Research paperSynergistic effects in the antiviral activity of the three Mx proteins from gilthead seabream (Sparus aurata)
Introduction
Type I interferon (IFN I) response is a key component of the innate immune system against viral infections in vertebrates (Samuel, 2001). IFN I induces hundreds of genes, some of which encode direct antiviral effectors, such as the Mx proteins. Mx proteins belong to the dynamin superfamily of high molecular weight GTPases, which are involved in intracellular membrane remodelling and intracellular trafficking (Kochs et al., 2005). They form homo-oligomers and self-assemble into ring-like and helical structures, which are critical for GTPase activity, protein stability, and viral recognition (Haller et al., 1998, Haller et al., 2007). Mx proteins interfere with viral replication at different stages, and, although their precise antiviral mechanisms are still unknown, there are evidences indicating that they rely on a direct interaction between the Mx protein and a specific viral target, that needs to be identified in each case (Haller and Kochs, 2011).
Mx proteins usually appear in different isoforms. Two Mx genes have been reported in amphioxus and in several mammalian species, including humans, whereas three genes have been described in rat (Li et al., 2009). Interestingly, a large variability in the number of Mx isoforms (from 1 to 7) has been disclosed in 15 fish species (Novel et al., 2013). Furthermore, Mx isoforms from the same fish species can differ in their antiviral activity mechanisms and/or antiviral specificity range (Fernández-Trujillo et al., 2011a, Fernández-Trujillo et al., 2013, Tafalla et al., 2007, Zenke and Kim, 2009). However, the putative synergy in the antiviral activity of Mx isoforms from the same species has never been addressed.
Three Mx proteins, Mx1, Mx2, and Mx3, corresponding to three different genes, have been identified in gilthead seabream (Sparus aurata) (Fernández-Trujillo et al., 2011a), which is one of the most important species in Southern Europe aquaculture. This fish species is highly resistant to viral diseases, being asymptomatic carrier of several viruses pathogenic to other fish species such as Viral Nervous Necrosis Virus (VNNV), Infectious Pancreatic Necrosis Virus (IPNV), and Viral Haemorrhagic Septicaemia Virus (VHSV) (Castric et al., 2001, Esteban et al., 2008, Pérez-Prieto et al., 2001). Actually, the only viral disease affecting gilthead seabream is the lymphocystis disease (LCD), caused by the Lymphocystis Disease Virus (LCDV) (García-Rosado et al., 1999).
The antiviral activity of each gilthead seabream Mx protein has been tested against IPNV, VHSV, LDCV and the European sheatfish virus (ESV), having shown that Mx1 presents activity against IPNV, VHSV and LCDV; Mx2 against IPNV, LCDV and ESV; and Mx3 against IPNV and VHSV (Fernández-Trujillo et al., 2011b, Fernández-Trujillo et al., 2013). Thus, in order to get more insight into the antiviral activity of gilthead seabream Mx proteins, the aim of the present study was to test if these three Mx proteins show synergy in their antiviral activity. For that, four in vitro experimental systems stably expressing combinations of the Mx proteins have been developed (Mx1+2, Mx1+3, Mx2+3 and Mx1+2+3 expressing cells). The antiviral activity in these cell lines was tested against IPNV, VHSV, ESV and LCDV, and compared with the antiviral activity in cells expressing each Mx separately.
Section snippets
Cell culture and viral isolates
The CHSE-214 cell line (Fryer et al., 1965) was grown at 20 °C in L-15 Leibovitz (L-15) medium (Invitrogen) supplemented with 10% foetal bovine serum (FBS, Invitrogen), 2% L-glutamine, and 1% antibiotic/antimycotic solution (100 U/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL fungizone, Invitrogen). The stable Mx expression systems were cultured under the same conditions.
The fish viruses used in this study were: (i) IPNV III-1 genotype (reference strain IPNV Sp), Birnaviridae family, with
Characterisation of the Mx-expressing cell populations
Several clonal populations stably expressing each Mx combination were obtained (Mx1+2, Mx1+3, Mx2+3 and Mx1+2+3). The cellular morphology, growth rate, as well as the transcription level of the recombinant Mx genes, were evaluated in these clones, and, for further experiments, the clonal population of each Mx combination showing standard morphology and growth rate as well as the least differences regarding the transcription level of the different Mx genes, were selected. All the selected lines
Discussion
The three Mx proteins from gilthead seabream have a wide antiviral spectrum, including viruses with different genomic composition. In addition, they differ in their antiviral specificities (Fernández-Trujillo et al., 2011b, Fernández-Trujillo et al., 2013). Therefore, it was tempting to consider a synergistic activity for the three Mx proteins, which could be responsible for the high resistance to viral infections showed by gilthead seabream. Our results evidence interactions between the three
Acknowledgments
The authors want to thank C.P. Dopazo, University of Santiago de Compostela, Spain and K. Way, CEFAS Weymouth Laboratory, UK, for the isolates of VHSV and ESV used in this work. This study has been funded by the project AGL2011-27181 from the Ministerio de Educación y Ciencia, Spanish Government, co-funded by FEDER from E.U.
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2022, Molecular ImmunologyCitation Excerpt :Different fish Mx isoforms display different antiviral ranges, but notably, fish Mx isoforms show interactions that have not been observed in mammals. Gilthead seabream (Sparus aurata), possessing three Mx isoforms (Mx1, Mx2, and Mx3), separately showed antiviral activity; however, these isoforms also interacted with each other, synergistically or antagonistically, depending on the virus species tested (Fernández-Trujillo et al., 2015). In Epinephelus coioides, Mx has been shown to limit GNNV replication by binding the coat protein of the virus to the effector domain of Mx (Chen et al., 2008).
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2017, Fish and Shellfish ImmunologyCitation Excerpt :Whether this protection is due to Mx proteins alone or in combination with other antiviral proteins is uncertain as poly IC induces the type I interferon pathway way that upregulates a battery of antiviral interferon stimulated genes [96]. However, it is likely that the induction of Mx proteins does have a role in protecting ASHe cells from VHSV infection as Mx proteins from Japanese flounder (Paralichthys olivaceus), Solea senegalensis, and gilthead seabream (Sparus aurata) can suppress VHSV replication [97–99]. In summary, ASHe and BAASf cells were susceptible to infection by all four fish RNA viruses: CSV, IPNV, VHSV IVa and VHSV IVb.
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2016, Developmental and Comparative ImmunologyCitation Excerpt :Gilthead seabream (Sparus aurata) expressing Mx1, Mx2, and Mx3 individually showed antiviral activity; however, these Mxs also interacted with each other, synergistically or antagonistic, depending on the virus that was infecting the cells (Fernández-Trujillo et al., 2015). It is hypothesized that interactions between different Mx isoform combinations create different oligomer conformations; if different isoforms are present in the oligomer ring it could affect the virus-targeting domain and alter its antiviral activity (Fernández-Trujillo et al., 2015). As with mammals, fish Mx proteins have different mechanisms of action.