this system. GCHV, Poly I:C and CAB IFN-containing supernatant can induce the expression of Gig1 and Gig2 at mRNA level. In this study, we further analyzed the expression regulation and antiviral function of Gig1 and Gig2.
Gig1 is the most abundant gene found in our subtractive cDNA library. It is still a protein with no recognizable domains and functions. Gig1 protein was successfully produced in prokaryotic expression system. By immunizing rabbit with this prokaryotic expressed and purified Gig1 protein, the specific anti-Gig1 antiserum was generated, and then used to confirm that Gig1 is a structural gene. Real-time PCR and Western blot analyses shown that UV-inactivated GCHV and Poly I:C could up-regulated the expression of Gig1 both at mRNA and protein levels. Indirect immunofluorescence and Western blot analyses shown that Gig1 mainly localized in the cytoplasm. In grass carp, Gig1 was ubiquitously distributed in all tested tissues with a constitutively weak expression both at mRNA and protein levels, and Poly I:C was able to induce an increased expression of Gig1 in several immune tissues. Over-expressed Gig1 protein in CAB cells and Epithelioma papulosum cyprinid (EPC) cells could inhibit virus replication and protect cells. Through searching public databases, we found Gig1 was a fish specific gene. Above all, Gig1 is a fish specific antiviral protein which can be induced by IFN.
Gig2 is another function un-known gene found in our subtractive cDNA library. Gig2 protein was successfully produced in prokaryotic expression system. By immunizing rabbit with this prokaryotic expressed and purified Gig2 protein, the specific anti-Gig2 antiserum was generated, and then used to confirm that Gig2 is a structural gene. Western blot analysis shown that UV-inactivated GCHV, Poly I:C and prokaryotic expressed IFN protein could induce the expression of Gig2 protein. In grass carp, Gig2 was ubiquitously distributed in all tested tissues both at mRNA and protein levels, and Poly I:C was able to induce an increased expression of Gig2 in several immune tissues. An 859 bp upstream region of Gig2 promoter was amplified by gene walking. Gig2 promoter contains three IFN-stimulated response elements (ISREs), five -IFN activated sites (GASs), 9 GAAA/TTTC motifs, a TATA-box and a Sp1 binding site. Promoter-reporter system was used to analyze the activity of Gig2 promoter. We found Poly I:C, IFN and over-expressed IRF7 could induce the activity of Gig2 promoter. In a word, Gig2 is a novel IFN inducible gene, which may have an important function in cellular antiviral immune response. Besides IRF7 might directly upregulate the expression of Gig2 in the context of Poly I:C induction or IFN induction.
Four Gig2 homologues were obtained when further screening our subtractive cDNA library. The full length cDNA sequences of the four Gig2 homologues were then obtained by RACE-PCR. UV-inactivated GCHV could induce the expression of all four Gig2 homologues, which indicated that they might play an important role in antiviral immune response. Gig2 and Gig2 homologues may be members of a gene family which may play an important role in antiviral immune response.
Taken together, the results generated in the present study will be essential for further clarifying fish IFN mediated signal pathway, fish IFN mediated antiviral mechanism in detail, and promoting researches on the antiviral drugs and development of the antiviral breeding technology in fish.