INTERACTION OF ANTI-LIPOPOLYSACCHARIDE FACTOR ISOFORM 3 FROM BLACK TIGER SHRIMP Penaeus monodon WITH WHITE SPOT SYNDROME VIRUS PROTEINS

Abstract

Antimicrobial peptides (AMPs) play a vital role in combating microbial pathogens. Among AMPs identified in Penaeus monodon, only anti-lipopolysaccharide factor isoform 3 (ALFPm3) has been reported to exhibit activity against white spot syndrome virus (WSSV); however, the mechanism(s) involved are still not clear. To better understand the ALFPm3 function in WSSV response, ALFPm3-interacting proteins including WSSV186, WSSV189, WSSV395, WSSV458 and WSSV471 proteins from WSSV have been previously identified by yeast two hybrid assay. In vitro pull-down assay has been confirmed the interactions between the recombinant ALFPm3 protein (rALFPm3) and the recombinant WSSV189 (rWSSV189) and recombinant WSSV471 (rWSSV471) proteins. In this study, the binding of the rALFPm3 protein and the recombinant WSSV458 protein (rWSSV458) was also confirmed. Pre-incubation of rWSSV189, rWSSV458, rWSSV471 proteins with the rALFPm3 protein interfered the neutralization effect of the rALFPm3 protein on WSSV in vivo was revealed. The decrease in the % survival of shrimp injected with WSSV pre-treated with the mixture of each rWSSV protein and rALFPm3 protein compared to those injected with WSSV pre-treated with the rALFPm3 protein only, was observed. However, the information on WSSV189, WSSV458 and WSSV471 proteins are limited. Here, the expression of the WSSV189, WSSV458 and WSSV471 proteins were determined in gills and hemocytes of WSSV-infected shrimp and the increase in their expression upon WSSV infection was observed. WSSV458 has been identified recently as a WSSV tegument protein. In the present study, Western blot analysis was employed to study localization of WSSV189 and WSSV471 proteins in the WSSV virion, fractions of envelope and nucleocapsid proteins of WSSV. The results indicated that both of them were envelope proteins. Immunoelectron microscopy using WSSV189 and WSSV471 specific antibodies was also performed and approved the location of both WSSV proteins on the WSSV envelope. Taken together, the results indicated that the ALFPm3 performs its anti-WSSV action by binding to the WSSV structural proteins, WSSV189, WSSV458 and WSSV471 proteins and possibly others.