Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio

Abstract

The interferon (IFN) family consisting of alpha IFN (IFN-alpha), IFN-beta, IFN-omega, IFN-delta, IFN-kappa, and IFN-tau is a large group of cytokines involved in the innate immune response against various microorganisms. Genes for IFN have been cloned from a variety of mammalian and avian species; however, IFN genes from lower-order vertebrates have not been forthcoming. Here, we report the cloning and characterization of the IFN gene from the zebrafish, Danio rerio. Zebrafish IFN (zfIFN) is 185 amino acids in length, with the first 22 amino acids representing a putative signal peptide. Treatment with the known IFN inducer polyinosinic acid-polycytidylic acid (poly[I]-poly[C]) resulted in an increase in zfIFN mRNA transcripts. zfIFN was also able to activate the IFN-inducible Mx promoter when cotransfected with a plasmid containing the zebrafish Mx promoter upstream of a luciferase reporter gene. To demonstrate antiviral activity, zebrafish cells were transfected with zfIFN and challenged with a fish rhabdovirus. A 36% reduction in plaque number was seen in zfIFN-transfected cells, compared to cells transfected with a control vector. Phylogenetic analysis has shown zfIFN to be approximately equally divergent from avian and mammalian IFN, consistent with its origin from an IFN present in the most recent common ancestor of these divergent lineages. A putative IFN from puffer, Fugu rubripes, was also found when zfIFN was used to search the fugu genome database, demonstrating that zfIFN can be used to find additional fish IFN genes. These results demonstrate that zebrafish can be used as an effective model for studying innate immunity and immune response to infectious disease.

FIG. 1.

Nucleotide sequence of the zfIFN gene (lowercase) and deduced amino sequence of zfIFN (uppercase). The two cysteine residues are boxed, and the highly conserved residues are circled. Phe56 is marked by a triangle, and the putative signal peptide is underlined.

FIG. 2.

Induction of the Mx promoter construct by zfIFN expression. ZF4 cells were either induced with 25 μg of poly(I)-poly(C)/ml, transfected with zfIFN or an empty vector, or untreated. After 24 h, cells were lysed and luciferase activity was measured. The experiment was performed in triplicate, with each bar representing the mean of three samples. Error bars, standard deviations.

FIG. 3.

Quantitation of zfIFN mRNA upon stimulation with poly(I)-poly(C). ZFL cells were either induced with 25 μg of poly(I)-poly(C)/ml or uninduced, and total cellular RNA was harvested at selected time points. Each bar represents the mean of three replicates. Error bars, standard deviations.

FIG. 4.

Plaque reduction in cells transfected with zfIFN and infected with SHRV. ZF4 cells were either induced with 25 μg of poly(I)-poly(C)/ml, transfected with zfIFN or the empty pcDNA3 vector, or untreated. All cells received equal amounts of liposome. After 16 h, all cells were challenged with SHRV for 24 h before termination by staining in crystal violet and ethanol. (A) pcDNA3 transfection; (B) liposome only; (C) poly(I)-poly(C) induction plus liposome; (D) zfIFN transfection. (E) Graph of plaque reduction numbers. The first four bars represent experiments done with a virus titer of 10² TCID₅₀/ml; the second four bars represent those done with a virus titer of 10³ TCID₅₀/ml. The experiments were performed in triplicate. Bars represent the means of three replicates. Error bars, standard deviations.

FIG. 5.

Alignment of zfIFN with other vertebrate IFNs. Dashes, gaps inserted into the alignment. Shaded residues represent identities among species. Accession numbers are shown in parentheses.

FIG. 5.

Alignment of zfIFN with other vertebrate IFNs. Dashes, gaps inserted into the alignment. Shaded residues represent identities among species. Accession numbers are shown in parentheses.

FIG. 5.

Alignment of zfIFN with other vertebrate IFNs. Dashes, gaps inserted into the alignment. Shaded residues represent identities among species. Accession numbers are shown in parentheses.

FIG. 5.

Alignment of zfIFN with other vertebrate IFNs. Dashes, gaps inserted into the alignment. Shaded residues represent identities among species. Accession numbers are shown in parentheses.

FIG. 6.

Phylogenetic relationships among zfIFN and representative IFN-α, -β, -δ, -ω, and -τ from mammals and birds. Phylogenetic analyses were performed using evolutionary-distance (shown) and maximum-parsimony algorithms. Bootstrap proportions (bp) are presented as the fractional percentage of 100 replicates (top, evolutionary distance; bottom maximum parsimony). Dashes, bp of <50%. Best trees recovered by both algorithms were identical with respect to all well-supported nodes (bp > 70%). The aligned sequence set included 31 sequences of 212 equally weighted positions (185 were parsimony informative). Alignment gaps are treated as missing data. GenBank accession numbers are shown in parentheses.