Proteomics analysis reveals a critical role for the WSSV immediate-early protein IE1 in modulating the host prophenoloxidase system

Abstract

White spot syndrome virus (WSSV) is a large, enveloped, double-stranded DNA virus that threatens shrimp aquaculture worldwide. So far, the mechanisms of WSSV-host interactions are ill-defined. Recent studies have revealed that IE1, an immediate-early protein of WSSV, is a multifunctional modulator implicated in virus–host interactions. In this study, the functions of IE1 were further explored by identifying its interacting proteins using GST-pull down and mass spectrometry analysis. A total of 361 host proteins that potentially bind to IE1 were identified. Bioinformatics analysis revealed that the identified IE1-interactors wereinvolved in various signaling pathways such as prophenoloxidase (proPO) system, PI3K-AKT, and MAPK. Among these, the regulatory role of IE1 in shrimp proPO system was further studied. The Co-immunoprecipitation results confirmed that IE1 interacted with the Ig-like domain of Penaeus vannamei proPO or proPO-like protein (hemocyanin). Additionally, we found that knockdown of IE1 reduced viral genes expression and viral loads and increased the hemocytes’ PO activity, whereas recombinant IE1 protein inhibited the PO activity in a dose-dependent manner. Finally, we demonstrated that WSSV could suppress the hemocytes’ PO activity at the early infection stage. Collectively, our current data indicate that IE1 is a novel viral regulator that negatively modulates the shrimp proPO system.

Applying genetic technologies to combat infectious diseases in aquaculture

“…Knockdown of IE-1 reduced viral load and WSSV gene expression, while recombinant IE-1 inhibited host prophenoloxidase in a dose-dependent manner. 531 Metabolomic studies of gill, haemolymph and hepatopancreas have revealed clearly different profiles in WSSV infected and non-infected shrimp, consistent with changes in osmoregulation in the gills, upregulation of the glutathione pathway, increased production of an antimicrobial peptide itaconic acid in the hemolymph and a shift from aerobic to anaerobic metabolism as previously described. 532 Interestingly, some of the changes in the haemolymph (increased TCA intermediates and decreased amino acids) were similar to those in haemolymph of P. vannamei challenged with Vibrio parahaemolyticus 533 while some molecules were specific for each pathogen such as itaconic acid in WSSV-challenged shrimp and increased phosphoenolpyruvic acid (PEP) in shrimp exposed to V. parahaemolyticus, suggesting that there are pathogenspecific innate immune responses in shrimp.…”

“…In P. vannamei, GST-pull down and mass spectrometry analysis were used to investigate the interaction of the immediate-early WSSV protein (IE-1) with shrimp proteins, finding 361 host proteins that could potentially bind to IE. 531 Most of these proteins were involved in signalling pathways such as the prophenoloxidase (proPO), PI3K-AKT, MAPK, focal adhesion, and cell cycle systems. Knockdown of IE-1 reduced viral load and WSSV gene expression, while recombinant IE-1 inhibited host prophenoloxidase in a dose-dependent manner.…”

Comparative Transcriptome Analysis Reveals That WSSV IE1 Protein Plays a Crucial Role in DNA Replication Control

“…IE1, which is an IE protein of WSSV, is a multifunctional regulator that plays critical roles in virus–host interactions. It not only functioned as a transcription factor [ 22 ], but also interacted with various shrimp cellular proteins (i.e., Rb, STAT, JNK, β-catenin, Chibby, and proPO) to manipulate the host cellular functions for viral genome replication [ 18 , 19 , 23 , 24 , 25 , 26 ]. In this study, RNAi coupled with transcriptome sequencing were conducted to explore the downstream genes that are regulated by IE1 directly or indirectly.…”

“…High-Five (BTI-TN-5B1-4) cells were seeded on a 24-well cell culture plate and maintained in Express Five SFM medium (Invitrogen, Waltham, MA, USA) overnight. For plasmid transfection, the IE1 expression plasmid pIZ-V5-IE1 (1 µg), which was constructed in our previous study [ 26 ], was transfected into cells using the FuGENE HD transfection reagent (Promega, Madison, WI, USA) following manufacturer’s instructions. An equal amount of empty vector pIZ-V5-His (Invitrogen, Waltham, MA, USA) was transfected and used as a negative control.…”

“…For instance, IE1 was demonstrated to act as a transcription factor with transactivation, dimerization, and DNA-binding activity [ 22 ]. Moreover, several studies reported that IE1 could bind to many host cellular proteins such as the retinoblastoma protein (Rb) [ 23 ], STAT [ 19 ], JNK [ 18 ], β-catenin [ 24 ], Chibby [ 25 ], and prophenoloxidase (proPO) [ 26 ], thereby hijacking cellular functions or suppressing the host immunity to promote virus multiplication. The foregoing data indicate that IE1 is a multifunctional regulator that is critical for WSSV pathogenesis.…”