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Protective roles of grass carp Ctenopharyngodon idella Mx isoforms against grass carp reovirus
Limin Peng1, Chunrong Yang, Jianguo Su
1College of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling, Shaanxi, China.
Background:
Myxovirus resistance (Mx) proteins are crucial effectors of the innate antiviral response against a wide range of viruses, mediated by the type I interferon (IFN-I) signaling pathway. However, the antiviral activity of Mx proteins is diverse and complicated in different species.
Methodology/Principal Findings:
In the current study, two novel Mx genes (CiMx1 and CiMx3) were identified in grass carp (Ctenopharyngodon idella). CiMx1 and CiMx3 proteins exhibit high sequence identity (92.1%), and low identity with CiMx2 (49.2% and 49.5%, respectively) from the GenBank database. The predicted three-dimensional (3D) structures are distinct among the three isoforms. mRNA instability motifs also display significant differences in the three genes. The spatial and temporal expression profiles of three C. idella Mx genes and the IFN-I gene were investigated by real-time fluorescence quantitative RT-PCR (qRT-PCR) following infection with grass carp reovirus (GCRV) in vivo and in vitro. The results demonstrated that all the four genes were implicated in the anti-GCRV immune response, that mRNA expression of Mx genes might be independent of IFN-I, and that CIK cells are suitable for antiviral studies. By comparing expression patterns following GCRV challenge or poly(I:C) treatment, it was observed that GCRV blocks mRNA expression of the four genes. To determine the functions of Mx genes, three CiMx cDNAs were cloned into expression vectors and utilized for transfection of CIK cells. The protection conferred by each recombinant CiMx protein against GCRV infection was evaluated. Antiviral activity against GCRV was demonstrated by reduced cytopathic effect, lower virus titer and lower levels of expressed viral transcripts. The transcription of IFN-I gene was also monitored.
Conclusions/Significance:
The results indicate all three Mx genes can suppress replication of grass carp reovirus and over-expression of Mx genes mediate feedback inhibition of the IFN-I gene.
Insights
Three grass carp Myxovirus resistance (Mx) genes were identified and found to suppress grass carp reovirus replication. Overexpression of these Mx genes also inhibits the type I interferon (IFN-I) gene, highlighting their role in innate immunity.
Area of Science:
- Aquaculture immunology
- Innate antiviral immunity
- Fish molecular biology
Background:
- Myxovirus resistance (Mx) proteins are key components of the type I interferon (IFN-I) signaling pathway, crucial for innate antiviral defense.
- The antiviral functions of Mx proteins vary significantly across different species.
- Understanding Mx protein diversity and function is essential for fish health in aquaculture.
Purpose of the Study:
- To identify and characterize novel Myxovirus resistance (Mx) genes in grass carp (Ctenopharyngodon idella).
- To investigate the expression patterns and antiviral functions of these Mx genes against grass carp reovirus (GCRV).
- To explore the relationship between Mx gene expression and the type I interferon (IFN-I) pathway during viral infection.
Main Methods:
- Identification and sequence analysis of two novel grass carp Mx genes (CiMx1 and CiMx3).
- Real-time fluorescence quantitative RT-PCR (qRT-PCR) to analyze spatial and temporal expression of Mx and IFN-I genes following GCRV infection.
- Transfection of grass carp kidney (CIK) cells with recombinant CiMx proteins to evaluate antiviral activity against GCRV.
Main Results:
- Two novel Mx genes, CiMx1 and CiMx3, were identified in grass carp, showing high sequence identity to each other and distinct 3D structures and mRNA instability motifs compared to CiMx2.
- All four investigated genes (three CiMx and one IFN-I) were involved in the anti-GCRV immune response, with Mx gene expression potentially independent of IFN-I.
- GCRV infection was found to inhibit the mRNA expression of all four genes, while recombinant CiMx proteins conferred significant protection against GCRV infection in CIK cells.
Conclusions:
- All three identified grass carp Mx genes (CiMx1, CiMx2, and CiMx3) possess the ability to suppress grass carp reovirus replication.
- Overexpression of Mx genes can induce feedback inhibition of the type I interferon (IFN-I) gene, suggesting a regulatory role in the antiviral response.
