Cloning and analysis of antiviral activity of a barramundi (Lates calcarifer) Mx gene☆
Introduction
The type I interferon (IFN) system is one of the most important mechanisms for antiviral defense. In the type I IFN response, many IFN-related proteins are induced for establishing an antiviral state, such as double-stranded RNA-activated protein kinase (PKR), the 2′, 5′-oligoadenylate synthetase, and Mx proteins [1]. Mx proteins belong to the dynamin superfamily [2] and contain a tripartite GTP-binding domain essential for the antiviral activity [3]. The first Mx protein was found in mice and named because of its resistance to orthomyxovirus influenza A [4]. Mx genes were subsequently found in higher vertebrates, including human [5], some livestock species [6], [7], [8], and birds [9], [10]. Moreover, some Mx proteins have been shown to inhibit virus replication [11], [12], [13], [14].
The lack of information on fish IFN and the difficulty of IFN detection due to its small production and short half-life, led to the use of Mx gene expression as an indicator of IFN activity. Until now, Mx remains useful as a sensitive and more stable marker of the IFN response. In fish, the first Mx gene was identified from perch (Perca fluviatilis L.) [15]. Following, piscine Mx genes have been cloned and characterized in rainbow trout (Oncorhynchus mykiss Walbaum) [16], Atlantic salmon (Salmo salar L.) [17], Atlantic halibut (Hippoglossus hippoglossus L.) [18], Japanese flounder (Paralichthys olivaceus) [19], fugu (Takifugu rubripes) [20], gilthead sea bream (Sparus aurata) [21], channel catfish (Ictalurus punctatus) [22], and orange-spotted grouper (Epinephelus coioides) [23]. However, only few papers have demonstrated the role of fish Mx proteins in IFN response. For example, the Japanese flounder Mx can inhibit the replication of hirame rhabdovirus and viral hemorrhagic septicemia virus (VHSV) [24], while the Atlantic salmon Mx1 can inhibit the replication of infectious pancreatic necrosis virus (IPNV) [25].
Fish nodavirus, nervous necrosis virus (NNV), and fish iridovirus are two major viral pathogens among many species of cultured marine fish in Taiwan [26], [27]. The mortality of NNV-infected fish at the larval stage could be as high as 80–100%, and most of the survivors became persistently infected [28]. The information about the mechanism of NNV persistent infection has been very limited until the NNV-persistently infected cell line BB was established [29], and a negative control cell line cBB was obtained by treating BB cells with NNV-specific antiserum [30]. Mx mRNA was detected in the BB cells, but not in the NNV-free cBB cells. In the present study, a barramundi Mx cDNA was cloned from the cBB cells, and the expression of Mx gene in cBB cells was analyzed after poly I:C transfection and virus infection. The antiviral activities against fish nodavirus, birnavirus and iridovirus in poly I:C-transfected cBB cells were examined, and the level of viral replication was further analyzed in cBB cells whose Mx gene expression was down-regulated by siRNA.
Section snippets
Cell lines and viruses
The cBB cells were cultured with L-15 medium supplemented with 10% fetal bovine serum (FBS), and incubated at 28 °C. Three fish viruses used in the study were: (1) fish nodavirus, B00GD, an NNV isolate of infected barramundi [26]; (2) fish birnavirus, IPNV-SP; and (3) fish iridovirus, TGIV, an isolate from infected grouper [27]. The GF-1 (Grouper Fin-1) cell line [31], the RTG-2 cell line, and the GF-3 (Grouper Fin-3) cell line were respectively used for nodavirus, birnavirus, and iridovirus
Sequence analysis of the barramundi Mx gene
The 5′ and 3′ RACE sequence overlapped the 718 bp fragment of barramundi Mx gene sequence by 76 bp and 178 bp respectively. The three parts of the sequences were aligned to give a full length cDNA sequence of 2214 bp, which contains an ORF of 1875 bp, encoding a 624 amino acid polypeptide with a predicted molecular weight of 71.4 kDa. The amino terminal contains a tripartite GTP-binding motif and a dynamin family signature. The carboxyl terminal contains a putative leucine zipper motif conserved in
Discussion
In this study, a cDNA clone of barramundi Mx gene was constructed from the extracted mRNA of cBB cells after NNV infection. The motif characteristics of Mx proteins, including the GTP-binding site, the dynamin family signature, and the leucine zipper are all present in barramundi Mx. GTP-binding motif is important for antiviral activity because GTP binding induces a conformational change of the Mx protein that allows specific recognition of viral targets [34]. Mutations in the GTP-binding
Acknowledgements
The authors would like to thank Dr. Oystein Evensen for providing IPNV-Sp isolate and Dr. Chou H.Y. for providing TGIV isolate. This work is financially supporting by the National Science Council of the Republic of China under the Contract No. NSC 94-2311-B-002-024.
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2020, Fish and Shellfish ImmunologyCitation Excerpt :The interferon (IFN)-mediated defense mechanism is one of the major protective systems against various type of viruses in vertebrates. Type I IFNs (α/β) are mainly responsible for the antiviral defense system; they induce an antiviral state in the cellular compartments by stimulating the expression of IFN-stimulated genes such as RNA-specific Adenosine Deaminase (ADAR1), the interferon-induced GTP-binding protein Mx, protein kinase (PKR), and 2′,5′-oligoadenylate synthetase [1,2]. The IFN-inducible proteins mediate the catalysis of RNA degradation, RNA editing, and modulation of protein phosphorylation from double-stranded RNA.
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Statement: The corresponding author (Dr. Chi S.C.) has signed permission from another author (Wu Y.C.) to act on their behalf.