Functional Study Of Ubiquitination And SUMOylation In Crustacean Immune Responses

Shrimp farming is an impotrant export industry in China However, the outbreak of white spot syndrome virus (WSSV) since1992leads to huge economic losses to our country and even the world. Therefore, the identification and functional analysis of host proteins involved in viral infection and replication are significant to the control of shrimp diseases Ubiquitination and SUMOylation need activating enzyme E1, conjugating enzyme E2and ligase enzyme E3to modify target proteins on specific sites. Though spacial structure and enzymatic reaction of SUMOylation are similar with ubiquitination, they result in quite different meanings in biology The study foucs on ubiquitin conjugating enzyme of prawn and SUMO conjugating enzme of crayfish. We investigate the roles ubiquitination and SUMOylation during viral infection and replication, and propose to illustrate the molecular mechanism of shrimp and virus interactions.1. FcUbc of Chinese white shrimp mediates ubiquitination of WSSV RING domain proteins and inhibits viral replication.We identified a ubiquitin conjugating enzyme E2from Fenneropenaeus chinensis named FcUbc The full length of FcUbc cDNA is967bp with open reading frame (ORF) of447bp The deduced peptide encoded148amino acids and contains a ubiquitin conjugating catalytic domain (UBCc). FcUbc RNAs and proteins were detected in prawn hepatopancreas and intestine. The recombinant FcUbc and the mutant FcUbc lacking catalytic residue were expressed and purified in Escherichiacoli.Subsequently, rFcUbc injected into WSSV-infected shrimp reduced the mortality, and greatly suppressed viral replication. Pull-down assay confirmed that FcUbc combined to four viral RING proteins, which function as potential E3ligase; but the mFcUbc lost the binding ability. More importantly, that FcUbc mediates ubiquitination of RING domains of WSSV277and WSSV304is illustrated by assays in vitro and on Drosophila S2cells. Furthermore, overexpression of FcUbc on S2cells enhanced ubiquitination of viral WSSV277and WSSV304. These results indicate FcUbc mediates ubiquitination of WSSV RING domain proteins and inhibits viral replication ultimately.2. SUMO conjugating enzyme of red swamp crayfish, UBC9mediates SUMOylation of WSSV IE protein and facilitates viral replicationA small ubiquitin-related modifier (SUMO) and SUMO conjugating enzyme UBC9were identified from Procambarus clarkii. The full length of UBC9cDNA is912bp with ORF of483bp coding160amino acids, and the active site is cysteine93. At the same time, that of SUMO cDNA is960bp with ORF of283bp coding93aa, and contains double glycine residues at C-terminal as the active site. Sequence alignment and phylogenetic tree display that UBC9and SUMO are conserved in evolution, and UBC9and SUMO of crayfish show high similarity with these of yeast and human. UBC9and SUMO constitutively expressed in every tissue of crayfish and increase after WSSV challenge. Crayfish UBC9, SUMO, mUBC9, mSUMO and active SUMO-GG are expressed in E. coliThe viral genes replication was enhanced in crayfish injected with UBC9or SUMO, but not with mUBC9and mSUMO. We analyzed the molecular mechanism of crayfish SUMOylation prompts WSSV replication. Pull-down assay confirmed crayfish UBC9combined to three of WSSV IE proteins, while only one of them, WSV051was SUMO-modified by UBC9in vitro. The decreased expressions of UBC9or SUMO by RNAi, leaded to serious inhibition on viral late genes and down-regulation on ie genes Moreover, the suppressions were removed by subsequent injection of recombinanl UBC9or SUMO, and the gene expressions of WSSV were rescued by UBC9or SUMO proteins. In a word, the study demonstrates that crayfish SUMOylation pathway can be employed by WSSV to modify its IE proteins, which facilitates viral genes transcription and replication.