Abstract
The myxovirus resistance (Mx) proteins belong to interferon-induced dynamin GTPase and play pivotal role in the inhibition of replication of numerous viruses. These antiviral proteins are released in usual or diseased condition to prevent the viral attack and to carry regular cellular activities like endocytosis and trafficking of nucleoproteins into the nucleus. The invasion of virus up-regulates the expression of Mx transcripts and double-stranded RNA mimic like polyinosinic polycytidyilic acid (Poly I:C). To understand the tissue-specific expression profiling and mechanism of GTP recognition of Mx protein from Labeo rohita (rohu), the full-length gene was cloned, sequenced and characterized through various Bioinformatics tools for the first time. The Mx cDNA was comprised of 2297 bp, and the open reading frame of 1938 bp encodes polypeptide of 631 amino acids. The coding sequence of Mx protein possess the signature motif of dynamin superfamily, LPRG(S/K)GIVTR, the tripartite guanosine-5/triphosphate (GTP)-binding motif (GXXXSGKS/T, DXXG and T/NKXD) and the leucine zipper motifs at the C-terminal end, well conserved in all interferon-induced Mx protein in vertebrates. Western blotting confirmed the molecular weight of Mx protein to be 72 kDa. After the intraperitoneal challenge of L. rohita with a Poly I:C, up-regulation of Mx protein was observed in brain, spleen, liver, kidney, intestine, heart, muscle, and gill. Ontogeny study displayed pronounced expression of Mx protein in all stages of the developmental of Rohu after Poly I:C induction. However a persistent expression of Mx transcript was also observed in Rohu egg as well as milt without induction with Poly I:C. Higher expression of Mx gene was observed on 96 h where it was 6.4 folds higher than the control. The computational modelling of Mx protein portrayed the tripartite N-terminal G-domain that binds to GTP, the bundle-signaling element (BSE) which interconnects the G-domain to the elongated stalk domain and C-terminal helical stalk domain. In agreement with the experimental studies, a series of conserved residues viz., Gln52, Ser53, Ser54, Leu68, Pro69, Gly71, Gly73, Thr76, Asp151, Gly154, Thr220, Lys221, Val251, Cys253, Arg254, and Gly255 were computed to be indispensable for tight anchoring of GTP within binding cavity of G-domain. The binding free energy calculation study depicted that the van der Waals and electrostatic terms contributs significantly to molecular recognition of GTP. Collectively, our study provides mechanistic insights into the tissue-specific expression profiling and GTP binding mechanism of Mx protein from Labeo rohita, which is expected to drive further research on several cellular events including viral resistance and endocytosis in the near future.
Highlights
The Innate immune system is the first line of defense system against the invasion of virus’s in vertebrates
A tripartite guanosine-5′-triphosphate (GTP)-binding motif, GXXXSGKS/T (GDQSSGKS) 35–42 amino acid position, DXXG (DLPG)[151–154] aa and T/NKXD (TKPD) 220–223 aa motifs typical to dynamin family were found in myxovirus resistance (Mx) protein of Rohu
The IFN-induced Mx proteins belonging to the evolutionary conserved dynamin-like large GTPases family exhibits a wide spectrum antiviral activity against many viruses
Summary
The Innate immune system is the first line of defense system against the invasion of virus’s in vertebrates. Two Mx proteins (MxA and MxB), are found to express, and part of the interferon response to viral infection. Through both share high percentage of homology, but they have shown the difference in their antiviral activities, signifying that these closely related proteins have distinct mechanisms of action. The Mx gene is induced by Type-I Interferon, the main cytokine mediating the innate immune antiviral function. Our study provides mechanistic insights into the structure-function mechanism and GTP recognition by Mx protein along with signal transduction cascade that leads to the up-regulation Mx protein upon induction by Poly I:C in different tissues in important fish like Rohu
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