Characterization, Expression Pattern and Antiviral Activities of Mx Gene in Chinese Giant Salamander, Andrias davidianus.

Yanan Liu,Nan Jiang,Yuding Fan,Yiqun Li,Yongze Zhou,Lingbing Zeng

doi:10.3390/ijms21062246

Abstract

Mx, Myxovirus resistance is an important interferon-stimulated protein that mediates antiviral responses. In this study, the expression and activities of Chinese giant salamander, Andrias davidianus Mx gene, AdMx, were investigated. The AdMx cDNA sequence contains an open reading frame (ORF) of 2112 nucleotides, encoding a putative protein of 703 aa. Meanwhile, AdMx possesses the conserved tripartite GTP binding motif and a dynamin family signature. qRT-PCR analysis revealed a broad expression of AdMx in vivo, with the highest expression levels in brain, kidney and spleen. The AdMx expression level in kidney, spleen and muscle significantly increased at 6 h after Chinese giant salamander iridovirus (GSIV) infection and peaked at 48 h, while that in muscle cell line (GSM) was not noticeably up-regulated until 72 h post infection. Additionally, a plasmid expressing AdMx was constructed and transfected into the Chinese giant salamander GSM cells. The virus load and gene copies in AdMx over-expressed cells were significantly reduced compared with those in the control cells. Moreover, compared to the control cells, a lower level of virus major capsid protein (MCP) synthesis in AdMx over-expressed cells was confirmed by Western blot. These results collectively suggest that Mx plays an important antiviral role in the immune responses against GSIV in Chinese giant salamander.

Highlights

Mx is a kind of antiviral protein induced by type I interferon (IFN), which formed the antiviral mechanism together with other IFN-stimulated proteins during virus infection [1]
a Chinese giant salamander Mx (AdMx) is composed of 703 amino acids residues with a calculated molecular mass of 79.09 kDa and a theoretical isoelectric point of 5.33
Similar to other Mx genes, AdMx possesses a putative hydrophobic signal peptide at the N-terminus, a GTPase domain containing a triplet GTP-binding region (GDQSSGKS, DLPG and TKPD) and GTPase effector domain GED at the C-terminus (610–701 aa) (Figure 1). a dynamin family signature with the sequence of LPRGSGIVTR was observed in Chinese giant salamander Mx

Summary

Introduction

Mx is a kind of antiviral protein induced by type I interferon (IFN), which formed the antiviral mechanism together with other IFN-stimulated proteins during virus infection [1]. In Chinese giant salamanders, Mx expression was significantly up-regulated in the IFN over-expressed cells infected by Chinese giant salamander iridovirus (GSIV) [15]. These studies indirectly indicate that the Mx gene of amphibians may have a viral inhibitory effect. The expression patterns of AdMx both in vivo and in vitro were profiled and the antiviral effect against GSIV in Chinese giant salamander muscle (GSM) cells was examined.

Identification and Molecular Characterization of AdMx

Discussion

Materials and Methods

Cloning of AdMx

Sequence Alignment and Phylogenetic Analysis

Construction of AdMx Plasmid and Transfection

AdMx Subcellular Localization

Western Blot

Statistical Analysis