GSK3β Plays a Negative Role During White Spot Syndrome Virus (WSSV) Infection by Regulating NF-κB Activity in Shrimp Litopenaeus vannamei

Glycogen synthase kinase-3 (GSK3), a cytoplasmic serine/threonine-protein kinase involved in a large number of key cellular processes, is a little-known signaling molecule in virus study. In this study, a GSK3 protein which was highly similar to GSK3 beta homologs from other species in Litopenaeus vannamei (designated as LvGSK3 beta) was obtained. LvGSK3 beta was expressed constitutively in the healthy L. vannamei, at the highest level in the intestine and the lowest level in the eyestalk. White spot syndrome virus (WSSV) reduced LvGSK3 beta expression was in immune tissues including the hemocyte, intestine, gill and hepatopancreas. The inhibition of LvGSK3 beta resulted in significantly higher survival rates of L. vannamei during WSSV infection than the control group, and significantly lower WSSV viral loads in LvGSK3 beta-inhibited L. vannamei were observed. Knockdown of LvGSK3 beta by RNAi resulted in increases in the expression of LvDorsal and several NF-kappa B driven antimicrobial peptide (AMP) genes (including ALF, PEN and crustin), but a decrease in LvCactus expression. Accordingly, overexpression of LvGSK3 beta could reduce the promoter activity of LvDorsal and several AMPs, while the promoter activity of LvCactus was increased. Electrophoretic mobility shift assays (EMSA) showed that LvDorsal could bind to the promoter of LvGSK3 beta. The interaction between LvGSK3 beta and LvDorsal or LvCactus was confirmed using co-immunoprecipitation (Co-IP) assays. In addition, the expression of LvGSK3 beta was dramatically reduced by knockdown of LvDorsal. In summary, the results presented in this study indicated that LvGSK3 beta had a negative effect on L. vannamei by mediating a feedback regulation of the NF-kappa B pathway when it is infected by WSSV.