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

Highlights

• There is a functional interaction between gilthead seabream Mx proteins.

• Seabream Mx proteins synergise in their anti-ESV activity.

• Seabream Mx proteins interfere negatively in their anti-IPNV and anti-VHSV activity.

• Seabream Mx proteins interaction does not affect their anti-LCDV activity.

Abstract

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.

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.