Inhibition of translation initiation factor eIF4A is required for apoptosis mediated by Microplitis bicoloratus bracovirus.

Apoptotic hemocytes induced by Microplitis bicoloratus parasitism have been reported, and M. bicoloratus bracovirus (MbBV) is known to be the apoptosis inducer. However, the mechanism how MbBV regulates apoptosis remains unclear. eIF4A, one of translation initiation factors, was found from a Spodoptera litura transcriptome, the expression of which in the parasitized hemocytes of S. litura was inhibited in RT-qPCR analysis. The western blot also illustrated eIF4A at 6-day post-parasitization was inhibited in hemocytes. For testing interaction of MbBV-eIF4A-apoptosis, a cDNA clone encoding 1,266 bp of eIF4A was obtained from S. litura hemocytes and sequenced. Then, a 48 kDa V5-fusion protein of the eIF4A was detected by using the anti-V5 antibody at 72-h post-transfection in the High Five cells, which is located in the cell cytoplasm. In vitro, overexpression of eIF4A rescued the apoptotic High Five cells induced by MbBV. Conversely, in vivo, loss of eIF4A proteins by dsRNA feeding increased apoptosis of hemocytes. Furthermore, RNAi and parasitism significantly increased apoptosis of hemocytes in S. litura. These findings suggested that MbBV inhibited the expression of eIF4A, which was required for apoptosis mediated by MbBV. This study will contribute to biological pest control and enhance our understanding of molecular mechanisms underlying polydnavirus-parasitoid-host interaction.

Microplitis bicoloratus bracovirus modulates innate immune suppression through the eIF4E-eIF4A axis in the insect Spodoptera litura.

Eukaryotic initiation factor 4E (eIF4E) is regulated during the innate immune response. However, its translational regulation under innate immune suppression remains largely unexplored. Microplitis bicoloratus bracovirus (MbBV), a symbiotic virus harbored by the parasitoid wasp, Microplitis bicoloratus, suppresses innate immunity in parasitized Spodoptera litura. Here, we generated eIF4E dsRNA and used it to silence the eIF4E gene of S. litura, resulting in a hallmark immunosuppressive phenotype characterized by increased apoptosis of hemocytes and retardation of head capsule width development. In response to natural parasitism, loss of eIF4E function was associated with similar immunosuppression, and we detected no significant differences between the response to parasitism and treatment with eIF4E RNAi. Under MbBV infection, eIF4E overexpression significantly suppressed MbBV-induced increase in apoptosis and suppressed apoptosis to the same extent as co-expression of both eIF4E and eIF4A. There were no significant differences between MbBV-infected and uninfected larvae in which eIF4E was overexpressed. More importantly, in the eIF4E RNAi strain, eIF4A RNAi did not increase apoptosis. Collectively, our results indicate that eIF4E plays a nodal role in the MbBV-suppressed innate immune response via the eIF4E-eIF4A axis.

A secreted-Cu/Zn superoxide dismutase from Microplitis bicoloratus reduces reactive oxygen species triggered by symbiotic bracovirus.

In the parasitoid/polydnavirus/host system, polydnaviruses protect larva development in the host hemocoel by suppressing the host immune response. However, the negative effects on the parasitoid and the strategy of the parasitoid to deal with this disadvantage are still unknown. Microplitis bicoloratus bracovirus induces granulocyte apoptosis to suppress immune responses, resulting in an apoptotic haemolymph environment in which immature M. bicoloratus larva develop. Here, we determined the transcriptional profiles of immature M. bicoloratus across five time-points throughout the immature developmental process from egg to third instar. Dynamic gene expression pattern analysis revealed clear rapid changes in gene expression characteristic of each developmental stage, indicating faster sequential unambiguous functional division during development. Combined with the proteome of the host haemolymph, immature parasitoids likely secreted a Cu/Zn superoxide dismutase to reduce reactive oxygen species generation by symbiotic bracovirus. These data established a basis for further studies of parasitoid/host interactions and identified a novel positive self-protection mechanism for the parasitoid.

[Screening serum response special antibodies of U251 cell line from surface display phage antibody library].

U251 cell is a sensitive cell line to serum, which stops at G0 phase of cell cycle in no-serum medium, and recovers growth when the serum is added into no-serum medium. The cell can express corresponding proteins in different phase of cell cycle. Therefore it is very signification for the study of cell cycle regulation mechanism that explores these proteins. In this paper, the mouse antibody phage display library was added into the bottle in which the serum starvation U251 cells had been cultured, and the special antibody phages were absorbed. Then the absorbed antibody phages were amplified by adding E. coli TG1 and helper phage M13K07. Amplified antibody phages were added into bottle in which the serum cultured cell after serum starvation (follow named as serum recovered cells) were incubated, so that the cell absorbed the no-special antibody phages for the serum starvation cell and the special antibody phages were in supernatant. The remaining no-special antibody phages in the supernatant were discarded by repeating above program 3-4 times. The pure special antibody phages were gotten, and amplified by adding the host cell E. coli TG1 and helper phage M13K07. Then the host bacterium infected special antibody phage was spread on the plate medium with ampicillin, and the monoclonal antibody phages were gotten. Using same as above program, the monoclonal antibody phages absorbed specially for serum recovered U251 cells were obtained when the serum recovered cells instead of serum starvation cells and serum starvation cells instead of serum recovered cells. In this study, ninety-six positive monoclonal antibody phages that absorbed specially the serum starvation cells and eighty-two positive monoclonal antibody phages that absorbed specially the serum recovered cells were obtained. By using cell immunochemistry assay, two special signification antibodies were obtained. one (No.11) was the strong response in serum starvation cells, the other (No.2) was the strong response in serum recovered cells. The antibody No.2 had the distinctive response to the serum recovered cells in different incubation time (15min, 30min, 1h, 2h, 4h, 8h, 12h and 48h) after serum starvation. The results showed that No.2 antibody would be useful to research the factors of cell cycle regulation and apply to tumor diagnosis.