Antiviral Functions Of Ctenopharyngodon Idella HMGB2 Proteins And The Induction Mechanism Of Their Dynamic Subcellular Localization

High-mobility group box 2(HMGB2) is a chromatin-associated nonhistone protein, involving in many physiological process, such as transcriptional regulation, DNA repair, recombination, differentiation and extracellular signaling. Recent study demonstrated that HMGB2 can interact with DNA and act as “universal sentinels” in nucleic acid-mediated innate immunity response. However, nothing is known about the function of piscine HMGB2 in antiviral immune response up to now. Grass carp(Ctenopharyngodon idella) is an important fresh water economic aquaculture species in China. But hemorrhagic disease induced by grass carp reovirus(GCRV) causes a great economic loss for grass carp cultivation industry. This study has cloned to full-length c DNA sequences of HMGB2(named Ci HMGB2 a and Ci HMGB2b) of grass carp, investigated their antiviral function and revealed the regulation mechanism of Ci HMGB2 a and Ci HMGB2 b dynamic localization in response to GCRV infection. The main results were listed as follow: 1. Both Ci HMGB2 a and Ci HMGB2 b encode proteins with 213 amino acids and containing two N-terminal box domain and an acidic C-terminal tail domain. 2. Ci HMGB2 a and Ci HMGB2 b were widely expressed in grass carp, but up-regulated in spleen and head kidney tissues post GCRV infection. 3. In C. idella kidney(CIK) cells, over-expressions of Ci HMGB2 a and Ci HMGB2 b induced the up-regulations of Ci TRIF, Ci My D88, Ci IPS-1 and Ci Mx1, but inhibited Ci IFN-I. Cytopathic effect(CPE) indicated that Ci HMGB2 a and Ci HMGB2 b possess significantly antiviral activity against GCRV and inhibited the virus replication in CIK cells. 4. Subcellular localization indicated that both Ci HMGB2 a and Ci HMGB2 b are localized in nucleus of CIK cells under normal condition. While GCRV infection induced the nucleocytoplasmic shuttling of Ci HMGB2 a and Ci HMGB2 b with different degrees. 5. Live Cell Imaging System uncovered that GCRV-induced the nucleocytoplasmic relocation of Ci HMGB2 b occurred in cells with three states: karyotheca rupture, apoptosis and proliferation. Western blot analyses detected extracellular Ci HMGB2 b at 6 h and 12 h post GCRV infection, which demonstrated the passive release and active secretion of Ci HMGB2 b. 6. N-terminal box domain of Ci HMGB2 b was nuclear retention, but the C-terminal tail was nucleocytoplasmic distribution. Comparative analyses of the dynamic relocation of Ci HMGB2 a and Ci HMGB2 b chimeric proteins proved that the intramolecular interaction between HMG-boxes and C-tail domains mediated the nucleocytoplasmic translocation of HMGBs. This study uncovered the regulation mechanism of Ci HMGB2 a and Ci HMGB2 b subcellular localization and their essential roles in antiviral immune response, which lays a foundation for the further studies of regulation of Ci HMGB2 a and Ci HMGB2 b to antiviral immune network.