| Viperin is known to be an antiviral protein which could be strongly induced in a variety of cell types by different cellular factors, such as type Ⅰ Ⅱ and Ⅲ IFNs, DNA and RNA viral proteins, viral mimic polyriboinosinic polyribocytidylic acid (polyⅠ:C) and polysaccharide. All viperin homologues possess a hypervariable N-terminal domain with an amphipathic a-helix that is known to participate in endoplasmic reticulum (ER) localization, a conserved domain of the radical S-adenosylmethionine (SAM) superfamily proteins in the central region, which contains a C×3×2C motif required for the formation of a [4Fe-4S] cluster and a highly conserved C-terminal region that is known to be critical for their antiviral activity against a number of viruses. Viperin has now been identified in various species from lower vertebrates to mammals, while, whether viperin of invertebrates, like that of vertebrates, has antiviral activity is completely unclear. In this study, a cDNA of Branchiostoma japonicum viperin homologue with 1540 bp in length is obtained by assembling the overlapping cDNA fragments, which contains an open reading frame (ORF) of 1074 bp, a 5’-untranslated region (UTR) of 71 bp and a 3’-UTR of 395 bp. Sequence alignment indicates that the deduced protein shares 60.7% to 87.8% overall sequence identity with the viperin homologues of human, chimpanzee, mouse, platypus, chicken, zebra finch, anole lizard, frog, zebrafish, tilapia, elephant shark and Florida amphioxus. The analysis by SMART program shows the deduced protein has a conserved domain of elongator protein 3 (Elp3)/radical SAM superfamily (residues 68-277) containing the highly conserved CxxxCxxC motif in the form of 79CNYKCGFC86, which is characteristic of viperin homologues. All these data show that the cDNA codes for the viperin homologue of B. japonicum, Bjvip. This is further supported by the 3D modeling of BjVip, which is closely similar to the 3D structure of human and zebrafish viperin homologues. In site models, codon-by-codon substitution analyses have revealed that all of the positively selected sites are located at the N-terminal amphipathic α-helix domain, indicating that the persistent antagonistic nature of surrounding infectious viral pathogens might be the likely cause for such adaptive evolutionary changes of certain amino acids in fish and amphioxus viperin antiviral protein. In branch-specific models, the free-ratio model rejects the one-ratio model, that means the co ratios are extremely variable among lineages. While in the two-ratio model, no positive selection branch is detected with P<0.05, suggesting that in these branches, they do not evolving under positive selection. Synteny analysis of viperin homologues of representative species shows that amphioxus viperin, RSAD2, is immediately linked to CMPK2, and analogously, all vertebrate RSAD2, except that of platypus, is immediately linked to CMPK2. This indicates that the close CMPK2-RSAD2 linkage is highly conserved throughout chordate evolution. The expression pattern of Bjvip shows that Bjvip is predominantly expressed in the hepatic caecum, hind-gut, gill and muscle, and at a lower level in the the ovary, testis and notochord, indicating that Bjvip is expressed in a tissue-specific fashion. Challenge with poly (I:C) results a significantly enhanced expression of Bjvip in all the tissues tested, including the hepatic caecum, hind-gut, gill and muscle, suggesting that the expression of Bjvip is subjected to the stimulation of the viral mimic poly (I:C). In vitro, the result shows that the relative amount of LCDV in the cells transfected with pcDNA3.1/GFP/Bjvip plasmid is significantly lowered at 48 and 72 h post infection, suggesting that BjVip is capable of killing LCDV or inhibiting its propagation. In vivo, purified rBjVip can markedly lower the relative quantity of WSSV in hepatopancreas and muscles of the shrimps at 8 h to 24 h post injection. Collectively, these data suggest that rBjVip has an antiviral activity against WSSV. |
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