Interferon-inducible Mx proteins in fish

Summary: Mx proteins are members of a family of hiterferon-inducible genes expressed when cells are treated with double-stranded RNA or virus infection. These proteins are important components of the antiviral response and form the first line of the body's defense against virus infections. The exact mechanism of action for these proteins has not been discovered, but mice missing the Mx genes are extremely sensitive to influenza virus infection. Mammals have between two and three Mx genes whose functions may vary with regard to the inhibition of a specific virus, cellular localization, and activity. The cDNA of three rainbow trout Mx proteins has been cloned and a comparison of their sequences with that of avian and mammalian species reveals striking conservation of domains. They all maintain the tripartite ATP/GTP binditig domain and the dynamin family signature in the amino terminal half of the protein. In the carboxyl terminal half of the Mx proteins are che Iocalization signals and the leucine zipper motifs which account for the trimerization of Mx in the cell. Like the rat and human Mx proteins, the different trout Mx proteins exhibit distinctly different immunohistochemical staining patterns in cells transfected with plasmids expressing RBTMx1, RBTMx2, or RBTMx3, To date, the antiviral function of the trout Mx proteins has not been satisfactorily established.     

Mx genes show weaker primary response to virus than other interferon-regulated genes.

Some interferon (IFN)-regulated genes are induced as a primary response to virus as well as secondarily through virus-induced IFN. Here we investigated whether this dual control mechanism would also regulate the activity of the human and mouse Mx genes that encode proteins with intrinsic antiviral potentials. To distinguish between a primary response to virus and a secondary response to virus-induced IFN, we studied virus-induced Mx gene expression in cell lines that lack a functional IFN system and in cells with blocked protein synthesis. In contrast to the two IFN-regulated human genes ISG56 and ISG15, the human MxA gene showed almost no primary response to Newcastle disease virus (NDV) or influenza virus. Similarly, direct activation of the mouse Mx1 gene by NDV or influenza virus was not significant in mouse embryo cells or explanted peritoneal macrophages. A moderate primary Mx1 response to NDV was observed in the permanent cell line L1210. Lack of a strong IFN-independent Mx response to virus indicates that this mode of gene regulation does not play a significant role in Mx-mediated resistance to viral disease.     

Expression regulation and functional characterization of a novel interferon inducible gene Gig2 and its promoter

Grass carp hemorrhagic virus (GCHV)-induced gene 2 (Gig2) is a novel gene previously identified from UV-inactivated GCHV-treated Carassius auratus blastulae embryonic (CAB) cells, suggesting that it should play a pivotal role in the interferon (IFN) antiviral response. In this study, a polyclonal anti-Gig2 antiserum was generated and used to study the inductive expression pattern by Western blot analysis, showing no basal expression in normal CAB cells but a significant up-regulation upon UV-inactivated GCHV, polyinosinic:polycytidylic acid (Poly I:C) and recombinant IFN (rIFN). However, constitutive expression of Gig2 is observed in all tested tissues from grass carp (Ctenopharyngodon idellus), and Poly I:C injection increases the relative amount of Gig2 protein in skin, spleen, trunk kidney, gill, hindgut and thymus. Moreover, the genomic sequence covering the whole Gig2 ORF and the upstream promoter region were amplified by genomic walking. Significantly, the Gig2 promoter contains three IFN-stimulated response elements (ISREs), nine GAAA/TTTC motifs and five γ-IFN activating sites (GAS), which are the characteristics of genes responsive to both type I IFN and type II IFN. Subsequently, the complete Gig2 promoter sequence was cloned into pGL3-Basic vector, and its activity was measured by luciferase assays in the transfected CAB cells. The Gig2 promoter-driven construct is highly induced in CAB cells after treatment with Poly I:C or rIFN, and the functional capability is dependent on IFN regulatory factor 7 (IRF7), because its activity can be stimulated by IRF7. Collectively, the data provide strong evidence that Gig2 is indeed a novel IFN inducible gene and its expression is likely dependent on IRF7 upon Poly I:C or IFN.     

Nucleotide sequences of genome segments S6, S7 and S10 of Dendrolimus punctatus cypovirus 1

The nucleotide sequences of genome segments S6, S7 and S10 of Dendrolimus punctatus cypovirus 1 Hunan I (DpCPV-HN(I)) and DpCPV-HN(I)-Se(3) (DpCPV-HN(I) passed three times in Spodoptera exigua) were determined. Segment S10 was 944 nucleotides in length and encoded a polyhedrin of 248 amino acids (28,439 Da). Only two nucleotide mutations were found between DpCPV-HN(I) S10 and DpCPV-HN(I)-Se3 S10, and the deduced amino acid sequences of the polyhedrin proteins were identical. Segment S7, 1 501 nucleotides, encoded a protein of 448 amino acids ( approximately 50 kDa; p50). Thirty-one nucleotide mutations were found between DpCPV-HN(I) S7 and DpCPV-HN(I)-Se3 S7, but these resulted in only four amino acid changes. DpCPV-HN(I) S6 encoded a protein of 561 amino acids (63,688 Da; p64). The amino acid sequence of p64, had a high leucine content (10%), and contained a leucine zipper motif and one ATP/GTP-binding site motif.     

Molecular characterisation and structural analysis of an interferon homologue in sea bass (Dicentrarchus labrax L.)

The interferons (IFNs) are a large family of soluble cytokines involved in the immune response against viral pathogens. Three families of IFNs have been identified in mammals (type I, type II and type III) and, recently, homologues of type I and type II genes have been found in various teleost fish species. In this paper we report the cloning of a cDNA encoding an type I IFN molecule from sea bass (Dicentrarchus labrax L.), its expression analysis and gene structure and, finally, its 3D structure obtained by template-based modelling. The sea bass IFN cDNA consists of 1047bp that translates in one reading frame to give the entire molecule containing 185 amino acids. The analysis of the sequence revealed the presence of a putative 22 amino acid signal peptide, two cysteine residues and three potential N-glycosylation sites. The sea bass IFN gene contains four introns as with other type I IFN teleost genes, except medaka that contains three introns. Real time PCR was performed after poly I:C stimulation of DLEC cell line to investigate the expression of sea bass IFN and Mx and an induction was observed for both genes. The predicted 3D structure of sea bass IFN is characterized by an "all-alpha" domain that shows an "up-down bundle" architecture made of six helices (ABB'CDE). The two cysteine residues present in the sequence (i.e. Cys(23) and Cys(126)) are in a position and at a distance that suggest the possible formation of a disulfide bridge that may stabilize the structure. Our results will give the opportunity to investigate more in detail antiviral immune responses in sea bass and add to studies on the evolution of the IFN system in teleosts and vertebrates more generally.     

Identification and characterization of rat Dact1 and Dact2 genes in silico

DACT1 (DAPPER1), Frizzled receptors, MUSK receptor, VANGL1, VANGL2, PRICKLE1, PRICKLE2, DAAM1, Casein kinases, MARK3 (PAR1), PP2C, AXIN1, AXIN2, NKD1, NKD2, FRAT1, FRAT2 and CXXC4 are WNT signaling molecules associating with Dishevelled family proteins. Human DACT1 is the ortholog of Xenopus Dapper and Frodo, and human DACT2 (DAPPER2) is the paralog of human DACT1. Here, we identified and characterized rat Dact1 (Dapper1) and Dact2 (Dapper2) genes by using bioinformatics. Rat Dact1 gene, consisting of four exons, was located within AC136677.3 genome sequence. Rat Dact2 gene, consisting of four exons, was located within AC139434.3 genome sequence. Dact1 was mapped to rat chromosome 6q24, and Dact2 gene to rat chromosome 1q12. Rat Dact1 (778 aa) showed 93.7, 82.9, 60.3, 58.7 and 48.6% total-amino-acid identity with mouse Dact1, human DACT1, Xenopus Dapper, Xenopus Frodo and zebrafish dact1, respectively. Rat Dact2 (768 aa) showed 86.6, 59.6 and 38.3% total-amino-acid identity with mouse Dact2, human DACT2 and zebrafish dact2, respectively. Dact1 orthologs were more evolutionarily conserved than Dact2 orthologs. Seven DAPH domains (DAPH1-DAPH7), originally identified as the regions conserved between human DACT1 and DACT2, were conserved among mammalian Dact1 orthologs and Dact2 orthologs. DAPH2 domain, corresponding to the Leucine zipper motif, was located within the coiled-coil region. DAPH3 domain was the Serine rich region. DAPH7 domain was the C-terminal PDZ binding region. This is the first report on the rat Dact1 and Dact2 genes.     

Interferon-induced rat Mx proteins confer resistance to Rift Valley fever virus and other arthropod-borne viruses.

Mx proteins belong to the interferon (IFN)-induced antiviral defense. The rat genome contains three Mx genes, ratMx1, ratMx2, and ratMx3. The Mx gene products differ in their subcellular localization and antiviral specificity. The nuclear ratMx1 protein confers resistance to influenza A virus, and the cytoplasmic ratMx2 is active against vesicular stomatitis virus (VSV), whereas the cytoplasmic ratMx3 protein is antivirally inactive. To investigate the antiviral potential of the rat Mx proteins against arboviruses, a phylogenetically diverse group of viruses that frequently infect rodents, we studied the replication of LaCrosse virus (LACV). Rift Valley fever virus (RVFV) (both family Bunyaviridae), and Thogoto virus (THOV) (family Orthomyxoviridae). To that end, we used transfected Vero cells constitutively expressing one of the rat Mx proteins. We observed that the antiviral activity of rat Mx proteins against these arboviruses correlates with their intracellular localization: ratMx1 is active against THOV, which replicates in the nucleus, whereas ratMx2 inhibits bunyaviruses that replicate in the cytoplasm. The results indicate that rats have evolved two Mx proteins to efficiently control viruses with different replication strategies.     

Comparative genomics on Fzd7 orthologs

WNT signaling pathway networks with Hedgehog and Notch signaling pathways during carcinogenesis and embryogenesis. FZD7 is up-regulated in gastric cancer, esophageal cancer, and hepatocellular carcinoma (HCC). Here we identified and characterized rat Fzd7 gene by using bioinformatics. Rat Fzd7 gene was identified within AC136379.2 genome sequence. The 5'-flanking region and exonic region were well conserved among mammalian Fzd7 orthologs. Nucleotide position 153000-152216 of AC136379.2 genome sequence was identified as the evolutionarily conserved promoter region of rat Fzd7 gene, and nucleotide position 2273-3046 of AC069148.6 genome sequence as the evolutionarily conserved promoter region of human FZD7 gene. Match program revealed that PAX4-binding site was conserved among rat Fzd7, mouse Fzd7 and human FZD7 promoters. Rat Fzd7 (572 aa) was a seven-transmembrane-type Wnt receptor, which showed 99.3, 96.9, 87.4, 85.5, 79.5 and 79.0% total-amino-acid identity with mouse Fzd7, human FZD7, chicken fzd7, Xenopus fzd7, zebrafish fzd7a and fzd7b, respectively. Frizzled (Fz) domain within the N-terminal extracellular region, Leucine zipper motif around the fifth transmembrane (TM5) region, Dishevelled (Dvl)- and Magi3-binding motifs within the C-terminal cytoplasmic region were conserved among vertebrate Fzd7 orthologs. Leucine zipper motif around the TM5 region of Fzd7 orthologs was disrupted in FzE3 aberrant cDNA due to multiple cloning artifacts or sequencing errors. These facts indicate that experimental data obtained by using FzE3 cDNA do not always reflect the functions of Fzd7 orthologs.