Molecular Cloning And Function Analysis Of Antiviral-related Proteins In Shrimp

The Chinese white shrimp (Fenneropenaeus chinensis) cultivation is an important economic activity in China. However, the aquaculture of shrimp has suffered vast economic losses because of serious viral diseases such as white spot syndrome virus (WSSV). So investigating the mechanism of antiviral immunity will help us to control those viral diseases.1. Fc-TRBP interacts with Fc-eIF6 directly, and both are involved in the antiviral RNA interferenceThe Trans-activation response RNA-binding protein (TRBP) plays an important role in many biological processes. In these studies, three new cDNAs of the TRBP family were cloned from Fenneropenaeus chinensis, and designated Fc-TRBP 1-3. Recombinant expressed Fc-TRBPl in Escherichia coli was used for panning of a T7 phage display library constructed using Chinese shrimp hepatopancreas. From this panning, eukaryotic initiation factor 6 (eIF6) was isolated and sequenced. After cloning and recombinantly expressing Fenneropenaeus chinensis-derived Fc-eIF6, the interaction between Fc-TRBP and Fc-eIF6 was confirmed using pull-down assays and far western analysis. Expression of Fc-TRBPs was detected in many tissues, with its expression level increasing in heart, gill and intestine in the early stages of infection by the white spot syndrome virus (WSSV), and increasing in most tissues following a challenge with Vibrio anguillarum. Western blot studies confirmed the increased expression of Fc-TRBP in gill after WSSV infection. The expression pattern of eIF6 was also analyzed. Infection analysis show the replication of WSSV was reduced after injection with Fc-TRBP.Then we show that the double strand RNA bind domain (dsRBD) B and C of TRBP homologue from Marsupenaeus japonicus (Mj-TRBP) mediated the interaction of TRBP and eIF6. Gel-shift assays reveal the N-terminal dsRBD of Mj-TRBP binds to double strand RNA (dsRNA) with a high affinity, while TRBP might be homo-dimerized by the C-terminal region and increase the affinity to dsRNA. RNAi against either Mj-TRBP or Mj-eIF6 impairs the dsRNA induced sequence-specific RNAi pathway and facilitates the proliferation of WSSV. These results indicate that TRBP and eIF6 may be components of the RNA-induced silencing complex (RISC), and thereby play a crucial role in the anti-viral defense response of shrimp.2. Cathepsin C is involved in the innate antiviral immunity.Cathepsin C (Cath C) is a lysosomal cysteine protease that belongs to the papain superfamily. Cath C is capable of activating many chymotrypsin-like serine proteases and is reported to be a central coordinator for the activation of many serine proteinases in immune and inflammatory cells. In this study, Cath C cDNA was cloned from Fenneropenaeus chinensis (Fc). The complete cDNA of Fc-Cath C in Chinese white shrimp was found to be 1445-base pairs (bp) long. It contained an open reading frame (ORF) 1356-bp long and encoded a 451-amino acid residue protein, including a 17-amino acid residue signal peptide. Real-time PCR analysis results indicated that Fc-Cath C mRNA was transcribed in the hepatopancreas and upregulated after stimulation by the Vibrio anguillarum and the white spot syndrome viruses (WSSVs). Replication of the WSSV increased after the injection of Fc-Cath C antiserum. These results imply that Fc-Cath C might play an important role in the antiviral immune response of shrimp.3. Molecular cloning and function analysis of TCTPThe translationally controlled tumor protein (TCTP) is a multi-functioning protein that performs vital roles, particularly in various complicated life processes. In this study, a new TCTP cDNA was cloned from Fenneropenaeus chinensis and hence was designated as Fc-TCTP. Its length is 711 bp, and it is characterized by 507-bp open reading frame (ORF) that encodes a deduced 168-amino acid protein, including a TCTP domain. Moreover, this study analyzed the expression patterns of this gene when it responds to infection specifically with Vibrio anguillarum and the white spot syndrome virus (WSSV). Based on the results, Fc-TCTP was present in all the analyzed tissues. Additionally, Fc-TCTP's expression level decreased after having been infected by bacteria, but was upregulated in the hepatopancreas after having been exposed to WSSV. Likewise, the Fc-TCTP protein was upregulated during its exposure to the virus. These results suggest that Fc-TCTP could well be involved in the antiviral response in F. chinensis.