Review
Early antiviral response and virus-induced genes in fish

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Abstract

In fish as in mammals, virus infections induce changes in the expression of many host genes. Studies conducted during the last fifteen years revealed a major contribution of the interferon system in fish antiviral response. This review describes the screening methods applied to compare the impact of virus infections on the transcriptome in different fish species. These approaches identified a “core” set of genes that are strongly induced in most viral infections. The “core” interferon-induced genes (ISGs) are generally conserved in vertebrates, some of them inhibiting a wide range of viruses in mammals. A selection of ISGs – PKR, vig-1/viperin, Mx, ISG15 and finTRIMs – is further analyzed here to illustrate the diversity and complexity of the mechanisms involved in establishing an antiviral state. Most of the ISG-based pathways remain to be directly determined in fish. Fish ISGs are often duplicated and the functional specialization of multigenic families will be of particular interest for future studies.

Introduction

Fish are able to mount strong and protective immune responses to many viruses, as evidenced by vaccine efficacy. The specific protection is most often afforded by neutralizing antibodies, for example, for rhabdoviruses (Boudinot et al., 1998, Corbeil et al., 1999) although T cells and NK cells probably play an important role in the clearing of infected cells. The first antiviral defense is mediated by innate immunity in particular by interferons and interferon-induced genes and by intrinsic cellular mechanisms. More than 200 interferon-responsive genes (ISGs) have been described in mammals to date (Der et al., 1998, Welsh and Sen, 1997), illustrating the diversity of the cellular pathways triggered by virus infections.

In mammals, interferons were described as protective against viral infection 50 years ago (Isaacs and Lindenmann, 1957), and they have been classified in three types according to their biological and structural properties: type I (IFN α, β, ω, ɛ, κ), type II (IFN-γ) and type III (IFN-λ, formerly IFN-like cytokine) (Pestka et al., 2004). Type I and III are directly induced by virus infection, and promote the transcription of antiviral genes through a single pathway. This pathway involves Jak kinases (Jak1 and Tyk2) and transcription factors belonging to the STAT family (STAT-1, -2, -3 and -4) (Zhou et al., 2007). IFN-γ induces the transcription of antiviral genes through a specific receptor constituted by two subunits IFNGR-1 and -2 and through a slightly different Jak-STAT pathway (Boehm et al., 1997).

In fish, the induction of a secreted mediator with IFN properties has been known for years (de Kinkelin and Dorson, 1973), but the first IFN sequences were only reported in the early 2000s from genomic and EST databases (Robertsen et al., 2003, Lutfalla et al., 2003, Altmann et al., 2003). Fish IFNs have been often compared to Type I mammalian IFNs (Robertsen, 2008) though they are encoded by genes with four introns (Lutfalla et al., 2003) and their receptor is closer to the type III interferon receptors (Levraud et al., 2007, Aggad et al., 2009). Additionally, the expression of fish interferons is subject to an additional level of regulation. Indeed, in zebrafish and rainbow trout viral infection triggers activation of an alternative IFN isoform with an effective signal peptide (Levraud et al., 2007, Purcell et al., 2009), while the normal promoter usage leads to the expression of a non-secreted isoform in absence of stimulation. In this review fish virus-induced IFNs “IFNϕ” will be named according to Stein et al. (2007) nomenclature. Multiple IFNϕ are found in fish studied so far, which are induced differentially depending on the inflammatory contexts (Lopez-Munoz et al., 2009). In zebrafish, IFNϕ1 ensures a slow and powerful induction of antiviral genes while IFN ϕ2 and 3 lead to rapid and transient responses to the virus (Aggad et al., 2009, Lopez-Munoz et al., 2009). Thus, IFNϕ1 exerts a better protection against IHNV than IFNϕ2. However, the specificity of the transcriptional response triggered by the fish multiple interferons is still poorly understood. Besides the IFNfs, an IFN-γ orthologue has also been identified in fish (Zou et al., 2004, Igawa et al., 2006, Milev-Milovanovic et al., 2006).

Besides the characterization of fish IFNs, other studies have focused on virus- and interferon-induced genes (ISGs). Many of these genes can be considered as the effectors of the interferon response, and some of them possess a documented antiviral activity. Tetrapods and fish share a number of ISGs that are remarkably conserved, indicating that the interferon system is an ancient and fundamental part of the immune system of gnathostomes. However, many fish ISGs remain poorly characterized, and a strong antiviral activity has only been demonstrated for a few of them. Additionally, since teleost fish constitute the most diverse class of vertebrates, a variety of new antiviral genes and antiviral strategies probably arise in the different fish families during a long evolutionary time marked by multiple global genome duplications (Volff, 2005).

To characterize the antiviral response mechanisms, different approaches were followed to directly identify genes induced by the virus infections using different strategies. The first part of this review will briefly describe how these strategies identified the interferon system as the main target of the virus-induced transcriptional response in fish. The second part will examine selected virus- and interferon-induced genes involved in the key pathways activated by viral infection.

Section snippets

Towards a comprehensive description of the transcriptional response to viruses in fish

Fish virus and interferon induced genes were first identified by PCR using primers designed on conserved sequences. For example, Mx – a protein from the dynamin family with a tripartite GTP domain and a leucine zipper – was cloned in rainbow trout (Oncorhynchus mykiss) (Trobridge and Leong, 1995) and in other teleost species (including Atlantic salmon (Salmo salar) (Robertsen et al., 1997), Atlantic halibut (Hippoglossus hippoglossus) (Jensen and Robertsen, 2000) and fugu (Takifugu rubripes) (

PKR, an interferon-induced regulator of protein synthesis

The double stranded RNA-dependent protein kinase or protein kinase R (PKR) is probably the best known of the interferon-induced proteins. Though recently characterized (Clemens et al., 1993) it has been known for decades that a protein with dsRNA-dependent kinase activity is involved in the cellular response to viral infections and is induced by interferons (Roberts et al., 1976, Sen et al., 1978). This protein kinase was first cloned in human in 1990 (Meurs et al., 1990). PKR has been

vig-1/viperin, a member of SAM superfamily suppressing diverse viruses

The transcripts cig5 and vig1 were identified in mRNA differential display screening for HCMV- and VHSV-induced genes in human and rainbow trout, respectively (Boudinot et al., 1999, Zhu et al., 1997). They encode for a protein highly conserved in vertebrates, with a MoaA motif suggesting a role in the synthesis of enzymatic cofactor (Fig. 1) (Boudinot et al., 1999). This motif places this protein into the large SAM superfamily of enzymes (Sofia et al., 2001, Duschene and Broderick, 2010).

Mxs, GTPases with antiviral activity

Mx – for orthomyxovirus resistance gene – was first identified (Horisberger et al., 1983, Haller et al., 1980) as a factor explaining high resistance to orthomyxoviruses in the inbred mouse strain AG2 (Lindenmann, 1964). Several Mx genes were found, that were induced by type I and type III interferons (Haller et al., 1980) and presented a strong antiviral activity against the avian influenza virus (Staeheli et al., 1986, Arnheiter et al., 1990). Homologues of Mx1 and Mx2 were soon found in rat,

ISG15, an ubiquitin-like antiviral protein

Isg15 (IFN-stimulated gene 15) is induced to a very high level by type I IFNs in mammals (Dao and Zhang, 2005). It was first identified more than 20 years ago as a 15 kDa-protein induced by IFNs in human and bovine cells (Korant et al., 1984). The gene was cloned in human (Blomstrom et al., 1986), and appeared to be highly similar to ubiquitin. The ISG15 protein was conjugated with other proteins, and was considered as an ubiquitin-like protein with similar properties (Haas et al., 1987, Loeb

finTRIMs and other teleost TRIM proteins: newcomers in fish antiviral activity

The superfamily of tripartite proteins (TRIM) is defined by the N-terminus TRIM or RBCC motif, which comprises in the following order a RING domain, one or two B-box domains and an associated coiled-coil domain (Fig. 1) (Reymond et al., 2001). The RBCC motif is usually followed by one or two domains that determine(s) the partners of the TRIM protein and the pathway in which it is involved. TRIM proteins are present in vertebrates and invertebrates, but their diversity seems to be greatly

vig2, a salmonid-specific ISGs with elusive functions

While the ISGs described above are generally well conserved among teleosts, or even among vertebrates, others can be unique to a fish family. vig2, a gene identified by differential display PCR in leukocytes incubated with VHSV (Boudinot et al., 2001), constitutes a good example of such a ISG with restricted species range. vig2 was observed in several transcriptome analyses after virus infection in salmonids (for example, Purcell et al., 2006). vig2 is strongly induced by interferon in both

Conclusion

Many mammalian ISGs have been retrieved in fish and are likely to play similar antiviral functions through vertebrates. The main components of the interferon system were highly conserved during evolution of fishes and tetrapods. Hence, this pathway probably appeared in early vertebrate evolution and became the primary antiviral system. These “core” components of the interferon system are generally active against a broad spectrum of viruses (Table 1). Such antiviral activity has been proved for

Acknowledgements

We thank Sylvie Quiniou for discussions and proofreading of the manuscript. This work has been funded by INRA.

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