State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, People’s Republic of China.
Characterization and Virus-Induced Expression Profiles of
the Porcine Interferon-ω Multigene Family
Xin Zhao, Gong Cheng, Weiyao Yan, Mingqiu Liu, Yao He, and Zhaoxin Zheng
Interferon-ω is a member of the type I interferon family. In this work, 8 functional porcine interferon-ω genes and
4 pseudogenes present on porcine chromosome 1 were identifi ed in the porcine genome database by BLAST scan-
ning. Their genetic and genomic characteristics were investigated using bioinformatics tools. Then the PoIFN-ω
functional subtype genes were isolated and expressed in BHK-21 cells. The PoIFN-ω subtypes possessed about
104 to 105 units of antiviral activity per milliliter. PoIFN-ω7 had the highest antiviral activity, about 20 times that
of PoIFN-ω4, which had the lowest antiviral activity. Differential expression of the subtypes was detected in PK15
cells and porcine peripheral blood mononuclear cells (PBMCs) in response to pseudorabies virus and poly(I).
poly(C). Expression of PoIFN-ω2/-ω6 was up-regulated to the greatest extent by virus infection.
ing antiviral, antiproliferative, antitumor, and immuno-
modulatory activities. They are classifi ed into type I IFNs
and type II IFNs. In contrast to the type II family, whose
sole member is IFN-γ, the type I interferons are a heteroge-
neous group comprising IFN-α, IFN-β, IFN-ω, IFN-κ, IFN-ε,
IFN-δ, IFN-τ, and the newly discovered subtype IFN-ζ (limi-
tin) (Pestka and others 1987; Flores and others 1991; Oritani
and others 2000; LaFleur and others 2001; Oritani and oth-
ers 2001; Hardy and others 2004). In addition, the IFN-λ sub-
types, also called interleukin-28A (IL-28A), IL-28B, and IL-29,
are known in humans and other mammals; they mediate
antiviral protection through a distinct receptor complex but
a similar signaling pathway to type I IFNs (Kotenko and
others 2003; Sheppard and others 2003).
Interferon-ω, fi rst found in humans, is a type I interferon
(Hauptmann and Swetly 1985). The human IFN-ω gene
cluster, which is composed of 6 sequences but only a sin-
gle functional gene, is located on human chromosome 9. It
has about 60% amino acids identity with HuIFN-α (Hardy
and others 2004). The functional IFN-ω gene is not found
in the mouse or rat, but has been identifi ed in many other
mammals, including felines, porcines, bovines, and rab-
bits (Hauptmann and Swetly 1985; Mege and others 1991;
Charlier and others 1993; Rodriguez and others 1998; Yang
and others 2007).
nterferons (IFNs) are a family of functionally related
cytokines that evoke a range of cellular responses includ-
Most of the members of the type I IFN gene family have
been identifi ed in pigs: IFN-α, IFN-β, IFN-ω, and IFN-δ
(Lefevre and Labonnardiere 1986; Amadori and others 1987;
Mege and others 1991; Lefevre and Boulay 1993); all are
located on porcine chromosome 1. The porcine interferon-α
(PoIFN-α) gene family is reported to have 16 distinct mem-
bers, comprising 14 functional genes and 2 pseudogenes. Six
of the functional genes have C-terminal truncations of 8 resi-
dues, and they have much less antiviral activity than the full-
length subtypes (Cheng and others 2006). The porcine IFN-ω
multigene family was fi rst described by Mege and his col-
leagues. They discovered and analyzed 3 functional PoIFN-ω
genes and 2 pseudogenes (Mege and others 1991).
In this work, we scanned the porcine genome database
and identifi ed 8 functional PoIFN-ω genes and 4 pseudo-
genes. We examined their genetic and genomic character-
istics using bioinformatics tools, isolated the subtype genes
from PK15 cells (porcine kidney cell), and cloned them into
pcDNA3 vector to analyze their antiviral activity and acid
stability. Their expression profi les in PK15 cells and PBMCs
in response to viral infection were also observed.
Materials and Methods
Cells and viruses
Porcine kidney cells (PK15) and baby hamster kidney
cells (BHK-21) were cultured in Dulbecco’s modifi ed Eagle’s
JOURNAL OF INTERFERON & CYTOKINE RESEARCH
Volume 29, Number 10, 2009
© Mary Ann Liebert, Inc.
ZHAO ET AL.688
Antiviral activity and pH 2 stability
The antiviral activity of IFN-ω preparations was tested in
the PK15 cells/pseudorabies virus (PRV) system. In brief, the
cells were seeded in fl at-bottom 96-well plates and grown to
95% confl uence, and serial dilutions of IFN-ω were added to
the wells. After 24 h, 100× TCID50 of virus was added. After
a further 24 h, antiviral activity was calculated using the
Reed–Muench method. One unit is the highest dilution that
reduced cell number by 50%. To investigate acid stability,
the supernatants of the transfected BHK-21 cell were incu-
bated at pH 2 for 24 h at 4°C, as described previously (van
Pesch and Michiels 2003; Cheng and others 2006), and then
assessed in the antiviral test.
Detection of induced expression of the
PK15 cell monolayers in 6-well plate were incubated with
PRV 10× TCID50 or 2 μL of 2 mg/mL poly(I).poly(C) and 6
μL lipofectamine reagent for 4 h. Then the virus and poly(I).
poly(C) were removed and 800 μL aliquots of DMEM were
added. After another 20 h, the cells were collected and stored
in Trizol reagent at −70°C.
Peripheral blood mononuclear cells (PBMC) of Shanghai
White Pig were isolated and incubated with PRV 10× TCID50
in 6-well plate for 24 h. Then the cells were collected and
stored in Trizol reagent at −70°C.
cDNA was synthesized from the viral or poly(I).poly(C)-
induced cells, and PCR was performed using the cDNA as
template together with consensus PoIFN-ω primers (the for-
ward primer TCCCCCAGGAGATGGTGGA, and the reverse
primer CAGGTAGAGATGGATTCCCT). The PCR products
were purifi ed and cloned into the pMD18-T vector. Positive
clones were chosen at random and sequenced. And the data
were analyzed using the DNAStar program (Cheng and oth-
Nucleotide sequence accession numbers
The nucleotide sequence inferred in this work, and
their Genbank accession numbers, are as follows: porcine
interferon-ω1 (EU797615), porcine interferon-ω2 (EU797616),
porcine interferon-ω3 (EU797617), porcine interferon-ω4
(EU797618), porcine interferon-ω5 (EU797619), porcine
interferon-ω6 (EU797620), porcine interferon-ω7 (EU797621),
porcine interferon-ω8 (EU797622), porcine interferon-α1
(DQ249000), porcine interferon-α12 (DQ249003), por-
cine interferon-γ (AY293733.1),
(NM_002177.1), human interferon-ω15 pseudogene (K03013),
human interferon-ω19 pseudogene (NG_005641), chimpan-
zee interferon-ω1 (XM_528554), rhesus monkey interferon-ω1
(XM_001108113), bovine interferon-ω1(AF238610), bovine
interferon-αII pseudogene (M60898), sheep interferon-ω1
(M73245), rabbit interferon-ω20
interferon-ω44 (S68997), feline interferon-ω1 (DQ420220),
feline interferon-ω2 (DQ420221), horse interferon-ω1
(M14544), horse interferon-ω2 (M14545).
Genomic loci of the PoIFN-ω multigene family
BLAST analysis in the porcine genome database revealed
that the PoIFN-ω cluster is distributed over ~700 kb of the
medium (DMEM) containing 10% heat-inactivated fetal
bovine serum (FBS) at 37°C with 5% CO2. Pseudorabies virus
(PRV) used for viral challenge was kindly donated by Dr. Du
(Institute of Biotechnology, Zhejiang Academy of Agricultural
Sciences). PK15 cells were used to grow PRV and determine
viral titers, and 50% tissue culture infective doses (TCID50)
were calculated using the Reed–Muench formula.
Sequence analysis and multiple alignments
Using the BLASTN algorithm, we identifi ed PoIFN-ω
gene fragments in the pig genome database generated by
the porcine genome sequencing project. The genomic loci
of these subtypes were located on the map using porcine
genomic sequence data (http://pre.ensembl.org/index.
html). Sequences were aligned using the DNAStar program
(DNASTAR Inc., WI) and signal peptides were predicted
with SignalP (http://www.cbs.dtu.dk/services/SignalP/),
while putative N- and O-glycosylation sites were predicted
with the NetNGlyc and NetOGlyc Web sites, respectively
(http://www.cbs.dtu.dk/services/NetNGlyc/ and http://
Multiple sequence alignments were obtained using
Clustal X, and the phylogenetic tree was generated using
PHYLIP 3.6 program by the maximum-likelihood (ML)
method. PoIFN-γ was chosen as out-group.
Sequence isolation and clone construction
Subtype-specifi c primers for the porcine IFN-ω subtypes
were designed from the sequences in the porcine genomic
database and all the subtypes were amplifi ed by PCR from
PK15 genomic DNA. The PCR products were inserted into
pMD18-T vector (TaKaRa, Kyoto, Japan) and further cloned
into pcDNA3 vector at XhoI and EcoRI restriction sites.
The primers used for the PoIFN-ω gene extraction are
as follows: PoIFN-ω1, forward primer: GGTCTCAGCCA
GCATCCGTAATTCTTC; PoIFN-ω2/-ω3/-ω5/-ω6, forward
primer: CTAGACACCCATCTCAGCCAGGCC, backward
primer: GACAAAAGATGAATGTGCTAGGATG; PoIFN-ω7,
forward primer: ATGGCCTTCATGCTCTCTCTACTGACA,
backward primer: TCAAGGTGACCCCAGGTGTT.
BHK-21 (baby hamster kidney) cells were transfected with
4 μg plasmid and 6 μL Lipofectamine 2000 (Invitrogen, Life
Technologies) in wells of a 6-well plate. Six hours after trans-
fection, the suspensions were removed and 800 μL DMEM
with 10% FBS was added. After 48 h, supernatants and the
cells were separately collected.
RNA isolation and reverse transcription
Total cellular RNA was prepared using Trizol reagent. It
was dissolved in 100 μL RNase-free ddH2O and treated with
DNase I (RNase-free) (New England Biolabs, Beverly, MA)
for 30 min. cDNA was synthesized with MMLV Reverse
Transcriptase (Invitrogen, Carlsbad, CA) and oligo-(dT)18
(Takara Kyoto, Japan).
PoIFN-훚 FAMILY AND THEIR EXPRESSION PROFILES
using maximum-likelihood (ML) analysis. Porcine IFN-γ
was selected as out-group. The tree revealed that the
C-terminally deleted subtype PoIFN-ω1 is closely related
to the fi ve subtypes from PoIFN-ω2 to PoIFN-ω6, while
PoIFN-ω7 and PoIFN-ω8 belong to another branch of the
tree (Fig. 2). Furthermore, the genes predicted by the phy-
logenetic analysis to have a closer evolutional relationship
are also closer together on chromosome 1, implying a recent
duplication event (Krause and Pestka 2005).
Isolation and expression of the PoIFN-ω subtypes
The coding sequences of all the 6 different functional
PoIFN-ω subtypes were isolated by PCR from the genomic
DNA of PK15 cells. These PoIFN-ω sequences obtained from
the PK15 genome share 99.1%–100.0% similarities with their
counterparts in the porcine genomic sequences generated
by the porcine genome sequencing project. The sequences
obtained were cloned into pcDNA 3 vector at XhoI and EcoRI
The PoIFN-ω recombinant plasmids were transfected into
BHK-21 cells. PoIFN-α12 and pcDNA 3 empty vector served
as positive and negative controls, respectively (Cheng and
others 2006). After 48-h transfection, supernatants were col-
lected to measure antiviral activity and pH 2 stability. Real-
time quantitative PCR was performed using cDNA from
the transfected cells to measure the transfection effi ciency.
The result demonstrated that the transfection effi ciencies of
the 4 PoIFN-ω recombinant plasmids were similar (data not
Antiviral activity and pH 2 stability
In order to detect the antiviral activity, the supernatants
were collected after 48-h transfection. The PoIFN-ω sub-
types all had signifi cant antiviral activities. Among them,
PoIFN-ω7 had the most antiviral activity. PoIFN-ω1, which
is 11 AA shorter than the other PoIFN-ω subtypes at the
C-terminal, exhibited similar antiviral activity with others
(Table 3). The supernatants from mock-transfected cells or
cells transfected with empty vector were performed as the
negative control. No antiviral activity was detected in the 2
control groups (data not shown).
Acid stability at pH 2 was one of the fi rst characteristics
described in type I IFNs (Isaacs and others 1957). To test the
long arm of chromosome 1. It consists of 12 genes: 8 func-
tional genes and 4 pseudogenes. They were designated
PoIFN-ω1 to PoIFN-ω8 and PoIFN-ωψ1 to PoIFN-ωψ4, in the
order of their chromosome loci. Table 1 gives the chromo-
somal loci of these PoIFN-ω genes.
Sequence alignment of PoIFN-ω
Alignment of the PoIFN-ω genes showed that the 8 func-
tional subtypes share 88.7%–100.0% identity at the nucleo-
tide level and 80.6%–100.0% identity at the amino acid level
(Table 2). The PoIFN-ω2/-ω6 and PoIFN-ω3/-ω5 pairs are
identical in sequence but are considered different genes due
to their different chromosomal locations. Although 7 of the 8
PoIFN-ω functional genes (PoIFN-ω2 to PoIFN-ω8) are trans-
lated into 190 amino acid (AA) prepeptides, PoIFN-ω1 only
has 179 AA owing to an 11 AA C-terminal deletion. All 8
functional PoIFN-ω genes have putative signal peptides of
23 AA, so that their mature peptides comprise 167 and 156
(PoIFN-ω1) amino acids, respectively. Cysteines at positions
1/99 and 29/134 of the mature peptides are predicted to form
disulfi de bonds. The only putative N-glycosylation site (N-X-
S/T) is found at residue 133 of PoIFN-ω8 (Fig. 1).
A phylogenetic tree of the interferon-ωs of different spe-
cies was generated with the PHLIP (version 3.1) program
Table 1. The Genome Location of the PoIFN-ω Subtypes
Gene name Genome location (5′–3′, nt) Length (nt)
Table 2. Similarities of Protein and Genetic Sequences Between Porcine IFN-ω Subtypes
AA PoIFN-ω1 PoIFN-ω2 PoIFN-ω3 PoIFN-ω4 PoIFN-ω5 PoIFN-ω6 PoIFN-ω7 PoIFN-ω8 HuIFN-ω1 PoIFN-α12
Abbreviations: NA, nucleic acid; AA, amino acid.
ZHAO ET AL.690
acid-resistance of the PoIFN-ω subtypes, we used samples
the same as used in the antiviral experiments and exposed
them to pH 2 for 24 h before measuring antiviral activity.
All the IFN-ωs retained antiviral activities after the acid
treatment, with at most a 1.5- to 6-fold loss of activity. The
C-terminal truncation of PoIFN-ω1 had no effect on its acid
Expression of the PoIFN-ω subtypes after viral
Differences in expression of the PoIFN-ω subtypes in
PK15 cells after PRV or poly(I).poly(C) induction were
detected using the RT-PCR cloning–sequencing procedure
with consensus primers (Cheng and others 2007). Untreated
PK15 cells served as the negative control and no obvious
expression of PoIFN-ω was detected in this group. PoIFN-ω1,
PoIFN-ω2/-ω6, PoIFN-ω3/-ω5, and PoIFN-ω8 were up-reg-
ulated in response to PRV, and PoIFN-ω1, PoIFN-ω2/-ω6,
PoIFN-ω3/-ω5, PoIFN-ω7, and PoIFN-ω8 were detected in
the poly(I).poly(C)-treated cells. Expression of PoIFN-ω2/-ω6
was up-regulated the most. PoIFN-ω4 was not detected after
either treatment (Fig. 3).
To further analyze the virus-triggered IFN response of
primary pig cells, we detected the differences in expression
of the PoIFN-ω subtypes in Shanghai White Pig PBMCs after
PRV induction. All the PoIFN-ω subtypes were detected in
this analysis. But consistent with the result on PK15, PoIFN-
ω2/-ω6 was up-regulated the most (Fig. 3).
In this report, we have identifi ed 8 interferon-ω func-
tional genes and 4 pseudogenes in the porcine genome and
isolated the functional genes from PK15 cells. Among them,
Porcine IFN-ω 2
Rhesus monkey IFN-ω1
Porcine IFN-ω 4
Porcine IFN-ω 3
Porcine IFN-ω 1
FIG. 2. Phylogenetic tree of the porcine interferon (IFN)-ω
multigene family with IFN-ω sequences from other mamma-
lian species. Porcine IFN-γ was selected as out-group. The phy-
logenetic tree was generated with PHLIP (version 3.1) using
maximum-likelihood analysis. “ψ“ stands for pseudogene.
FIG. 1. Multiple sequence alignments of por-
cine interferon-ω (PoIFN-ω) subtypes. The
2 pairs of subtypes (PoIFN-ω2/-ω6, PoIFN-
ω3/-ω5) identical in sequence are shown as
1, respectively. The dots (.) in the consensus
sequence are nonconserved sites. The pre-
dicted signal peptide cleavage site is indi-
cated. The cysteine residues involved in the
disulfi de bridges are boxed and their positions
on the mature peptide are marked under the
boxes. The positions of variant residues in 1
or 2 subtypes are shown in gray, and the sub-
stitutions occurring in multiple subtypes are
labeled by arrows above the fi gure. The pre-
dicted N-glycosylation site (Asn-X-Ser/Thr) in
PoIFN-ω8 is outlined.
PoIFN-훚 FAMILY AND THEIR EXPRESSION PROFILES
immediate-early IFNs further induce IRF-7, which is essen-
tial for priming the transcription of other subtypes (Sato
and others 2000; Paun and Pitha 2007). The differential
expression profi le of PoIFN-ω subtypes reveals that PoIFN-
ω2/-ω6 is the subtype with the highest expression in the
fi rst 24 h after viral challenge. It may therefore play a crit-
ical role in early defense against viral invasion and in the
induction of subsequent immune responses. Analysis of the
the PoIFN-ω1 has an 11 residue C-terminal deletion and the
sequences of the pairs PoIFN-ω2/-ω6 and PoIFN-ω3/-ω5 are
X-ray crystallography of human interferon-α2b and
ovine interferon-τ revealed that type I interferons are com-
posed of fi ve α-helices (Radhakrishnan and others 1996;
Radhakrishnan and others 1999). Alignment suggests that
all the PoIFN-ω subtypes have a gap of 5 AA in the loop
between helix C and helix D (Fig. 4).
Previous researches were performed on the C-terminal
tails of type I interferons: deletion of the last fi ve residues of
IFN-α2 resulted in only a 2-fold reduction in binding affi nity
to IFNAR2 (Slutzki and others 2006), and loss of the 6 AA tail
of ovine interferon-τ did not affect its biological activity. In
contrast, an 8 AA C-terminal deletion of PoIFN-α resulted in
a reduction of 1 or 2 orders of magnitude in antiviral activ-
ity. In this report, we showed that the absence of 11 AA at
the C-terminus of PoIFN-ω1 did not greatly affect its anti-
viral activity. Alignment of their sequences revealed that
interferon-ωs and interferon-τs have 6 AA longer C-termini
than interferon-αs (data not shown). Furthermore, structural
modeling suggests that the arginine at position 161 (R161)
of type I interferon C-terminus have an electrostatic effect
with IFNAR2 (Radhakrishnan and others 1996; Viscomi
1997; Kumaran and others 2007). Based on these fi ndings by
various research groups, we presume that residues 159–161
of type I interferons (Fig. 4) play a critical role in ligand–
receptor binding and thus effect the antiviral activity.
Viral-induced expression of type I interferons takes
place in two stages: fi rst, viral infection activates constitu-
tively expressed IRF-3 (interferon regulator factor) by phos-
phorylation cascades and thus mediated the expression of
the interferons termed immediate-early IFNs. Then, the
Table 3. Antiviral Activity of the PoIFN-ω Subtypes
Before and After Acid Treatment
Antiviral activity after pH 2
treatment (×104 U/mL)
1.58 ± 0.47
5.46 ± 2.08
4.57 ± 0.17
0.91 ± 0.05
22.75 ± 0.00
19.79 ± 1.81
17.24 ± 0.98
0.32 ± 0.00
1.93 ± 0.01
2.03 ± 0.55
0.14 ± 0.00
7.94 ± 0.00
13.08 ± 0.13
3.16 ± 0.15
n = 22
n = 21
n = 17
PK 15/Poly I.C
PoIFN-ω4 PoIFN-ω7 PoIFN-ω8
PoIFN-ω1 PoIFN-ω2/ω6 PoIFN-ω3/ω5 PoIFN-ω4 PoIFN-ω7 PoIFN-ω8
PoIFN-ω1 PoIFN-ω2/ω6 PoIFN-ω3/ω5 PoIFN-ω4 PoIFN-ω7 PoIFN-ω8
FIG. 3. Differential expression profi les of the porcine
IFN-ω (PoIFN-ω) subtypes after viral induction. The sub-
types expressed were identifi ed by the RT-PCR cloning–
sequencing procedure. (A) PRV-infected PK15 cells. (B)
Poly(I).Poly(C)-induced PK15 cells. (C) PRV-infected PBMCs.
“n” is the total number of clones detected.
Helix D Helix E
FIG. 4. Sequence alignment of PoIFN-ω1/PoIFN-ω4 with PoIFN-α1/PoIFN-α12 and HuIFN-ω1. The 91–172 AA residues of
the mature proteins are shown. Conserved residues are marked with underlinings. The fi ve amino acid gap is boxed. The
putative regions of the D and E helices are indicated above the sequences.
ZHAO ET AL. 692
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virus-responsive elements (VREs) of the PoIFN-ω subtypes
showed that none have an intact VRE as described for human
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Martin and others 2002). Furthermore, it is reported to have
antitumor and immunoregulatory effects (Nieroda and oth-
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I IFN has focused on the individual IFN subtypes, which
differ greatly in their expression and biological functions
(Fung and others 2004; van Pesch and others 2004; Cheng
and others 2006; Cheng and others 2007; Yang and others
2007). The fi ndings of the present work will be useful for
further study of the roles of the PoIFN-ω subtypes in defense
against infectious diseases, particularly viral infections, and
should facilitate research on the porcine immune system.
We thank Dr. Julian D. Gross for critically reading the
manuscript. This work was supported by a RFDP grant
to Z.Z. (20030246016) and the High-Tech Project (863)
Author Disclosure Statement
No competing fi nancial interests exist.
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Drs. Zhaoxin Zheng and Weiyao Yan
State Key Laboratory of Genetic Engineering
220 Handan Road
People’s Republic of China
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