Insect immunity is innate and consists of cellular and humoral immune responses. Cellular immune response usually requires hemocyte-spreading behavior, which is accompanied by cytoskeletal rearrangement. A glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), catalyzes an oxidation reaction of glyceraldehyde-3-phosphate to 1,3-biphosphoglycerate in the cytosol. Another function of GAPDH in mammalian cell is to bind C-terminal α-tubulin to facilitate cytoskeletal arrangement. An immunoprecipitation (IP) of viral protein, CpBV-CrV1, against hemocyte protein lysate revealed that CpBV-CrV1 binds to GAPDH, identified by MALDI-TOF analysis. RNA interference (RNAi) of GAPDH significantly suppressed cellular immune response, but neither RNAi of hexokinase nor aldolase suppressed the cellular immune response. A common molecular motif of CpBV-CrV1 and a-tubulin at C-terminal region supported the IP analysis. To test the role of α-tubulin motif in CpBV-CrV1, point mutations of CpBV-CrV1 were applied and resulted in loss of the biological activity of CpBV-CrV1. Furthermore, an immunofluorescence assay indicates CpBV-CrV1 colocalized with a-tubulin in hemocytes collected from Plutella xylostella parasitized by Cotesia plutellae possessing C. plutellae bracovirus (CpBV). This result suggests that GAPDH plays a critical role in hemocyte-spreading behavior during immune challenge, and it is a molecular target of the pathogenic virus.

Polydnavirus are well known to interfere with the host endocrine system, causing immune suppression and other physiological disorders. The Cotesia rubculla polydnavirus gene products, CrV1, are known to be a potent immunosuppressive agent. CrV1 protein express within 12 h after viral infection at oviposition during deposition of parasitoid eggs and are mainly secreted in to host hemocyte, where it functions like phagocytosis and cell spreading. This study identified its homolog in CpBV and analyzed its molecular characteristics motif called “coiled-coil. A point mutation of Alanine to Proline of CpBV-CrV1 could lose the coiled-coil motif from in silico assay. The coiled type CpBV-CrV1 could inhibit host cellular immunity, however, interestingly the mutant CpBV-CrV1 lacking in coiled-coil motif completely lost the immunosuppressive activity. This study suggests that the coiled-coil motif is functional to inhibit host cellular immune responses. RNA interference against CrV1 significantly loses the inhibitory activity and thus further supporting the immunosuppressive activity of CrV1. In this study we also have analyzed the localization of CrV1 by transient transfection in HiFive Cell lines by in situ hybridization.