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
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).
References (52)
- et al.
Molecular cloning of double-stranded RNA inducible Mx genes from Atlantic salmon (Salmo salar L.)
Dev Comp Immunol
(1997) - et al.
Cloning and analysis of expression of Mx cDNA in Japanese flounder, Paralichthys olivaceus
Dev Comp Immunol
(2000) - et al.
Cloning and analysis of expression of a gilthead sea bream (Sparus aurata) Mx cDNA
Fish Shellfish Immunol
(2004) - et al.
Cloning and characterisation of a channel catfish (Ictalurus punctatus) Mx gene
Fish Shellfish Immunol
(2004) - et al.
Cloning and characterization of an Mx gene and its corresponding promoter from the zebrafish, Danio rerio
Dev Comp Immunol
(2004) - et al.
Molecular characterisation of a turbot Mx cDNA
Fish Shellfish Immunol
(2005) - et al.
Cloning of an orange-spotted grouper (Epinephelus coioides) Mx cDNA and characterisation of its expression in response to nodavirus
Fish Shellfish Immunol
(2006) - et al.
In vitro inhibition of fish rhabdoviruses by Japanese flounder, Paralichthys olivaceus Mx
Virology
(2003) - et al.
An Mx1 promoter-reporter system to study interferon pathways in rainbow trout
Dev Comp Immunol
(2004) - et al.
DNA vaccines as a tool for analysing the protective immune response against rhabdoviruses in rainbow trout
Fish Shellfish Immunol
(2002)
Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus
Mol Immunol
Protection of rainbow trout against infectious hematopoietic necrosis virus four days after specific or semi-specific DNA vaccination
Vaccine
Immunity induced shortly after DNA vaccination of rainbow trout against rhabdoviruses protects against heterologous virus but not against bacterial pathogens
Dev Comp Immunol
DNA vaccination against viral haemorrhagic septicaemia (VHS) in rainbow trout: size, dose, route of injection and duration of protection-early protection correlates with Mx expression
Fish Shellfish Immunol
Combined DNA immunization with the glycoprotein gene of viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus induces double-specific protective immunity and nonspecific response in rainbow trout
Virology
Viral haemorrhagic septicaemia virus induces vig-2, a new interferon-responsive gene in rainbow trout
Fish Shellfish Immunol
Expression of the glycoprotein of viral haemorrhagic septicaemia virus (VHSV) on the surface of the fish cell line RTG-P1 induces type 1 interferon expression in neighbouring cells
Fish Shellfish Immunol
Quantitative expression profiling of immune response genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) infection or DNA vaccination
Fish Shellfish Immunol
Kinetics of Mx expression in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar L.) parr in response to VHS-DNA vaccination
Fish Shellfish Immunol
Comparative immune responses in Japanese flounder, Paralichthys olivaceus after vaccination with viral hemorrhagic septicemia virus (VHSV) recombinant glycoprotein and DNA vaccine using a microarray analysis
Vaccine
Cloning and expression analysis of rainbow trout Oncorhynchus mykiss interferon regulatory factor 1 and 2 (IRF-1 and IRF-2)
Dev Comp Immunol
Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus
Cell
Expression of the human MxA protein is associated with hyperphosphorylation of VSV P protein in human neural Cells
Virology
Inhibition of influenza C viruses by human MxA protein
Virus Res
Some properties of the Epithelioma papulosum cyprini (EPC) cell line from carp
Cyprinus carpio
Expression of genes related to the early immune response in rainbow trout (Oncorhynchus mykiss) after viral haemorrhagic septicemia virus (VHSV) infection
Dev Comp Immunol
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