In vitro and in vivo differential expression of rainbow trout (Oncorhynchus mykiss) Mx isoforms in response to viral haemorrhagic septicaemia virus (VHSV) G gene, poly I:C and VHSV

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Abstract

Three different Mx isoforms are known to be present in rainbow trout, however, to date, neither their mechanism of action nor their regulation have been established. Because most previous studies have focused only on one Mx isoform of the three present in rainbow trout, the expression of all isoforms was simultaneously studied in this work in response to the viral haemorrhagic septicaemia virus (VHSV) G gene, poly I:C or VHSV. Thus, RT–PCR assays were specifically designed to amplify each of the Mx1, Mx2 and Mx3 transcripts induced both in vitro (RTG-2 cell line and head kidney leucocytes) and in vivo (muscle, head kidney, spleen and liver). Regardless of the inducer used, in vitro results showed that while in RTG-2 cells Mx3 was predominantly induced, all three isoforms were similarly induced in head kidney leukocytes. In vivo, regardless of the inducer used a predominant expression of Mx3 transcripts was also observed in muscle but expression of all three Mx isoforms or predominantly Mx1 and Mx2 was found in head kidney and spleen. Mx expression in the liver was however more dependant on the inducer used. In conclusion, the results obtained demonstrated, for the first time, that both in vitro and in vivo the expression of the different Mx genes is differentially regulated. Moreover, this is also the first report showing Mx induction after cell transfection with a plasmid coding for the VHSV-G protein.

Introduction

Type I (α/β) interferon (IFN)-inducible Mx proteins are highly conserved large GTPases with homology to the dynamin superfamily [1], [2]. They have been found in all vertebrate species examined including mammals, birds and fish [3]. In addition to a conserved N-terminal tripartite GTP-binding domain, Mx family members share a central-interacting domain and a leucine zipper motif located in the C-terminal domain [2]. Interactions between all these domains are important for oligomerisation, GTPase activity and antiviral function.

In fish, the cDNAs of Mx proteins have been cloned and characterised in rainbow trout, Oncorhynchus mykiss [4], [5], atlantic salmon, Salmo salar [6], japanese flounder, paralichthys olivaceus [7], atlantic halibut, Hippoglossus hippoglossus [8], pufferfish, Takifugu rubripes, gilthead sea bream, Sparus aurata [9], channel catfish, Ictalurus punctatus [10], crucian carp, Carassius auratus [11], zebrafish, Danio rerio [12], turbot, Scophthalmus maximus [13] and orange-spotted grouper, Epinephelus coioides [14] However, evidence for antiviral activity has only been established for Mx proteins from japanese flounder and atlantic salmon [15], [16].

In rainbow trout, three different Mx genes, Mx1, Mx2 and Mx3, have been cloned and characterised [4], [5]. Mx1 and Mx3 are very similar in sequence, but Mx2 has a differential cysteine at position 324 and lacks the cysteine at position 16, there is a 13–15 amino acid insert at positions around 550 (with high probability to form coiled coils) and a nuclear localisation signal NLS (506KKRK510) [17]. Moreover, previous results have shown that trout Mx1 and Mx3 were localised in the cytoplasm while Mx2 was localised in the nucleus [5]. On the other hand, the antiviral effect of their encoded proteins was assayed by transfection of salmon cells with plasmids coding for their genes, but no antiviral activity could be demonstrated for any of the isoforms [5]. To date, Mx1 is the only trout isoform known to be directly induced by IFN [18].

DNA vaccines using the G genes of IHNV (infectious hematopoietic necrosis virus) or VHSV (viral haemorrhagic septicaemia virus) are more effective than previously developed vaccines [19], [20], [21], [22]. These vaccines are characterised by inducing a long-term specific immunity which is preceded by a strong early non-specific protective response [23], [24], [25], [26] similar to that of natural infection induced in non-vaccinated fish [19], [27]. This strong early antiviral response, mostly elicited by the endogenous expression of the G protein [19], [20], [21], [22], [28], [29], [30], is now considered the basis of the high efficacy of these vaccines [2], [19], [21], [28].

Non-specific responses following administration of rhabdovirus G-gene DNA vaccines in fish have included early up-regulation and expression of different immune-related genes including type I IFN genes [21] and type I IFN-induced genes [21], [23], [28], [31], [32], [33], [34], [35]. Among those genes, Mx genes are of importance because a correlation between early protection and Mx3 gene expression has been observed in DNA vaccinated rainbow trout [27]. In all these studies, however, only one Mx isoform has been studied, either the Mx1 [21], [32] or Mx3 [21], [27], [31], [33].

Since differential antiviral activity has been demonstrated for murine, rat and human [3], [36], [37], [38], [39], [40] and suggested for carp [11] Mx proteins, we studied the expression pattern of the three trout Mx isoforms stimulated by different inducers. Thus, Mx expression was induced in vitro (RTG-2 cell line or head kidney leucocytes) and in vivo (muscle, head kidney, spleen and liver) by the VHSV-G gene, polyriboinosinic-polyribocytidylic acid (poly I:C) and VHSV and then analysed by using RT–PCR assays specifically designed to amplify each isoform. The results obtained demonstrated, for the fist time, that both in vitro and in vivo the expression of the different Mx genes is differentially regulated. Moreover, this is also the first report showing Mx induction after cell transfection with a plasmid coding for VHSV-G protein.

Section snippets

Cell cultures and virus

The fish cell lines EPC (epithelioma papulosum cyprinid) [41], purchased from the European collection of cell cultures (ECACC no. 93120820), and RTG-2 (rainbow trout gonad) [42], purchased from the American Type Culture Collection (ATCC CCL 55), were used in this work.

EPC cells were maintained at 28 °C in a 5% CO2 atmosphere with RPMI-1640 Dutch modified (Gibco, Invitrogen Corp., UK) cell culture medium containing 10% foetal calf serum (Sigma Chemical Co, St. Louis, MO), 1 mM pyruvate (Gibco), 2 

Expression of Mx1, Mx2 and Mx3 in RTG-2 cells

Prior to analysing the expression levels of trout Mx isoforms, the nucleotide sequences of PCR products obtained using the designed primer sets were determined to confirm their identities and relative locations within Mx1, Mx2 and Mx3 mRNA sequences previous reported in GenBank (GenBank accession numbers of trout Mx1, Mx2 and Mx3 mRNAs are U30253, U47945 and U47946 respectively). In all cases, a 100% identity with the sequence previously submitted was found, showing that only the targeted Mx

Discussion

To study possible differences in the expression of trout Mx isoforms, we have analysed in vitro and in vivo the expression of its three Mx genes in response to different well known IFN inducers such as the G gene of VHSV (pMCV1.4-G), poly I:C and VHSV. This is the first time all Mx isoforms are simultaneously studied in rainbow trout.

Only Mx3 transcripts were significantly induced in response to pMCV1.4-G in RTG-2 cells. Although the percentage of pMCV1.4-G transfected cells was only of 5–7%

Acknowledgements

Thanks are due to Beatriz Bonmati and Esther Sanchez for technical assistance. This work was supported by projects AGL2004-07404-C02-01-02/ACU and AGL2005-00339ACU (MEyC, Spain), project CPE03-016-C3 (INIA, Spain) and project GV-ACOMP-06/068 (Generalitat Valeciana, Spain).

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