Synergistic effects in the antiviral activity of the three Mx proteins from gilthead seabream (Sparus aurata).

Due to their direct antiviral activity, Mx proteins play a main role in the response mediated by type I interferon against viral infections. The study on gilthead seabream Mx proteins is especially interesting, since this species is unusually resistant to viral diseases, being asymptomatic carrier of several viruses pathogenic to other fish species. Gilthead seabream has three Mx proteins (Mx1, Mx2 and Mx3) that, separately, display antiviral activity against a wide range of viruses, showing interesting differences in their antiviral specificities. In this work, the possible synergy between the three Mx isoforms has been studied using in vitro systems consisting of CHSE-214 cells stably expressing two or the three gilthead seabream Mx proteins. The antiviral activity of these Mx combinations has been tested against the Infectious Pancreatic Necrosis Virus (IPNV), the Viral Haemorrhagic Septicaemia Virus (VHSV), the European Sheatfish Virus (ESV) and the Lymphocystis Disease Virus (LCDV). A synergistic effect of the Mx proteins was only detected against ESV, no synergy was observed against LCDV, and a negative interference was detected against the two RNA viruses tested, IPNV and VHSV, as viral replication was higher in cells expressing certain Mx combinations than in cells expressing these proteins separately. These results suggest a functional interaction between gilthead seabream Mx isoforms, which results in a higher or lower antiviral activity depending on the virus tested, thus supporting the idea of complex virus-host interactions and finely tuned mechanisms controlling the antiviral activity of Mx proteins.

Fernández-Trujillo MA ，García-Rosado E ，Alonso MC ，Álvarez MC ，Béjar J ... -  《-》

Mx1, Mx2 and Mx3 proteins from the gilthead seabream (Sparus aurata) show in vitro antiviral activity against RNA and DNA viruses.

Mx proteins are important components of the antiviral innate immune response mediated by type I interferon. Classically, these proteins have been considered to be triggered by viral RNA, thus showing activity against RNA viruses. Actually, three Mx proteins (SauMx1, SauMx2 and SauMx3) from gilthead seabream (Sparus aurata) have previously shown antiviral activity against a dsRNA virus: the infectious pancreatic necrosis virus (IPNV) in vitro. For further characterizing their antiviral spectrum, the activity of SauMx proteins were tested against three different viral pathogens of fish: the lymphocystis disease virus (LCDV, a dsDNA virus), a pathogen of gilthead seabream; the viral haemorrhagic septicaemia virus (VHSV, a ssRNA virus), to which gilthead seabream is considered a reservoir species; and the European sheatfish virus (ESV, a dsDNA virus), that has not been detected in gilthead seabream to date. Three clonal populations of CHSE-214 cells developed in a previous study, stably expressing SauMx1, SauMx2 and SauMx3, respectively, were challenged with the three viruses. Results combining cytopathic effects and virus yield reduction assays showed that SauMx1 protected the cells against VHSV and LCDV, SauMx2 protected against ESV and LCDV, and SauMx3 showed activity only against VHSV. This study, besides confirming the antiviral activity of the three gilthead seabream Mx proteins, is the first report of the protective effect of a fish Mx against DNA viruses. Additionally, it discloses a clear specificity between Mx proteins and virus targets, supporting the idea that the relationship between virus and Mx proteins is finely tuned.

Fernández-Trujillo MA ，García-Rosado E ，Alonso MC ，Castro D ，Álvarez MC ，Béjar J ... -  《-》

Gilthead seabream (Sparus aurata) Mx gene promoters respond differentially to IPNV and VHSV infections in RTG-2 cells.

The understanding of virus-host interactions relies on the knowledge of the regulatory mechanisms of the type I interferon (IFN I)-stimulated genes (ISGs). Among ISGs, those coding Mx proteins play a main role due to their direct antiviral activity. The study of these genes in gilthead seabream is interesting, since this species displays high natural resistance to viral diseases, being asymptomatic carrier of infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV). Gilthead seabream has three Mx genes (Mx1, Mx2, and Mx3), encoding proteins with a wide spectrum of antiviral activity. The structure of the three promoters (pMx1, pMx2 and pMx3) has been previously disclosed, and their response to poly I:C in RTG-2 cells characterized. To further analyze these promoters, their response to two viral infections has been evaluated in the present study. For that purpose, RTG-2 cells transiently transfected with the luciferase gene under the control of each promoter were inoculated with either IPNV or VHSV at two different doses. The highest and lowest fold induction values were recorded for pMx2 and pMx3, respectively. The promoter induction was always stronger after VHSV inoculation than in IPNV-inoculated cells. In addition, the higher dose of VHSV tested induced higher response of the three promoters, whereas in IPNV-infected cells the highest induction was recorded after inoculation with the lower viral dose. To further study the response of the Mx2 promoter, RTG-2 cells stably transfected with the luciferase gene under the control of pMx2 were stimulated with poly I:C and subsequently infected with IPNV or VHSV. Interestingly, IPNV infection inhibited the induction caused by poly I:C, suggesting an antagonistic activity of IPNV on Mx2 transcription. In contrast, VHSV infection did not alter the response triggered by poly I:C. These results highlight the specific regulation that controls the activity of each promoter, and support the existence of complex interactions between host cells, specific Mx promoters, and viruses, which are responsible for the final outcome of a viral infection.

González-Mariscal JA ，Fernández-Trujillo MA ，Alonso MC ，García-Rosado E ，Álvarez MC ，Béjar J ... -  《-》

Differential antiviral activity of Mx1, Mx2 and Mx3 proteins from gilthead seabream (Sparus aurata) against Infectious Pancreatic Necrosis Virus (IPNV).

Mx proteins are crucial effectors of the innate antiviral response mediated by the interferon type I signalling pathway. Recently, three Mx proteins, named SauMx1, SauMx2 and SauMx3, corresponding to three different genes, have been identified in the cultured marine species gilthead seabream (Sparus aurata). In this study, the three SauMx cDNAs were cloned into expression vectors and used for transfection of CHSE-214 cells. Monoclonal cell populations stably expressing each recombinant protein have been obtained and characterized. The protection conferred by each recombinant SauMx against Infectious Pancreatic Necrosis Virus (IPNV) infection has been in vitro evaluated, having found clear differences among them. According to the cytopathic effects and the virus yield reduction assays, only cells expressing SauMx2 and SauMx3 showed significant resistance to IPNV infection. Otherwise, quantitative RT real-time PCR assays suggested that each SauMx protein has a different target during the viral inhibition process. The differences observed among the three SauMx proteins are discussed in terms of their differential mechanism of action and antiviral specificity, suggesting, as a whole, to play a synergistic activity in the protection of gilthead seabream against IPNV.

Fernández-Trujillo MA ，García-Rosado E ，Alonso MC ，Borrego JJ ，Alvarez MC ，Béjar J ... -  《-》

Cloning and characterization of the Mx1, Mx2 and Mx3 promoters from gilthead seabream (Sparus aurata).

Mx proteins are main effectors of the antiviral innate immune response mediated by type I interferon (IFN I). Actually, diverse Mx proteins from fish proved highly active against fish viruses, standing out among them the Mx1, Mx2 and Mx3 from gilthead seabream (Sparus aurata), a species exhibiting a natural resistance to viral diseases. In this study, the structure and functional activity of their corresponding promoters (pMx1, pMx2 and pMx3) have been assessed. The three promoters present an identical 3' region of 157 bp, exhibiting a single canonical interferon-stimulated response element (ISRE), which is indispensible for the poli:IC induction of pMx1 and pMx3, while not for that of pMx2. In the remaining part of the three promoters other regulatory motifs were identified, as gamma IFN activated sites in variable number (1, 4 and 2 in pMx1, pMx2 and pMx3, respectively), as well as several independent GAAA elements or ISRE core sequences (13, 15 and 12 in pMx1, pMx2 and pMx3, respectively). The structural dissimilarities shown by the three promoters parallels with the differences observed in their response profiles, in terms of the time course of the induction, and basal and induced expression levels of each promoter. Altogether, these findings indicate that the expression of Mx1, Mx2 and Mx3 genes from the gilthead seabream might be specifically regulated, in accordance with the functional role of each Mx protein in the successful antiviral response shown by this species.

González-Mariscal JA ，Gallardo-Gálvez JB ，Méndez T ，Álvarez MC ，Béjar J ... -  《-》