Research ArticleInterferon induced Mx protein from Indian snow trout Schizothorax richardsonii (Gray) lacks critical functional features unlike its mammalian homologues
Graphical abstract
Introduction
Type I interferons mediate signaling via JAK/STAT pathway and lead to the induction of variety of proteins due to stimulation of interferon stimulated genes (ISGs) (Schneider et al., 2014). Among ISGs, Mx is a well characterized type I interferon induced protein responsible for antiviral host defense (Haller et al., 2007). Mx proteins are usually present in all vertebrates and are known to appear in one to three isoforms (Verhelst et al., 2013). The ability of Mx in conferring selective resistance against influenza viruses has been well demonstrated besides blocking viral spread (Staeheli et al., 1986a, Staeheli et al., 1986b; Arnheiter et al., 1990). It is established that Mx can induce antiviral state against a number of viruses across different animal species (Haller et al., 2007; Verhelst et al., 2013). Mx proteins possess distinguishing antiviral activities and can be localized either in the nucleus or cytoplasm. Nuclear forms for example, mouse Mx1, protects against influenza (Krug et al., 1985; Staeheli et al., 1986a, Staeheli et al., 1986b; Grimm et al., 2007; Zimmermann et al., 2011) and Thogo virus (Haller et al., 1995) that are known to replicate in the nucleus while the cytoplasmic form, like mouse Mx2, are known to inhibit the replication of viruses in the cytoplasm like the vesicular stomatitis virus and hantavirus (Jin et al., 1999, Jin et al., 2001; Zurcher et al., 1992). The nuclear Mx possess a nuclear localization signal important to shuttle the Mx protein into the nucleus besides binding to the viral capsid protein (Zurcher et al., 1992; Schulte et al., 2015).
Mx proteins belong to the family of GTPase that include dynamins (Praefcke et al., 1999; Haller and Kochs, 2002). They possess a tripartite GTP binding domain, a middle domain and a C terminal GTPase effector domain (Praefcke et al., 1999; Haller and Kochs, 2002) with a characteristic leucine zipper (Verhelst et al., 2013; Haller and Kochs, 2011). Sequence variations have been observed among different Mx proteins with C-terminal GTP-binding motif being more conserved which may account for functional differences between the Mx isoforms (Lee and Vidal, 2002).
Antiviral activity of Mx proteins has also been reported from fish against a number of fish viruses (Verhelst et al., 2013; Lee and Vidal, 2002; Caipang et al., 2003; Larsen et al., 2004; Kibenge et al., 2005; Wu and Chi, 2007). Usually one to three Mx isoforms have been reported in fish, except European eel, Anguilla anguilla, which has four (Staeheli et al., 1989; Trobridge et al., 1997; Huang et al., 2013; Nygaard et al., 2000; Robertsen et al., 1997; Plant and Thune, 2004; Chen et al., 2006). As there is a great variation in the Mx isoforms within different fish species, it was intriguing to unravel the characteristics of Mx gene from snow trout, Schizothorax richardsonii (Gray), an important food and game fish inhabiting one of the most difficult high altitudinal regions in the foothills of Indian Himalayas. This investigation is a first report on identification and molecular characterization of interferon induced Mx protein from any Indian Coldwater fish.
Section snippets
Fish collection and stimulation
Schizothorax richardsonii, Indian snow trout were collected at an attitude of 1184 m above sea level from Kalsa stream Chaafi. The coordinates of the site located in the Kumaon hills of Uttarakhand, India were 29.365 N, 79.586 E. Fish were acclimatized in laboratory aquariums for a week. Fish ranging in weight from 8 to 10 g were injected intraperitoneally with poly (I:C) at a dose of 200 μg per fish. Liver spleen and kidney were collected from the poly (I:C) treated fish 72 h post treatment.
Sequence analysis of Mx gene
The nucleotide sequence of two fragments obtained by PCR was analyzed and a partial coding region of interferon-induced Mx protein comprising 1472 nucleotides was submitted to GenBank (Accession No. KU529282). After the analysis of 5′ and 3′ RACE PCR products, a nucleotide sequence of 2518 bases was obtained. The obtained sequence has an open reading frame (ORF) of 1854 nucleotides with a 5′UTR of 45 nucleotides and a 3′UTR of 619 nucleotides. This complete coding sequence was submitted to
Discussion
Mx coding sequence of snow trout is 2518 nucleotides with an open reading frame of 1854 nucleotides, a 5′ UTR of 45 nucleotides and a 3′ UTR of 619 nucleotides. SrMx coding sequence has three instability motifs (ATTTA) with a polyadenylation signal (AATAAA) present 211 nucleotides upstream the poly (A) tail. SrMx protein consists of 617 amino acids which is one amino acid less as compared with the usually reported range between 618 and 646 amino acids in different fish species (
Conclusion
Mx protein of snow trout is a cytoplasmic form of Mx protein. It retains most of the structural features of its mammalian orthologues. However, it is still a question whether the functional characteristics of the protein are similar. Intriguingly, the Mx protein of snow trout neither possess the determinant of virus substrate selection of nuclear Mx isoform nor does it possess the viral selection determinant of cytoplasmic form of mammalian Mx. Absence of such sequence in snow trout Mx protein
Disclosure statement
There is no potential conflict of interest was reported by the authors.
Funding
The project was supported by the Indian Council of Agricultural Research under ICAR–National Fellowship Scheme (Edn./27/9/NF/2014-HRD).
Acknowledgement
The authors duly acknowledge the Indian Council of Agricultural Research and for providing the funding and necessary facilities to carry out this work.
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Both authors contributed equally.