Research paperCharacterization of tilapia (Oreochromis niloticus) viperin expression, and inhibition of bacterial growth and modulation of immune-related gene expression by electrotransfer of viperin DNA into zebrafish muscle
Introduction
Viperin is an anti-viral protein found in a wide range of organisms, from fish to mammals (Fitzgerald, 2011). It was first identified as a virus-induced gene (vig1) in fish upon infection of rainbow trout leukocytes with viral hemorrhagic septicemia virus (VHSV) (Boudinot et al., 2000). A unique interferon (IFN)-γ inducible gene named viperin (cig5) was identified following infection of human fibroblasts with human cytomegalovirus (HCMV) (Chin and Cresswell, 2001). All known viperin sequences encode an N-terminal amphipathic region (that bind membranes and induces membrane curvature), a CX3CX2C motif (present in the superfamily of SAM-dependent radical enzymes), and a highly conserved C-terminal region (Fitzgerald, 2011, Dang et al., 2010, Drin and Antonny, 2010). Viperin contains three conserved cysteine residues that are part of an iron-sulfur cluster involved in catalysis, and is thus considered a member of the SAM superfamily (Duschene and Broderick, 2010).
Viperin is usually expressed at low levels, but can be strongly induced by viruses, LPS, poly (I:C), and type I and (to a lesser extent) type II IFNs (Sun and Nie, 2004, Mattijssen and Pruijn, 2012). Viperin is one of the most highly induced IFN effector proteins (Zhu et al., 1997, Proud et al., 2008), which suggests that it may be induced by IFN stimulation or infection with viruses like Sendai virus (SV) (Severa et al., 2006), vesicular stomatitis virus (VSV) (Rivieccio et al., 2006), Japanese encephalitis virus (JEV) (Chan et al., 2008), and infectious pancreatic necrosis virus (Sun et al., 2011). LPS, double-stranded B-form DNA, and Edwardsiella tarda have also been reported to enhance viperin expression (Dang et al., 2010, Olofsson et al., 2005, Ishii et al., 2006).
Over-expression of viperin can inhibit HCMV, hepatitis C virus (HCV), SIN, and influenza A virus infection (Chin and Cresswell, 2001, Chan et al., 2008, Jiang et al., 2008, Zhang et al., 2007). Its anti-viral activity is in part mediated through reduced synthesis of late viral (HCMV) structural proteins (such as pp65, pp28, and glycoprotein B), which in turn perturbs viral assembly; consequently, viperin over-expression reduces viral protein synthesis (Chin and Cresswell, 2001). Over-expression of viperin also alters lipid raft conformation on plasma membranes, which inhibits the budding and release of influenza A virions from infected cells (Wang et al., 2007). It has also been suggested that viperin expression reduces protein secretion and modifies endoplasmic reticular (ER) membrane morphology (Hinson and Cresswell, 2009).
Is it possible that viperin also possesses anti-bacterial activity? To date, one paper has described differential regulation of Sciaenops ocellatus viperin expression during the host immune response to bacterial infection (Dang et al., 2010). However, the role of viperin in the anti-bacterial response remains unclear. In this study, we set out to establish whether over-expression of fish viperin in zebrafish muscle inhibits bacterial growth, through regulation of the immune response and immune-related gene expression. We cloned and analyzed the viperin gene from tilapia (Oreochromis niloticus), and found that its expression was affected by LPS and poly(I:C) treatment, and Vibrio vulnificus and Streptococcus agalactiae infection. Furthermore, resistance to an intramuscular challenge with Vibrio was increased by electroporation of plasmid DNA containing viperin under the control of the CMV promoter into zebrafish muscle. The effect of viperin DNA transfer was determined by measuring the number of viable Vibrio cells post-injection, and the messenger (m)RNA levels of certain cytokines.
Section snippets
Fish
Tilapia (O. niloticus) were bought from a fish farm in Tainan, Taiwan. Tilapia were maintained at room temperature in aerated fresh water, and grouper were maintained in seawater under the same condition. Fish were cultured at the Marine Research Station aquarium (Jiaushi, Ilan, Taiwan) for 14 days before bacterial infection or other experiments. Fish were anaesthetized with an anesthetic, ms222 (Sigma, St. Louis, MO, USA), before tissue removal and similarly invasive experiments.
Molecular cloning, sequencing, and phylogenetic analysis
Grouper and
Phylogenetic analysis of the tilapia (O. niloticus) viperin cDNA sequence
We used RT-PCR cloning and sequencing to obtain the tilapia (O. niloticus) viperin cDNA sequence (from ATG to TGA; Supplementary Fig. 1a). We also obtained the grouper (E. fuscoguttatus) viperin cDNA sequence (Supplementary Fig. 1b) by the same methods. We subsequently compared tilapia viperin to other vertebrate viperins, and aligned the conserved cysteine residues of viperin to build the phylogenetic tree shown in Fig. 2. Alignments of all viperin residues are shown in Supplementary Fig. 2.
Discussion
In this work, we describe the cloning, characterization, and functional analysis of a tilapia (O. niloticus) viperin gene, which was induced in vivo during infection with fish pathogens. Phylogenetic analysis revealed strong evolutionary relationships between tilapia viperin and its orthologues in S. ocellatus, C. kawamebari, and C. whiteheadi. This suggests that C. whiteheadi is the sister taxon to C. herzi plus C. kawamebari, corresponding to a previous publication (Chen et al., 2010).
Acknowledgment
This work was supported by a grant awarded to Dr. Jyh-Yih Chen from the Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan.
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