SUMO and SUMO-Conjugating Enzyme E2 UBC9 Are Involved in White Spot Syndrome Virus Infection in Fenneropenaeus chinensis.

Xiaoqian Tang,Xiuzhen Sheng,Jing Xing,Wei Li,Wenbin Zhan,Gao-Feng Qiu

doi:10.1371/journal.pone.0150324

Abstract

In previous work, small ubiquitin-like modifier (SUMO) in hemocytes of Chinese shrimp Fenneropenaeus chinensis was found to be up-regulated post-white spot syndrome virus (WSSV) infection using proteomic approach. However, the role of SUMO in viral infection is still unclear. In the present work, full length cDNAs of SUMO (FcSUMO) and SUMO-conjugating enzyme E2 UBC9 (FcUBC9) were cloned from F. chinensis using rapid amplification of cDNA ends approach. The open reading frame (ORF) of FcSUMO encoded a 93 amino acids peptide with the predicted molecular weight (M.W) of 10.55 kDa, and the UBC9 ORF encoded a 160 amino acids peptide with the predicted M.W of 18.35 kDa. By quantitative real-time RT-PCR, higher mRNA transcription levels of FcSUMO and FcUBC9 were detected in hemocytes and ovary of F. chinensis, and the two genes were significantly up-regulated post WSSV infection. Subsequently, the recombinant proteins of FcSUMO and FcUBC9 were expressed in Escherichia coli BL21 (DE3), and employed as immunogens for the production of polyclonal antibody (PAb). Indirect immunofluorescence assay revealed that the FcSUMO and UBC9 proteins were mainly located in the hemocytes nuclei. By western blotting, a 13.5 kDa protein and a 18.7 kDa protein in hemocytes were recognized by the PAb against SUMO or UBC9 respectively. Furthermore, gene silencing of FcSUMO and FcUBC9 were performed using RNA interference, and the results showed that the number of WSSV copies and the viral gene expressions were inhibited by knockdown of either SUMO or UBC9, and the mortalities of shrimp were also reduced. These results indicated that FcSUMO and FcUBC9 played important roles in WSSV infection.

Highlights

Small ubiquitin-like modifiers (SUMO) are a family of small proteins that could covalently attach to and detached from other proteins in cells to modify their functions
Total RNA of hemocytes and ovary were prepared from the white spot syndrome virus (WSSV) infected shrimps sampled at various time points as above, and Quantitative real-time RT-PCR (qRT-PCR) was performed to investigate the effects of WSSV infection on full length cDNAs of SUMO (FcSUMO) and FcUBC9 transcript levels respectively
Multiple sequences alignment of the deduced proteins showed that the two molecules both had significant homology with the ones from various species, and the amino acid sequence of FcSUMO was even completely identical to the SUMOs of L. vannamei and P. clarkia, indicating that they are highly evolutionarily conserved

Summary

Introduction

Small ubiquitin-like modifiers (SUMO) are a family of small proteins that could covalently attach to and detached from other proteins in cells to modify their functions. SUMO in hemocytes of Chinese shrimp Fenneropenaeus chinensis was found to be significantly up-regulated at both mRNA and protein levels post WSSV infection [13]. A recent research demonstrated that WSSV Immediate early (ie) proteins could be modified by crayfish SUMOylation, and the modification would benefit WSSV replication [1]. All these results implied that SUMO and UBC9 played important roles in WSSV infection. The potential roles of SUMO and UBC9 in WSSV infection were further investigated in vivo by RNA interference (RNAi)

Materials and Methods

Results

Discussion