Volume 5 Supplement 4
Porcine type I interferons: polymorphic sequences and activity against PRRSV
© Sang et al; licensee BioMed Central Ltd. 2011
Published: 3 June 2011
Type I interferons (IFN) are a heterogeneous group of cytokines central to innate and adaptive antiviral immune responses. We have recently reported that porcine type I IFNs comprise at least 39 functional genes with diverse antiviral activity against porcine reproductive and respiratory syndrome virus (PRRSV). Here we report that potential cytokine polymorphisms exist in several genes of porcine type I IFNs.
We have detected more than 100 potential polymorphic mutations, which include nucleotide substitutions and deletions, within the coding regions of porcine type I IFNs. Approximately 50% of the nucleotide changes were mutations that resulted in non-conserved amino acid substitution, as well as deletions that produced frame shifts in the open reading frames (ORFs). We have identified more than 20 polymorphic mutants that showed alterations in anti-PRRSV and anti-vesicular stomatitis virus (VSV) activity in vitro. In particular, some mutations in IFN-α2, IFN-α3, IFN-α8, IFN-α12 and IFN-ω5 significantly altered the antiviral activity of expressed proteins in comparison to the wild-type or variant with more similarity to the wild-type.
Multiple polymorphic isoforms potentially exist within subtypes of the porcine type I IFN family. Polymorphic mutations are more common in multiple-member subtypes than single-member subtypes, and most are found within the IFN-α subclass. Some polymorphic isoforms have altered amino acid composition and shifted ORFs, which show significantly different antiviral activity in vitro.
Type I interferons (IFNs) are a family of cytokines prominent in antiviral responses [1, 2]. Genetic mutations that result in dysfunction of type I IFN production and actions are ultimately associated with susceptibility to viral diseases [2, 3]. Conversely, viruses may antagonize or evade type I IFN actions resulting in successful infections . In this context, porcine reproductive and respiratory syndrome virus (PRRSV), a RNA virus that causes devastating losses in the global swine industry, suppresses type I IFN production [5, 6]; and diverse PRRSV strains manifest different abilities to suppress type I IFN expression . Furthermore, emerging evidence implicates that cytokine polymorphisms such as those in IFN-γ and interleukin (IL)-8 are key factors in breed- or genotype-associated viral resistance [8–10]. However, how type I IFN polymorphisms affect resistance to PRRSV infection is not known. We have recently reported that the porcine type I IFN family consists of at least 39 functional genes, which are classified into seven subclasses of 17 IFN-α, 11 IFN-δ, and 7 IFN-ω subtypes, and single-subtype subclasses consisting of IFN-αω, IFN-β, IFN-ε, and IFN-κ . We showed that members of different or the same porcine type I IFN subclasses have diverse expression profiles in various pig tissues and in vitro antiviral activities . Here we report that expressed proteins from multiple polymorphic isoforms within subtypes of type I IFNs have significantly altered antiviral activity, which may represent an important factor for determining the outcome of the host-virus interaction.
The isolation and analyses of IFN genes were conducted as previously described . In brief, coding regions of porcine IFNs were amplified from several cDNA and genomic DNA pools using a high-fidelity PCR with subtype-common cloning primers. PCR fragments, which flank six nucleotides before the start codon and stop codon of each IFN ORF, were purified and cloned into a pcDNA™ 3.3-TOPO® TA cloning/expression vector (Invitrogen, Carlsbad, CA) under the action of the vector CVM promoter. At least five clones for each IFN gene were sequenced and clones that showed the highest sequence identity (>97%) to the reference sequences (extracted from genomic draft sequences by Sanger Institute Porcine Project Team, which is available from http://www.ensembl.org/Sus_scrofa/index.html) were selected as polymorphic variants of a particular IFN for mutation definition and protein expression. Mutation determination was conducted by combined use of sequence analysis tools in Lasergene v8.1 (DNASTAR, Inc. Madison, WI) and Bioedit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). IFN peptides were collected and partially purified from supernatants of HEK293F cells transfected with individual IFN-expression plasmids. Authentic expression of individual IFN peptides was confirmed by gel electrophoresis; and peptide concentrations were adjusted to 2 μg/ml with the serum-free medium to ensure the antiviral differences were not a result of peptide levels in the antiviral assays (11). Antiviral activity of overexpressed IFN peptides was assayed as the inhibition of the cytopathic effect (CPE) of PRRSV and VSV on MARC-145 and PK-15 cells, respectively, in which 1 unit is defined as the highest dilution that reduced cell loss by 50% according to the Reed-Muench method .
Results and conclusions
Existence of multiple polymorphic isoforms in IFN-α, IFN-δ and IFN-ω subtypes
Polymorphic isoforms of porcine type I interferons (IFNs)
IFN polymorphic isoforms
Different from reference sequences1
NA → AA
Potential responsible mutations2
7 SNP 5 AA
5 SNP 3 AA
5 SNP 4 AA
7 SNP 6 AA
4 SNP 1 AA
8 SNP 5 AA
7 SNP 4 AA
8 SNP 2 AA
12 SNP 8 AA
12 SNP 8 AA
4 SNP 1 AA
L90-P, D101-N, A127-V, V174-A
10 SNP (ORF)
ORF shifted after 100 AA
1 SNP 1 AA
2 SNP 1 AA
3 SNP 1 AA
6 SNP (ORF)
d(stop) and added C-17 AA
Diverse activity of polymorphic type I IFNs
This work was supported in part by USDA NRICGP grants 2003-35204-13704 and 2006-35204-17337. We thank Danielle Goodband for her excellent technical support.
This article has been published as part of BMC Proceedings Volume 5 Supplement 4, 2011: Proceedings of the International Symposium on Animal Genomics for Animal Health (AGAH 2010). The full contents of the supplement are available online at http://www.biomedcentral.com/1753-6561/5?issue=S4.
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