Grass carp (Ctenopharyngodon idella)is an important species of freshwater aquaculture fish in China. However, grass carp reovirus (GCRV) can cause fatal hemorrhagic disease in yearling populations. Until now, a strategy to define the antigenic capacity of the virus's structural proteins for preparing an effective vaccine has not been available. In this study, some single-chain variable fragment antibodies (scFv), which could specifically recognize grass carp IgM, were selected from a constructed mouse naïve antibody phage display cDNA library. The identified scFv C1B3 clone was shown to possess relatively higher specific binding activity to grass carp IgM. Furthermore, ELISA analysis indicated that the IgM level in serum from virus-infected grass carp was more than two times higher than that of the control group at 5-7 days post infection. Moreover, Western blot analysis demonstrated that the outer capsid protein VP7 has a specific immuno-binding-reaction with the serum IgM from virus-infected grass carp. Our results suggest that VP7 can induce a stronger immune response in grass carp than the other GCRV structural proteins, which implies that VP7 protein could be used as a preferred immunogen for vaccine design.

No abstract

Hypoxia modulates senescence, but their physiological link remains unclear. Here, we found that eIF4E2, a hypoxia-activated translation initiation factor, interacted with GSK3β to maintain phosphorylation of p53, thus resisting senescence under hypoxia. RNA-binding protein RBM38 interacted with eIF4E to inhibit the translation of p53, but GSK3β-mediated Ser195 phosphorylation disrupted the RBM38-eIF4E interaction. Through investigation of RBM38 phosphorylation, we found that the eIF4E2-GSK3β pathway specifically regulated proline-directed serine/threonine phosphorylation (S/T-P). Importantly, peptides e2-I or G3-I that blocking eIF4E2-GSK3β interaction can inhibit the basal S/T-P phosphorylation of p53 at multiple sites, therby inducing senescence through transcriptional inhibition. Additionally, a nanobody was screened via the domain where eIF4E2 bound to GSK3β, and this nanobody inhibited S/T-P phosphorylation to promote senescence. Furthermore, hypoxia inhibited eIF4E2-GSK3β pathway by mediating S-Nitrosylation of GSK3β. Blocking eIF4E2-GSK3β interaction promoted liver senescence under hypoxia, thus leading to liver fibrosis, eventually accelerating N, N-diethylnitrosamine (DEN)-induced tumorigenesis. Interestingly, eIF4E2 isoforms with GSK3β-binding motif exclusively exist in mammals, which protect zebrafish heart against hypoxia. Together, this study reveals a mammalian eIF4E2-GSK3β pathway that prevents senescence by maintaining basal S/T-P phosphorylation of p53, which underlies hypoxia adaptation of tissues.