Characterization of the porcine alpha interferon multigene family

Gene 382 (2006) 28 – 38 www.elsevier.com/locate/gene

Characterization of the porcine alpha interferon multigene family Gong Cheng a , Weizao Chen a , Zuofeng Li b , Weiyao Yan a , Xin Zhao a , Jun Xie a , Mingqiu Liu a , Hao Zhang a , Yang Zhong b,c , Zhaoxin Zheng a,⁎ a

State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University,220 Handan Road, Shanghai 200433, PR China b Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, School of Life Science, Fudan University, Shanghai 200433, PR China c Shanghai Center for Bioinformation Technology, Shanghai 201203, PR China Received 17 April 2006; received in revised form 6 June 2006; accepted 12 June 2006 Available online 29 June 2006 Received by A. Bernardi

Abstract The availability of data on the pig genome sequence prompted us to characterize the porcine IFN-α (PoIFN-α) multigene family. Fourteen functional PoIFN-α genes and two PoIFN-α pseudogenes were detected in the porcine genome. Multiple sequence alignment revealed a Cterminal deletion of eight residues in six subtypes. A phylogenetic tree of the porcine IFN-α gene family defined the evolutionary relationship of the various subtypes. In addition, analysis of the evolutionary rate and the effect of positive selection suggested that the C-terminal deletion is a strategy for preservation in the genome. Eight PoIFN-α subtypes were isolated from the porcine liver genome and expressed in BHK-21 cells line. We detected the level of transcription by real-time quantitative RT-PCR analysis. The antiviral activities of the products were determined by WISH cells/Vesicular Stomatitis Virus (VSV) and PK 15 cells/Pseudorabies Virus (PRV) respectively. We found the antiviral activities of intact PoIFN-α genes are approximately 2–50 times higher than those of the subtypes with C-terminal deletions in WISH cells and 15–55 times higher in PK 15 cells. There was no obvious difference between the subtypes with and without C-terminal deletion on acid susceptibility. © 2006 Elsevier B.V. All rights reserved. Keywords: Multiple sequence alignment; C-terminal deletion; Virus-responsive elements; Structural analysis; Phylogenetic tree; Antiviral activity

1. Introduction The interferons (IFNs) are a family of proteins with antiviral, growth inhibitory and immunomodulatory activity. They were initially classified as Type I IFNs (virus-infected IFNs) and Type II IFNs (immune IFNs) (Sen and Lengyel, 1992). Type I interferons are a heterogeneous group comprising IFN-α, IFNβ, IFN-ω, IFN-κ, IFN-ε, IFN-δ and IFN-τ; there is only one Type II molecular species, IFN-γ. In humans, type I IFNs consist of multiple IFN-α subtypes, and single member each of IFN–β, IFN-ω, IFN-κ, IFN-ε (Flores et al., 1991; Pestka et al.,

Abbreviations: PoIFN-α, porcine interferon alpha; HuIFN-α, human interferon alpha; MuIFN-α, Mouse interferon alpha; BvnOmega, Bovine interferon omega; RabOmega, Rabbit interferon omega; VRE, Virus-responsive elements; LRT, the likelihood ratio test. ⁎ Corresponding author. Tel.: +86 21 65642504; fax: +86 21 65642504. E-mail address: [email protected] (Z.X. Zheng). 0378-1119/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2006.06.013

1987; Hardy et al., 2004; LaFleur et al., 2001). IFN-κ is expressed only in epidermal keratinocytes, has similar biological functions and binds to the same receptor as other type I IFNs. IFN-ε is expressed in cells and tissues with a reproductive function, such as the uterus and ovary. The same IFNs exist in mice, except for IFN-ω (van Pesch et al., 2004). A type I IFNlike cytokine called Limitin/IFN-like 1, which binds to the type I IFN receptor, has recently been identified in the mouse (Oritani et al., 2000; Oritani et al., 2001). Recently, a new interferon was designated as IFN-λs (IL-28 and IL-29), which utilizes a new receptor–ligand system to contribute to antiviral or other defenses by a mechanism similar to, but independent of, type I IFNs (Kotenko et al., 2003; Sheppard et al., 2003). So far, all type I IFNs except IFN-κ have been found in pigs. In ruminants and pigs, IFN-τ and -δ are produced by the trophectoderm and are involved in maternal recognition during pregnancy (Demmers et al., 2001; Lefevre et al., 1998; Roberts et al., 1999).

G. Cheng et al. / Gene 382 (2006) 28–38

The alpha-interferons are encoded by a family of closely related intronless genes in all mammalian species studied (Weissmann and Weker, 1986). They are mainly produced by virus-infected peripheral blood leukocytes, lymphoblastoid and myeloblastoid cell lines (Familletti et al., 1981). The human IFN-α gene cluster is distributed over approximately 400 kb on the short arm of chromosome 9, and consists of at least thirteen IFN-α genes and one IFN-α pseudogene (Diaz et al., 1994; Hardy et al., 2004). The mouse IFN-α genes cluster is located on the centromere–proximal region of chromosome 4, and so far fourteen functional IFN-α genes and three pseudogenes have been identified in the genome (Kelley et al., 1983; Kelley and Pitha, 1985; van Pesch and Michiels, 2003; van Pesch et al., 2004). The porcine IFN-α (PoIFN-α) gene family is located on chromosome 1 (Yerle et al., 1986). Previous examination of porcine genomic library identified at least ten potential porcine IFN-α genes or pseudogenes belonging to this multigene family, two of which were sequenced and denoted PoIFN-α1 and PoIFN-α2. Only porcine IFN-α1 was cloned and expressed in E. coli, and the expressed protein was found to possess antiviral activity (Lefevre et al., 1986; Lefevre et al., 1990a; Lefevre et al., 1990b; La Bonnardiere et al., 1994). Porcine IFNα was recently used as a genetic adjuvant for DNA vaccines and a therapeutic cytokine to protect animals from viral infection (Chinsangaram et al., 1999; Chinsangaram et al., 2001; Chinsangaram et al., 2003; Moraes et al., 2003; Valarcher et al., 2003). However, there is no data on the sequences of the porcine IFN-α multigene family, their phylogenetic classification and properties. In this study, we demonstrated that four working draft sequences (submitted by the Swine Genome Sequencing Project) contain 16 distinct IFN-α gene-related loci by scanning with the BLASTN algorithm. We performed multiple sequence alignments, phylogenetic analyses and predictions of expression profiles. Multiple sequence alignment revealed a C-terminal deletion of eight residues in six subtypes. Eight different subtype genes were amplified from porcine liver genomic DNA and our data suggested that the C-terminal deletion influences the antiviral activity in WISH cells and PK 15 cells. 2. Materials and methods 2.1. Cells and viruses Baby hamster kidney cells (BHK-21) and Pig kidney cells (PK 15) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Amnion-derived WISH cells were cultured in a mixture of Ham F12 and DMEM (F12/DMEM) (1:1 vol/vol) supplemented with 10% heat-inactivated FBS, 100 IU of penicillin per ml, and 100 μg of streptomycin (Invitrogen, Lifetechnologies) per ml (pH 7.4). Cultures were incubated at 37 °C with 5% CO2. Pseudorabies Virus (PRV) was kindly donated by Dr. Du (the Institute of Biotechnology, Zhejiang Academy of Agricultural Sciences) and Vesicular Stomatitis Virus (VSV) was donated by Dr. Pan (Wanxing Co., LTD., Shanghai). Those were used for viral challenge.

29

2.2. Isolation of porcine IFN-α genes and construction of plasmids Porcine IFN-α genes, including signal sequences, were amplified by PCR from porcine liver genomic DNA. Common primer sets were designed based on alignment of available PoIFN-α sequences in GenBank (GenBank accession nos. M28623, X57191, andNM_214393) from the National Center for Biotechnology Information (NCBI) website (www.ncibi. nlm.nih.gov). Forward primer: ATGGCCCCAACCTCAG; reverse primer: TCACTCCTTCTTCCTG. The PCR fragments were inserted into pMD18-T vector (TaKaRa, Kyoto, Jp). Twenty clones were sequenced and eight divergent IFN-α genes were isolated. The eight fragments were further cloned into pcDNA3 vector at EcoRI and XhoI restriction sites introduced into the PCR primers. 2.3. Transient cellular transfection BHK-21 cells were transfected with plasmids in a 24-well plate, using Lipofectamine 2000 (Invitrogen, Life Technologies) as directed by the manufacturer. Cell monolayers grown in a 24well plate were incubated for 5 h at 37 °C in 5% CO2 with 200 μl of DMEM containing 2 μg plasmid DNA and 6 μl lipofectamine reagent complex. After 5 h transfection, 200 μl of growth medium containing twice the normal concentration of serum was added without removing the transfection mixture. Supernatants containing interferon alpha were collected 48 h after transfection. 2.4. Real-time quantitative RT-PCR analysis For detection of the targeted gene expression in BHK-21, after 24 h transfection, total RNA was extracted from BHK-21 culture with Trizol reagent (Invitrogen), incubated for 30 min at 37 °C with Dnase RQ1 and subjected to the real-time quantitative RTPCR analysis. Briefly, the real-time quantitative RT-PCR was

Table 1 The loci of the PoIFN-α subtypes in working draft sequences Gene Name

GenBank numbers The location (nt) of the working draft

Length (nt)

Porcine interferon alpha-1 Porcine interferon alpha-2 Porcine interferon alpha-3 Porcine interferon alpha-4 Porcine interferon alpha-5 Porcine interferon alpha-6 Porcine interferon alpha-7 Porcine interferon alpha-8 Porcine interferon alpha-9 Porcine interferon alpha-10 Porcine interferon alpha-11 Porcine interferon alpha-12 Porcine interferon alpha-13 Porcine interferon alpha-14 Porcine interferon alpha-1ψ Porcine interferon alpha-2ψ

AC127471 AC127471 AC127471 AC130792 AC130792 AC130792 AC130792 AC138785 AC138785 AC138785 AC138785 AC135219 AC135219 AC135219 AC130792 AC138785

546 546 546 570 570 570 546 570 570 546 546 570 570 570 568 555

“ψ”stands for “pseudogene”.

24,747–25,292 42,316–42,861 53,279–53,824 129,757–130,326 49,595–49,026 30,116–29,547 173,769–174,314 92,627–93,196 76,425–76,994 138,485–139,030 26,810–273,55 197,875–197,306 163,577–163,008 184,892–184,323 1655–1088 105,906–106,456

30

G. Cheng et al. / Gene 382 (2006) 28–38

Table 2 Similarities of protein and genetic sequences between porcine IFN-α subtypes A.A.

IFN-α1 IFN-α2 IFN-α3 IFN-α4 IFN-α5 IFN-α6 IFN-α7 IFN-α8 IFN-α9 IFN-α10 IFN-α11 IFN-α12 IFN-α13 IFN-α14

N.A. IFN-α1

IFN-α2

IFN-α3

IFN-α4

IFN-α5

IFN-α6

IFN-α7

IFN-α8

IFN-α9

IFN-α10

IFN-α11

IFN-α12

IFN-α13

IFN-α14

\ 100.0 98.1 96.2 96.2 97.5 95.6 99.4 98.1 97.5 94.9 96.8 96.8 93.7

99.8 \ 98.1 96.2 96.2 97.5 95.6 99.4 98.1 97.5 94.9 96.8 96.8 93.7

99.2 99.4 \ 97.5 95.6 96.8 96.2 98.7 98.7 96.8 95.6 96.8 96.2 93.7

97.7 97.9 98.1 \ 93.7 96.8 96.2 96.8 96.8 94.3 95.6 98.1 96.8 96.2

97.7 97.5 97.3 97.0 \ 93.7 93.0 96.8 95.6 93.7 92.4 94.3 94.3 91.1

97.3 97.1 96.9 97.4 96.8 \ 96.8 96.8 95.6 96.2 96.2 98.7 98.1 95.6

97.9 97.7 97.9 97.9 97.1 97.9 \ 96.2 96.2 95.6 99.4 96.8 96.2 93.7

98.7 99.0 98.7 98.2 98.0 97.2 97.7 \ 98.7 96.8 95.6 97.5 97.5 94.3

98.1 98.3 98.5 98.2 97.6 96.8 97.5 98.8 \ 96.8 95.6 96.2 96.2 93.0

98.7 99.0 98.7 96.9 96.9 96.9 97.9 97.9 97.7 \ 94.9 94.9 95.6 93.0

97.7 97.5 97.7 97.7 96.9 97.7 99.8 97.5 97.3 97.7 \ 96.2 95.6 93.0

97.5 97.7 97.5 98.8 97.0 98.2 97.7 98.2 98.2 96.6 97.5 \ 98.7 96.8

97.5 97.7 97.5 98.2 97.2 98.0 98.1 98.4 97.6 97.5 97.9 98.6 \ 96.8

96.0 96.2 96.0 97.8 96.0 97.2 96.6 96.8 96.4 96.0 96.4 98.2 98.0 \

The values are given as percentages and maximum and minimum values are bold. N.A./A.A. denote nucleotide and amino acid respectively.

performed in a 96-well plate (BioRad, Hercules, CA) in a 20 μl reaction volume containing components of the SYBR® RT-PCR Kit (Perfect Real Time) (TaKaRa Code: DRR045S). The 20 μl reaction mixture contained 10 μl SYBR® master mix (2×), 0.4 μl of 0.2 μM forward primer, 0.4 μl of 0.2 μM reverse primer, 2.0 μl of 500 ng RNA sample and 7.2 μl of water. The RT-PCR thermocycling program consisted of 42 °C for 20 min, 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s, 50 °C for 10 s and 72 °C for 30 s. Forward primer: GGCTCTGGTGCATGAGAT GC; reverse primer: CAGCCAGGATGGAGTCCTCC. To confirm the specific amplification, melt-curve analysis of the RT-PCR products was performed according to the manufacturer's protocol. Fluorescence was measured following each cycle and displayed graphically by iCycler iQTM Real-Time PCR Detection System Software Version 3.0A (BioRad, Hercules, CA). 2.5. Antiviral activity The antiviral activity of PoIFN-α was tested on WISH cells/ Vesicular Stomatitis Virus (VSV) system (LaFleur et al., 2001) and PK 15 cells/Pseudorabies Virus (PRV) system (Pol et al., 1991) respectively. Briefly, the cells were seeded in flat-bottom 96-well plates and grown to 95% confluence, and serial dilutions of PoIFN-α were added to the wells. After 24 h, optimal concentrations of the virus were added. After a further 24 h, the values of antiviral activity were calculated. In WISH cells, the cell monolayers were stained with 1% crystal violet in 15% ethanol. They were scored by extracting the stained cells with 70% ethanol/1% acetic acid and measuring absorbance at 570 nm in an ELISA microplate reader. Antiviral activity was expressed as EC50 and calculated by Prism software. The antiviral activity of PoIFN-α in PK 15 cells was calculated

using Reed–Muench Method. One unit is the highest dilution that reduced the cells number by 50%. 2.6. pH 2 stability Supernatants from transfected BHK-21 cell were incubated at pH 2 for 24 h at 4 °C, as described previously (van Pesch et al., 2001), and the antiviral activities of the treated and untreated samples then compared. 2.7. Identification of porcine gene sequences and multiple alignments Using the BLASTN algorithm, we identified PoIFN-α gene fragments in the working draft sequences (submitted by Swine Genome Sequencing Project). Sequenced fragments were aligned, analyzed and then classified into subtypes using the MultAlin program (Corpet, 1988). The putative N-glycosylation site of porcine IFN-α genes was found using NetNGlyc website (http://www.cbs.dtu.dk/services/NetNGlyc). Secondary structure elements were predicted using the algorithms available from NPS (Combet et al., 2000) (http://www.npsa-pbil.ibcp.fr.). 2.8. Phylogeny reconstruction Multiple sequence alignment of PoIFN-α types was obtained using Clustal X (Thompson et al., 1997). CodonAlign 2.0 (http:// www.sinauer.com/hall/) was used to align the PoIFN-α gene codon sequences according to their protein sequence alignment. The sequences of five human IFN-α, six mouse IFN-α and two IFN-ω (Omega) of bovine and rabbit were retrieved from GenBank and their GenBank accession numbers are listed in

Fig. 1. Multiple sequence alignments of the sixteen porcine IFN-α (PoIFN-α). The C-terminal deletion is present in six functional IFN-α. The eight residues deleted are listed in the gray column. Cysteines that can form disulfide bonds are shown in yellow. Stop codons (denoted by “.”) and gaps (denoted by “–”) exist in the middle of the two pseudogenes. The positions of variant residues in individual subtypes are shown in gray, and the substitutions occurring in multiple subtypes are labeled by arrows above the figure. The putative N-glycosylation site (Asn-X-Ser/Thr) is present in the four PoIFN-α genes and those amino acids are boxed. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

G. Cheng et al. / Gene 382 (2006) 28–38

31

32

G. Cheng et al. / Gene 382 (2006) 28–38

Fig. 2. Multiple sequence alignment of the virus-responsive elements of 14 porcine IFN-alpha genes and 2 pseudogenes. The A and B modules boxed by dotted lines correspond to the IRF-7 binding site. The boxed C module corresponds to the IRF-3 biding site. All four modules are shown in yellow, and the “TATA” box is shaded in blue. The residues highlighted in gray are those that do not match the reported VRE consensus of human and mouse. In the −78 bp site, the nucleotides that do not correspond to the consensus TG sequence (GAATTGGAAAGC) are also shown. A gap of approximately 7 bp exists in the region between the C and D modules in six of the subtypes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Materials and methods section. Modeltest 3.7 (Posada and Crandall, 1998) was used to select an optimal data model. The model named K80 combined with Gamma (G) was selected with k = 2 and Ti/Tv = 1.4882. A phylogenetic tree was generated by the maximum likelihood (ML) method implemented in PAUP 4.0 b4a (Swofford, 1998). The IFN-ω of bovine and rabbit were chosen as outgroup. The accuracy of the tree topology was assessed by bootstrap analysis, with 100 replicates for the ML method. 2.9. Evolutionary rate and positive selection For examining possible evolutionary rate changes with speciation, we used the method of Li and Bousquet implemented in RRTree 1.1 (Marc and Dorothée, 2000) to compare substitution rates in monophyletic groups of sequences. The bovine IFN-ω was selected as outgroup and rabbit IFN-ω was excluded from the analysis. In addition, codon-substitution models implemented in the codeml program in the PAML package were used to detect the positively selected amino acid sites (Yang, 1997). 2.10. Nucleotide sequence accession numbers The following new sequences or sequences used in this article have been deposited in the GenBank database: human interferon alpha-1 (GenBank accession no. NM_024013), human interferon alpha-2 (GenBank accession no. NM_000605), human interferon alpha-6 (GenBank accession no. NM_021002), human interferon alpha-8 (GenBank accession no. NM_002170), human interferon alpha-13 (GenBank accession no. NM_006900), murine interferon alpha-1 (GenBank accession no. NM_010502), murine interferon alpha-2 (GenBank accession no. NM_010503), murine interferon alpha-4 (GenBank accession no. NM_010504), murine interferon alpha-5 (GenBank accession no. NM_010505), murine

interferon alpha-7 (GenBank accession no. NM_008334), murine interferon alpha-11 (GenBank accession no. NM_008333), bovine interferon Omega (GenBank accession no. AF238610), rabbit interferon Omega (GenBank accession no. S68999), porcine interferon alpha-1 (GenBank accession no. DQ249000), porcine interferon alpha-2 (GenBank accession no. DQ249002), porcine interferon alpha-3 (GenBank accession no. DQ248998), porcine interferon alpha-5 (GenBank accession no. DQ249001), porcine interferon alpha-8 (GenBank accession no. DQ248999), porcine interferon alpha-11 (GenBank accession no. DQ464064), porcine interferon alpha-12 (GenBank accession no. DQ249003), porcine interferon alpha-14 (GenBank accession no. DQ248997). 3. Results 3.1. Loci of the PoIFN-α genes and alignment of the PoIFN-α sequences Fourteen PoIFN-α genes and two PoIFN-α pseudogenes were identified in four different working draft sequences (GenBank Accession nos. AC127471, AC130792, AC135219, and AC138785). To distinguish them from previous designations (Lefevre et al., 1986; Lefevre et al., 1990a), these sequences were designated PoIFN-α1 to PoIFN-α14, and PoIFN-αψ1 to PoIFNαψ2, according to the BLAST order obtained from scanning the working draft sequences. Table 1 describes the loci of PoIFN-α genes in working draft sequences. Alignment of the sequences shows striking overall conservation of the various PoIFN-α subtypes, with 96.0%–99.8% identity at the nucleotide level and 91.1%–100% at the amino acid level (Table 2). The PoIFN-α intact functional genes encode 189 amino acid (AA) preproteins and the genes with C-terminal deletions encode 181 amino acid (AA) preproteins, both with putative signal

G. Cheng et al. / Gene 382 (2006) 28–38

33

Fig. 3. The phylogenetic relationships between the porcine, human and mouse IFN-α gene families were inferred by ML analysis. The porcine IFN-α genes with Cterminal deletions are marked by a ‘#’. The bovine and rabbit IFN-ω were selected as outgroups. Five human IFN-α genes, six murine IFN-α genes and two bovine and rabbit IFN-ω genes were retrieved from GenBank and their accession numbers are listed in Materials and methods section.

peptides of 23AA, so that the mature proteins probably comprise 166AA and 158AA. Eight of the sixteen chromosomal PoIFN-α genes contain intact open reading frames (ORF) (166AA). Each of other six PoIFN-α genes has a C-terminal stop codon, which deletes the eight amino acids (158AA). Several gaps and stop codons are present in the middle of the ORFs in the two remaining sequences and we presume that these are pseudogenes (Fig. 1). The first residue of the mature peptide is always a cysteine that is important for disulfide bond formation and structure stabilization. Another three cysteines involved in disulfide bond formation (residue 1 with 99, and 29 with 139) are conserved in all the subtypes (Fig. 1). The putative Nglycosylation site (Asn-X-Ser/Thr), which exists in mouse alpha interferon genes (van Pesch et al., 2004), is found in four PoIFN-

α subtypes (PoIFN-α7, PoIFN-α9, PoIFN-α10, PoIFN-α11). The positions of substituted residues in the various subtypes are as indicated (Fig. 1). 3.2. Analysis of the virus-responsive elements (VRE) in the porcine IFN-α sequences A multiple alignment of the virus-responsive elements (VRE) of the porcine IFN-α promoters is shown in Fig. 2. Four modules (A, B, C, and D) are reported to modulate the activity of IFN promoters in response to viral infection in humans and mice (Braganca et al., 1997). Module A and B correspond to the IRF-7 binding site (Lin et al., 2000) and module C to the IRF-3 binding site (Juang et al., 1998). In these modules, the conserved hexamer

34

G. Cheng et al. / Gene 382 (2006) 28–38

Table 3A Relative evolutionary rate analysis of porcine, human and porcine alpha IFNs Lineage1

Lineage2

Ks1

Ks2

P_Ks

Human Human Mouse

Mouse Porcine Porcine

0.477021 0.477021 0.732983

0.732983 0.644409 0.644409

0.00864686 0.0829322 0.486308

⁎

Ka1

Ka2

P_Ka

0.331166 0.331166 0.421185

0.421185 0.361425 0.361425

0.00404958 0.257658 0.101131

⁎

Ks, synonymous substitution rates; Ka, non-synonymous substitution rates.

GAAANN is related to the IRF-3/7 binding site (Naf et al., 1991; Morin et al., 2002). Substitutions in the sequences are believed to affect the activity of the VREs and to regulate their expression in response to different conditions. The shaded nucleotides in the PoIFN-α VREs are those that do not match the reported VRE consensus of human and mouse (Fig. 2). From the alignment, we identified four subtypes (PoIFN-α6, PoIFN-α8, PoIFN-α12 and PoIFN-α14) that possess intact putative IRF-binding modules. Another five subtypes including one pseudogene (PoIFN-α4, PoIFN-α7, PoIFN-α11, PoIFN-α13 and PoIFN-αψ1) do not contain precise A and B modules, since there are substitutions of one or two nucleotides in the hexamer GAAANN. The conserved sequences of ten functional genes and two pseudogenes (PoIFNα1, PoIFN-α2, PoIFN-α3, PoIFN-α4, PoIFN-α5, PoIFN-α7, PoIFN-α9, PoIFN-α10, PoIFN-α11, PoIFN-α13, PoIFN-αψ1 and PoIFN-αψ2) contain one or two nucleotide substitutions in the C module. Gaps of about 7 bp exist between the C and D modules in six subtypes (PoIFN-α4, PoIFN-α7, PoIFN-α11, PoIFN-α13, PoIFN-αψ1 PoIFN-αψ2); such gaps are not present in the VREs of human and mouse IFN-α (Fig. 2). Studies of the VREs of human and mouse IFNs have shown that the −78 (A/G) site is significant for interferon transcription. The TG bases of the GAATTGGAAAGC sequence in the C module are critical for IRF-3 responsiveness in the VRE of human IFN-α (Naf et al., 1991; Morin et al., 2002). In the virus-responsive elements of the porcine IFN-α promoters, substitutions at −78 bp (GAAGT(G/A) GAAAGT) may inactivate the domain and inhibit IRF-3 binding; hence this may be a key site (Fig. 2).

However, no significant differences in Ka and Ks were found between porcine and human, or between porcine and mouse. The codon-substitution models (M0, M3, M7 and M8) implemented in PAML were applied to detect positively selected sites. Following this, the likelihood ratio test (LRT) was conducted to detect the reliability of the different models. According to the results of the LRT shown in Table 3B, the M3 model was accepted and the M0 model rejected. That is to say, the selective pressure at different sites varies, following the recommendation of Yang (Yang, 2000). We also conducted an M7–M8 comparison, and the M7 model was accepted. According to the LRT results, there is no positive selection for amino acid site (Table 3B). 3.5. Structural analysis of porcine IFN-α subtypes The predicted structure and possible residues involved in the biological activity of porcine IFN-α subtypes were identified (Fig. 4). Based on the previous analysis of the residues involved in the biological activity of human IFN-α (Radhakrishnan et al., 1996), the results of aligning the primary structures of PoIFN-α and HuIFN-α enable us to predict which residues might impact the function of porcine IFN-α (Fig. 4). The biological activity positions at which HuIFN-α2b and/or MuIFN-α15 differ from those of PoIFN-α1/4 are boxed. The investigation of secondary structure indicates that porcine IFN-α has five putative alpha helices. The amino acids in helices A to E and the putative AB loops are well conserved between human and porcine IFN-α (Fig. 4) (Radhakrishnan et al., 1996).

3.3. Reconstruction of phylogeny 3.6. Detection of the targeted gene expression The phylogenetic relationships between porcine, human and mouse IFN-α types were inferred by means of ML analysis (Fig. 3). Porcine IFN-α with C-terminal deletions are marked with ‘#’ in the tree. The bovine and rabbit IFN-ω were selected as outgroups. This figure shows two features of the tree: first, IFN-α forms three species-specific clusters; second, three porcine IFN-α subtypes with C-terminal deletions (PoIFN-α7, PoIFN-α11 and PoIFN-αψ1) form a monophyletic group. These features are confirmed by maximum-parsimony (MP) and neighbor-joining (NJ) analyses with 1000 bootstrap replicates (data not shown). 3.4. Evolutionary rate and positive selection We calculated and compared lineage-specific synonymous (Ks) and non-synonymous (Ka) substitution rates with the RRTree program, and the results are given in Table 3A. Using bovine IFN-ω as outgroup, we detected significant differences in Ks and Ka between the human and mouse lineages (p b 0.05).

Eight divergent IFN-α genes were isolated from porcine liver genome DNA by PCR amplification and further cloned into the pcDNA3 vector. By sequence analysis using MultAlin program, they were classified into PoIFN-α1, 2, 3, 5, 8, 11, 12 and 14 respectively. The cloned sequences showed high conservation to Table 3B Detection of positive selection sites with PAML Model code

ν

Log2Δl likelihood(l)

LRT

Positive sites

M0 (one-ratio) M3 (discrete)

1 19 (K = 10) 2 4

− 3882.0 − 3831.5

101

p b 0.001

Exist

− 3835.2 − 3832.3

5.8

p N 0.05

None

M7 (beta) M8 (beta and omega)

ν, number of parameters in the ω distribution; K, number of site classes; LRT, likelihood ratio test.

G. Cheng et al. / Gene 382 (2006) 28–38

35

Fig. 4. Structure-based sequence alignment of porcine IFN-α1/4, human IFN-α2b, and murine IFN-α15. The porcine IFN-α1/4 sequences are from this paper. Human IFN-α2b is from Hardy, M. P. et al. (GenBank Accession no. NM_000605) and the murine IFN-α15 from van Pesch, V. et al. (GenBank Accession no. AY226993). The possible active site residues of these proteins are shown in light blue, and variant residues are boxed. Cysteine residues forming disulfide bonds are marked in yellow. The eight almost identical C-terminal residues of porcine IFN-α4 and human IFN-α2b are shown in gray. Secondary structure assignments are shown above the sequences; letters A to E refer to the helices in porcine IFN-α1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Real-time quantitative RT-PCR analyzing the targeted gene expression. BHK-21 cells were transfected with resulting plasmids. After 24 h, the cells were collected and total RNAs were extracted from cultures to subject to real-time RT-PCR. (A) One amplification plot of two separate experiments is shown. The y-axis represents the PCR baseline-subtracted RFU (relative fluorescence units). Cycle number is displayed on the x-axis. (B) Cycle threshold (CT) values are derived from the amplification profiles shown in (A).

36

G. Cheng et al. / Gene 382 (2006) 28–38

Table 4 The antiviral activity of PoIFN-α subtypes Groups

Antiviral activity for PK 15 cells (U/ml)a

Antiviral activity for WISH cells (U/ml)a

PoIFN-α1 PoIFN-α2 PoIFN-α3 PoIFN-α5 PoIFN-α8 PoIFN-α11 PoIFN-α12 PoIFN-α14

6.53 × 104 3.69 × 104 5.82 × 104 1.59 × 106 1.02 × 106 6.53 × 104 2.04 × 106 9.93 × 105

706.7 524.1 69.7 1558.3 2476.8 74.8 3610.0 2680.7

a

Highest dilution that reduced the cells number by 50%.

the PoIFN-α genes of the genome database, with 99%–100% identity at the nucleotide level (data not shown). PoIFN-α1, 2, 3 and 11 are subtypes with C-terminal deletions and the other four are intact genes. Then, the genes were cloned into pcDNA3 vector. For detection of the targeted gene expression, cells were transfected with the resultant plasmids. At 24 h post transfection, total RNA of cells were isolated and real-time quantitative RT-PCR analysis was performed. The level of target RNAs, as determined by RT-PCR, are similar in the cycle threshold (CT) values (the range from 15.5 to 18.1) (Fig. 5). The CT values of PoIFN-α1/11 are slightly higher than the average value and that of PoIFN-α8 is lower. Melt-curve analysis confirmed the specific amplification of RT-PCR products (data not shown). The results suggested that the expression of targeted genes were similar in mRNA level. 3.7. The effect of C-terminal deletion on the antiviral activity and pH 2 stability of PoIFN-α For assessing the influence of C-terminal deletion to PoIFNα properties, antiviral activity and pH 2 stability of eight subtypes were measured. No measurable activity could be

detected in the supernatants of control samples from mocktransfected cells or cells transfected with empty pcDNA3. The supernatants containing PoIFN-α were collected 48 h after transfection. The WISH cells/VSV system and PK 15 cells/PRV system were used to determine the antiviral activity respectively. The average values of antiviral activity of the intact subtypes are approximate 2–50 times higher than those of the subtypes with C-terminal deletion in WISH cells and 15–55 times higher in PK 15 cells (Table 4). The data showed that the C-terminal deletion reduced the antiviral activity in these eight porcine IFN-α subtypes. IFN-α has been shown to resist low pH. In order to test whether C-terminal deletion influences this property, the supernatants were incubated at pH 2 for 24 h, and their antiviral activity against VSV and PRV was measured. The porcine IFN-α all retained antiviral activity; the maximal loss of activity was approximately 2- to 6-fold in WISH cells and the reduction of the activity was in a similar range in PK 15 cells. There was no obvious difference between the subtypes with and without Cterminal deletion on acid susceptibility (Fig.6). 4. Discussion In this study we identified the sequences of fourteen PoIFNα genes and two PoIFN-α pseudogenes from working drafts of the genomic sequence (Swine Genome Sequencing Project) and eight PoIFN-α subtypes were isolated by PCR from the porcine liver genomic DNA. According to the sequence alignment, the lack of eight C-terminal residues in several functional PoIFN-α subtypes is proved. Our models of three-dimensional structures showed that the C-terminal deletions do not have any great influence on the central framework (data not shown). However, our data on antiviral activity are not consistent with the results of computer simulation: the subtypes with C-terminal deletions had less antiviral activity than the subtypes with intact genes in WISH cells and PK 15 cells. There are three explanations for

Fig. 6. The pH 2 stability of PoIFN-α subtypes. Eight PoIFN-α subtypes were cloned into pcDNA3 vector and the resulting plasmids were transfected into BHK21cells. After 48 h, the culture supernatants were collected and the antiviral activities of pH 2 treatment and untreatment were measured. The relation of before/after pH 2 treatment showed the acids stability of PoIFN-α subtypes. PoIFN-α1, 2, 3, 11 are subtypes with C-terminal deletion and PoIFN-α5, 8, 12, 14 are intact genes. The data is denoted by antiviral activity.

G. Cheng et al. / Gene 382 (2006) 28–38

that: First, the eight C-terminal residues could be part of the active protein domain and play an important role in the interaction with porcine IFN-receptors, despite the fact that the deletion does not lead to structural distortion; Second, the Ehelix may be shortened by the deletion and this may lead to other structural changes during the formation of the tertiary barrel-shape structure (Fig. 4); Third, the protein may be unstable due to the C-terminal deletion, or the protein with Cterminal deletion tend to lose activity. The analysis of porcine IFN-α virus-responsive element can help to predict the expression profiles of the various subtypes. It is reported that IFN genes are expressed by a two-step mechanism (van Pesch et al., 2004). Transcription of human and mouse IFN-α4 genes and IFN-β gene, termed immediate– early IFNs, depends on IRF-3, a constitutively expressed transcription factor that is activated immediately after viral infection by phosphorylation cascades (Juang et al., 1998). The transcription of additional IFN-α genes depends on IRF-7, whose expression itself depends on priming of the cells by exogenous IFN-α (Lin et al., 2000). Thus, viral infection is thought to induce the release of IFN-α4, which primes the cells and triggers the production of other IFN-α subtypes. Mouse IFN-α4 is the only type with an intact VRE (van Pesch et al., 2004). In this study, we identified four subtypes (PoIFN-α6, PoIFN-α8, PoIFN-α12, PoIFN-α14) with intact VREs (Fig. 2), suggesting that they may be more highly expressed after viral infection. Other PoIFN-α subtypes that do not contain the reported VRE consensus of human and mouse IFN-α (GAAANN), may be constitutively expressed or expressed under different conditions. Our phylogenetic analysis suggests that the IFN-α subtypes originated from common ancestral genes and that the duplications that gave rise to the current subtypes occurred after the divergence of pig, human and mouse. The eight C-terminal deletion PoIFN-α (including two pesudogenes) form a polyphyletic group. However, PoIFN-α7, PoIFN-α11 and PoIFN-αψ1 form a monophyletic group. This indicates that they may derive from a common ancestor. Two models are commonly used to predict the preservation of duplicated genes: the classical model and the duplication– degeneration–complementation (DDC) model (Force et al., 1999). The classical model suggests that the fate of most duplicated genes is to be fixed as pseudogenes and that there should be a corresponding acceleration of non-synonymous substitutions. However we did not observe such acceleration in the porcine cluster. Moreover, the codon-substitution models for positively selected amino acid sites were also rejected according to the results of LRT shown in Table 3B. Therefore, porcine IFN-α evolution does not follow the classical model, and the DDC model may be more appropriate. According to the latter, preserved duplicated genes subdivide the functions of the ancestral gene and the regulatory region degenerates with ensuing divergence in the spatial and temporal expression of the duplicated genes. In the present instance the porcine IFN-α with C-terminal deletions retained the normal activity of IFN-α though at a reduced level (Table 4) and there were many changes in the regulatory region (Fig. 2). The outcome of this is

37

that the duplicated genes avoid competing with the ancestral genes and are therefore maintained in the genome. That is to say, the duplicated genes with C-terminal deletions are not on the way to becoming pseudogenes but are preserved in the genome by an alternative strategy. The growing demand for specific porcine cytokine reagents indicates that many laboratories in different branches of immunology and physiology wish to study cytokine (IFN) responses using the pig as model (Kamstrup et al., 2001; Johansson et al., 2002; Magnusson et al., 2001). The present research on aligning the subtypes of porcine IFN-α, simulating their structures, analyzing their promoters, and identifying the effects of Cterminal deletion, should not only enhance understanding of the function of these versatile molecules in a novel animal model, but also facilitate further research on the role of the porcine IFN system. Acknowledgment We thank Julian D. Gross, Professor of Biochemistry at Oxford University for critically reading the manuscript. This work was supported by a grant from the National Programs for Science and Technology Development to Y.W. (2004BA519A38), an NSFC grant to M.L. (30300011), a SRFDP grant to Z.Z. (20030246016), the National Basic Research Project (973) (2003CB715900), the High-Tech Project (863) (2002AA23104) and a Fudan University grant to G.C. (CQH1322025).

References Braganca, J., et al., 1997. Synergism between multiple virus-induced factorbinding elements involved in the differential expression of interferon A genes. J. Biol. Chem. 272, 22154–22162. Chinsangaram, J., Piccone, M.E., Grubman, M.J., 1999. Ability of foot-andmouth disease virus to form plaques in cell culture is associated with suppression of alpha/beta interferon. J. Virol. 73, 9891–9898. Chinsangaram, J., Koster, M., Grubman, M.J., 2001. Inhibition of L-deleted foot-and-mouth disease virus replication by alpha/beta interferon involves double-stranded RNA-dependent protein kinase. J. Virol. 75, 5498–5503. Chinsangaram, J., Moraes, M.P., Koster, M., Grubman, M.J., 2003. Novel viral disease control strategy: adenovirus expressing alpha interferon rapidly protects porcine from foot-and-mouth disease. J. Virol. 77, 1621–1625. Combet, C., Blanchet, C., Geourjon, C., Deleage, G., 2000. NPS@: network protein sequence analysis. Trends Biochem. Sci. 25, 147–150. Corpet, F., 1988. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 16, 10881–10890. Demmers, K.J., Derecka, K., Flint, A., 2001. Trophoblast interferon and pregnancy. Reproduction 121, 41–49. Diaz, M.O., et al., 1994. Structure of the human type-I interferon gene cluster determined from a YAC clone contig. Genomics 22, 540–552. Familletti, P.C., McCandliss, R., Pestka, S., 1981. Production of high levels of human leukocyte interferon from a continuous human myeloblast cell culture. Antimicrob. Agents Chemother. 20, 5–9. Flores, I., Mariano, T.M., Pestka, S., 1991. Human interferon-ω (omega) binds to the alpha/beta receptor. J. Biol. Chem. 266, 19875–19877. Force, A., Lynch, M., Pickett, F.B., Amores, A., Yan, Y.L., Postlethwait, J., 1999. Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151, 1531–1545. Hardy, M.P., Owczarek, C.M., Jermiin, L.S., Ejdeback, M., Hertzog, P.J., 2004. Characterization of the type I interferon locus and identification of novel genes. Genomics 84, 331–345.

38

G. Cheng et al. / Gene 382 (2006) 28–38

Johansson, E., Wallgren, P., Fuxler, L., Domeika, K., Lefevre, F., Fossum, C., 2002. The DNA vaccine vector pcDNA3 induces IFN-alpha production in pigs. Vet. Immunol. Immunopathol. 87, 29–40. Juang, Y.T., et al., 1998. Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3. Proc. Natl. Acad. Sci. U. S. A. 95, 9837–9842. Kamstrup, S., Verthelyi, D., Klinman, D.M., 2001. Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides. Vet. Microbiol. 78, 353–362. Kelley, K.A., et al., 1983. Mapping of murine inferferon-α genes to chromosome 4. Gene 26, 181–188. Kelley, K.A., Pitha, P.M., 1985. Characterization of a mouse interferon gene locus I. Isolation of a cluster of four α-interferon genes. Nucleic Acids Res. 13, 805–823. Kotenko, S.V., et al., 2003. IFN-λs mediate antiviral protection through a distinct class II cytokine receptor complex. Nat. Immunol. 4, 69–77. La Bonnardiere, C., Lefevre, F., Charley, B., 1994. Interferon response in pigs: molecular and biological aspects. Vet. Immunol. Immunopathol. 43, 29–36. LaFleur, D.W., et al., 2001. Interferon-kappa, a novel type I interferon expressed in human keratinocytes. J. Biol. Chem. 276, 39765–39771. Lefevre, F., Bonnardiere, C. La, 1986. Molecular cloning and sequencing of a gene encoding biologically active porcine alpha-interferon. J. Interf. Res. 6, 349–360. Lefevre, F., Mege, D., Haridon, R.L., Bernard, S., Vaureix, C.D., Bonnardiere, C. La, 1990a. Contribution of molecular biology to the study of the porcine interferon system. Vet. Microbiol. 23, 245–257. Lefevre, F., Haridon, R.L., Cuesta, F.B., Bonnardiere, C. La, 1990b. Production, purification and biological properties of an Escherichia coli-derived recombinant porcine alpha interferon. J. Gen. Virol. 71, 1057–1063. Lefevre, F., Guillomot, M., D'Andrea, S., Battegay, S., La Bonnardiere, C., 1998. Interferon-delta: the first member of a novel type I interferon family. Biochimie 80, 779–788. Lin, R.T., Mamane, Y., Hiscott, J., 2000. Multiple regulatory domains control IRF-7 activity in response to virus infection. J. Biol. Chem. 275, 34320–34327. Magnusson, M., Johansson, E., Berg, M., Eloranta, M.L., Fuxler, L., Fossum, C., 2001. The plasmid pcDNA3 differentially induces production of interferon-alpha and interleukin-6 in cultures of porcine leukocytes. Vet. Immunol. Immunopathol. 78, 45–56. Marc, R.R., Dorothée, H., 2000. RRTree: relative-rate tests between groups of sequences on a phylogenetic tree. Bioinformatics 16, 296–297. Moraes, M.P., Chinsangaram, J., Brum, M.C., Grubman, M.J., 2003. Immediate protection of porcine from foot-and-mouth disease: a combination of adenoviruses expressing interferon alpha and a foot-and-mouth disease virus subunit vaccine. Vaccine 22, 268–279. Morin, P., et al., 2002. Preferential binding sites for interferon regulatory factors 3 and 7 involved in interferon-A gene transcription. J. Mol. Biol. 316, 1009–1022. Naf, D., Hardin, S.E., Weissmann, C., 1991. Multimerization of AAGTGA and GAAAGT generates sequences that mediate virus inducibility by mimicking an interferon promoter element. Proc. Natl. Acad. Sci. U. S. A. 88, 1369–1373.

Oritani, K., et al., 2000. Limitin: an interferon-like cytokine that preferentially influences B lymphocyte precursors. Nat. Med. 6, 659–666. Oritani, K., Kincade, P.W., Zhang, C., Tomiyama, Y., Matsuzawa, Y., 2001. Type I interferons and limitin: a comparison of structures, receptors, and functions. Cytokine Growth Factor Rev. 12, 337–348. Pestka, S., Langer, J.A., Zoon, K.C., Samuel, C.E., 1987. Interferons and their actions. Annu. Rev. Biochem. 56, 727–777. Pol, J.M., Broekhuysen-Davies, J.M., Wagenaar, F., La Bonnardiere, C., 1991. The influence of porcine recombinant interferon-alpha 1 on pseudorabies virus infection of porcine nasal mucosa in vitro. J. Gen. Virol. 72, 933–938. Posada, D., Crandall, K.A., 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817–818. Radhakrishnan, R., et al., 1996. Zinc mediated dimer of human interferon-alpha (2b) revealed by X-ray crystallography. Structure 4, 1453–1463. Roberts, R.M., Ealy, A.D., Alexenko, A.P., Han, C.S., Ezashi, T., 1999. Trophoblast interferons. Placenta 20, 259–264. Sen, G.C., Lengyel, P., 1992. The interferon system. A bird's eye view of its biochemistry. J. Biol. Chem. 267, 5017–5020. Sheppard, P., et al., 2003. IL-28, IL-29 and their class II cytokine receptor IL28R. Nat. Immunol. 4, 63–68. Swofford, D.L., 1998. PAUP ⁎. Phylogenetic Analysis Using Parsimony (*and Other Methods). Sinauer Associates, Sunderland. Version 4. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The Clustal-X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24, 4876–4882. Valarcher, J.F., Furze, J., Wyld, S., Cook, R., Conzelmann, K.K., Taylor, G., 2003. Role of alpha/beta interferons in the attenuation and immunogenicity of recombinant bovine respiratory syncytial viruses lacking NS proteins. J. Virol. 77, 8426–8439. van Pesch, V., Michiels, T., 2003. Characterization of interferon-alpha 13, a novel constitutive murine interferon-alpha subtype. J. Biol. Chem. 278, 46321–46328. van Pesch, V., van Eyll, O., Michiels, T., 2001. The leader protein of Theiler's virus inhibits immediate–early alpha/beta interferon production. J. Virol. 75, 7811–7817. van Pesch, V., Lanaya, H., Renauld, J.C., Michiels, T., 2004. Characterization of the murine alpha interferon gene family. J. Virol. 78, 8219–8228. Weissmann, C., Weker, H., 1986. The interferon genes. Prog. Nucleic Acid Res. Mol. Biol. 33, 251–300. Yang, Z., 1997. PAML: a program package for phylogenetic analysis by maximum likelihood. CABIOS 13, 555–556. Yang, Z., 2000. Phylogenetic Analysis by Maximum Likelihood (PAML). University College London, England. Version 3.0. Yerle, M., Gellin, J., Echard, G., Lefevre, F., Gillois, M., 1986. Chromosomal localization of leukocyte interferon gene in the pig (Sus scrofa domestica L.) by in situ hybridization. Cytogenet. Cell Genet. 42, 129–132.