Penaeus monodon GILT enzyme restricts WSSV infectivity by reducing disulfide bonds in WSSV proteins

Abstract

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is involved in the adaptive immune response via its effects on major histocompatibility complex (MHC)-restricted antigen presentation. In addition to antigen presentation, GILT exerts its antiviral activity by reducing disulfide bonds in proteins involved in viral infection and assembly, thereby inhibiting viral envelope-mediated infection and viral progeny production. In black tiger shrimp, Penaeus monodon GILT (PmGILT) was cloned and characterized, and found to be involved in the shrimp innate immune response and to exert neutralizing activity against white spot syndrome virus (WSSV) infection. However, the anti-WSSV mechanism of PmGILT in the shrimp innate immune response has not been defined. To explore the anti-WSSV activity of PmGILT, a yeast 2-hybrid (Y2H) assay was performed to identify WSSV proteins targeted by PmGILT. The assay revealed 4 potential PmGILT-interacting WSSV proteins: WSSV002, WSSV164, WSSV189, and WSSV471. Three of these 4 WSSV proteins (WSSV002, WSSV164 and WSSV189) were successfully produced and confirmed to interact with PmGILT in in vitro pull-down assays. WSSV189 and WSSV471 were previously identified as structural proteins, whereas WSSV164 is an immediate-early protein which has anti-melanization activity, and WSSV002 is an unknown. Because of the thiol reductase activity of PmGILT, WSSV164 and WSSV189, both of which are cysteine-containing WSSV proteins, were chosen for disulfide bond reduction assays. PmGILT reduced intrachain disulfide bonds in both WSSV proteins, suggesting that PmGILT exerts its anti-WSSV activity via its thiol reductase activity to disrupt the WSSV protein complex and restore the melanization activity of PmproPO1 and PmproPO2.