We previously reported a natural antisense (AS) RNA as an important modulator of human interferon-Alpha1 (IFNA1) mRNA levels. Here, we identified the guinea pig (Cavia porcellus) IFNA1 gene to enable a proof-of-concept experiment to be performed to confirm that the AS-mRNA regulatory axis exerts in vivo control over innate immunity. We selected a guinea pig model system for influenza virus infection because guinea pigs encode a functional M × 1 gene, an important anti-viral effector in the type I interferon pathway. We identified 15 guinea pig IFNA1 gene candidates upon bioinformatic analysis and selected the three candidates with the highest sequence homology to Homo sapiens, Mus musculus and Marmota himalayana IFNA1. The anti-viral activity of guinea pig IFN-Alpha1 protein against influenza virus A/Puerto Rico/8/34- or endomyocarditis virus-infection was then determined for the three gene candidates. We identified cpIFNA1 as the candidate with the highest sequence homologies and best anti-viral effects. cpIFNA1 will enable us to perform a proof-of-concept experiment to verify that IFN-Alpha1 AS increases cpIFNA1 mRNA levels, resulting in inhibition of influenza virus proliferation in vivo.
Type I interferons (IFNs) and their downstream effectors collectively limit viral proliferation and spread; therefore, IFN-Alpha-based treatments are widely used for the treatment of chronic viral infections. A large number of studies investigating IFN regulation have focused on the transcriptional activation of type I IFN genes and on IFN-Alpha protein function and the IFN-Alpha signal transduction cascade. However, regulation at the RNA level has received less attention (1).
During our studies on a novel cis-acting element that is responsible for the CRM1-dependent nuclear export of human interferon-Alpha1 (IFNA1) mRNA (2), we observed that deletion of this element from the stem-loop region of the IFNA1 mRNA selectively impaired the stability of the mRNA. This finding led us to identify IFN-Alpha1 antisense RNA (AS), a natural antisense transcript, transcribed from the opposite strand of the IFNA1 gene that acts as an important modulator of IFNA1 mRNA levels in both Sendai virus-infected human Namalwa lymphocytes and influenza virus A/PR/8/34 (PR/8 virus)-infected guinea pig (gp) 104C1 fetal fibroblasts by promoting IFNA1 mRNA stability (1).
In the present study we identified the guinea pig (Cavia porcellus) IFNA1 gene to enable a proof-of-concept experiment to be performed to confirm that the natural antisense transcript-mRNA regulatory axis exerts control over innate immunity in vivo. Mice are frequently employed as an animal model to investigate influenza A virus (IFAV) pathogenesis. However, standard laboratory mouse strains, such as BALB/c and C57BL/6, do not have a complete antiviral defense system because they carry defective alleles of the M × 1 gene (3, 4). The expression of M × 1, which encodes the Mx GTPase, is tightly controlled by type I IFN, and is a decisive antiviral factor that controls IFAV infections in mice (5). These characteristics preclude the use of these mice for evaluating prophylactic treatment with exogenous IFN. Furthermore, the mice do not shed the virus from the respiratory tract, preventing study of proliferation profiles in the respiratory tract of individual animals without euthanasia (6).
We, therefore, employed the guinea pig, which possesses a functional M × 1 gene and exhibits virus shedding and is, therefore, useful for investigating the IFN response against IFAV infection (6). Although we previously published the concordant expression profiles of gpIFNA1 mRNA/AS in 104C1 cells infected with PR/8 virus (1), we have not yet reported the identification and characterization of the Cavia porcellus IFNA1 gene (cpIFNA1).
In this report, we identified fifteen cpIFNA1 candidates upon bioinformatic analysis, all of which were localized in Scaffold 2 of the genome with comparable lengths of coding region and deduced amino acids. We selected and cloned the three IFNA1 candidates with the highest sequence homology against Homo sapiens, Mus musculus and Marmota himalayana IFNA1. The anti-viral activity of guinea pig IFNA1 candidate proteins in 104C1 cells against PR/8 virus- or endomyocarditis virus (EMCV)-induced cytopathic effects was then determined. We identified cpIFNA1 from the highest sequence homologies and the best anti-viral effects against the PR/8 and EMCV infections.
Gp104C1 cells (fetal fibroblasts; ATCC CRL-1405) and MDCK (Madin-Darby canine kidney) cells (ATCC CCL-34) were maintained as previously described (1). Mouse L cells (ATCC CCL-1) were maintained in Dulbecco’s minimum essential medium containing 10% heat-inactivated fetal calf serum (D10). L cells were infected with EMCV (ATCC VR-1762) at a MOI (multiplicity of infection) of 0.0.1 at 37ºC for 1 hour. The infected cells were incubated until the cytopathic effects were well advanced through 90% of the culture. The rodent-adapted PR/8 virus, influenza virus A/Puerto Rico/8/34 (A/PR/8/34, H1N1) (7), was propagated in the allantoic cavity of 10-day-old embryonated hen eggs and was employed to infect gp104C1 cells as previously described (1). The viral titers were measured with a plaque-forming assay using L cells for EMCV and MDCK cells for PR/8 virus.
Orthologs of IFNA family genes in the assembled guinea pig genome (http://www.broadinstitute.org/science/projects/mammals- models/guinea-pig/guinea-pig-genome-project; released in February 2008) were searched for by comparison with existing IFNA family genes of Homo sapiens, Mus musculus and Marmota himalayana (8), using the BLAT program hosted by the UCSC Genome Browser (http://genome.ucsc.edu). All potential hits were evaluated by the presence of an open reading frame using GENETYX-MAC software (version 15; GENETYX Co., Tokyo, Japan). Identification of cpIFNA family gene subtypes was conducted by phylogenetic analysis using ClustalW (9), available from the DNA Data Bank of Japan (http://clustalw.ddbj.nig.ac.jp).
CpIFNA1 gene candidates 1 to 3 were amplified by polymerase chain reaction (PCR) using genomic DNA from Hartley guinea pig lung tissue as a template. The gene-specific primers used are listed in Table 1. Each amplicon was digested with HindIII/XbaI and cloned into the HindIII/XbaI sites of pSI (10) to generate pSI-cpIFNA1 candidate 1 ~ 3 expression plasmids.
104C1 cells were subjected to magnet-assisted transfection (MATra; IBA, Goettingen, Germany) of pSI-cpIFNA1 candidate 1, 2 or 3, and pSV-Beta-galactosidase control vector (Promega, Madison, WI, USA), as described previously (1). Culture supernatants and cell lysates were collected 48 hours after infection. The enzymatic activity of Beta-galactosidase in lysates was measured according to the manufacturer’s instructions (Beta-Glo assay system; Promega) to normalize the transfection efficiency of each cpIFNA1 expression plasmid. The culture supernatants were kept frozen at − 80°C until the viral protection assay.
For the viral protection assay, 104 C1 cells were pre-incubated for 24 h with each culture supernatant described above. Cells were then challenged with EMCV at a MOI of 0.005. The PR/8 virus challenge was conducted as previously described (1). After incubation for another 24 h (EMCV) or 48 h (PR/8), the extents to which the test culture supernatants inhibited virus-induced cytopathic events were detected by viable cell counting, as previously reported (11). Briefly, the infected cells were incubated with equal amounts of 6 mM of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5- (2,4-disulfophenyl) -2H- tetrazolium salt and 0.4 mM 1-methoxy-5-methylphenazinium methylsulfate (Dojindo, Kumamoto, Japan). After addition of 1 N H2SO4 to stop further color development, the optical density (OD) in each culture well was measured with a microplate reader at both 450 nm and 655 nm. The net change in OD450nm-655nm in each well was calculated as (OD450nm-655nm of the test well) - (OD450nm-655nm of the wells treated with the vector alone-supernatant).
Results in the Figures are representative of at least three independent experiments with triplicate samples generating similar findings. Differences presented in the Figures were analyzed using Student’s t test.
The genes employed in this study have the following accession numbers: AB671739 (Cavia porcellus IFNA1), AB578886 (Homo sapiens IFNA1), NM_010502.2. (Mus musculus Ifna1), AY962656 (Marmota himalayana IFNA1).
Bioinformatic analysis revealed the presence of 15 Cavia porcellus IFNA gene family candidates, all of which were localized in Scaffold 2 of the genome with coding region length varying from 546 nucleotides to 564 nucleotides and the number of predicted amino acids varying from 181 to 187 (Table 2; see also Figure 1A for cpIFNA1 candidate 1 nucleotide sequence and deduced amino acid sequence). We then selected the three cpIFNA1 candidates with the highest sequence homology against Homo sapiens, Mus musculus and Marmota himalayana IFNA1, with candidate 1 showing the highest homology (Table 3, top).
Characterization of the Cavia porcellus IFNA1 candidate 1 nucleotide and deduced amino acid sequences. (A) The nucleotide sequence of IFNA1 candidate 1 from Cavia porcellus is shown. The deduced amino acid sequence is indicated below the nucleotide sequence. Nucleotide residues in the coding sequence are numbered with respect to the initiation codon. * denotes stop codon. (B) Comparison of amino acid sequences for the IFN-Alpha1 proteins. Amino acid sequences of the potential IFN-Alpha1 protein encoded by cpIFNA1 candidate 1 are aligned to IFNA1 sequences from Homo sapiens, Mus musculus and Marmota himalayana. Amino acid residues are numbered with respect to the initiation methionine of cpIFNA1 candidate 1 protein. The IFN-Alpha/Beta/Delta family signature is boxed. Gaps are shown by hyphens. “*” denotes perfect alignment. “:” and “.” denote sites belonging to groups exhibiting strong and weak similarities, respectively. Strong similarity corresponds to a PAM250 MATRIX score between amino acids of greater than 0.5., whereas weak similarity corresponds to a score of 0.5. or less. The multiple amino acid sequence alignment was examined by ClustalW (9), DNA Data Bank of Japan (http://clustalw.ddbj.nig.ac.jp).
The deduced amino acid sequences of these cpIFNA1 candidates showed that all of the candidate proteins harbor the conserved IFN-Alpha, Beta, Delta family signature (8, 12) in the C-terminal region (amino acids 146–164), indicating that these proteins are gpIFN-Alpha proteins (see Figure 1B for cpIFNA1 candidate protein 1). Moreover, the candidate 1 protein showed the highest homology to both the IFN-Alpha1 proteins and the IFN signatures from the three species examined (Table 3, top and bottom, respectively). Interestingly, all of the IFN-Alpha1 candidate proteins were most homologous to the IFN-Alpha1 protein from Marmota himalayana, which most closely related to Cavia porcellus.
The anti-viral activity of gpIFN-Alpha1 candidate proteins was then determined by their ability to protect guinea pig 104 C1 fetal fibroblasts against cytopathic events following PR/8 virus or EMCV infection. As shown in Figure 2, all of the cpIFNA1 candidates showed significantly improved cell viability after infection of 104 C1 cells pretreated with culture supernatants from cells transfected with each candidate gene expression plasmid relative to the mock-treated and mock-infected 104 C1 cells, except for cpIFNA1 candidate 3 against EMCV infection. The relative viability of PR/8 virus-infected cells varied from 88% (candidate 3) to 105% (candidate 1) compared with vector-control cells. EMCV-infected cells showed relative cell viability of 83% (candidate 3) to 104% (candidate 1), whereas the negative control cells showed 74% cell viability. Based on these biological data and the bioinformatic analysis, candidate 1 ‘cpIFNA1’ showed the best antiviral effects against PR/8 and EMCV infection and had the highest homologies to both the IFN-Alpha, Beta, Delta family signature and IFNA1 sequences.
Antiviral assay with cpIFNA1 candidates. 104C1 cells were transfected with pSI-cpIFNA1 candidate 1, 2 or 3. The culture supernatants were collected and added individually to 104C1 cells. The cells were then infected with either PR/8 virus (top, solid bars) or EMCV (bottom, empty bars). The net changes in OD450nm-655nm were measured and used to calculate cell viability. The average net change of mock-treated and mock-infected 104C1 cells (Mock) are presented as 100% relative cell viability. Values of three independent experiments are presented as the mean ± s.e.m. of four or five samples. Vector: pSI vector-transfected and virally infected 104C1 cells.
We previously identified and characterized human IFN-Alpha1 AS, a natural antisense transcript and a long non-coding RNA, which plays a critical role in the post-transcriptional regulation of IFNA1, and subsequently IFN-Alpha1 protein production (1). This regulatory function was mediated by transient duplex formation between IFN-Alpha1 AS and the mRNA, which resulted in stabilization of the IFNA1 mRNA.
The regulatory effect of the duplex formation was verified by transfection of an antisense oligoribonucleotide (asORN), which was designed from the IFN-Alpha1 AS domain that recognizes a single-stranded target structure within a conserved secondary structure element formed in the IFNA1 mRNA-coding region. The asORN raised the levels of IFNA1 mRNA a few fold higher compared with levels in ncasORN-transfected cells, whereas neither the asORN nor ncasORN had any effect on IFN-Alpha1 AS expression (1).
To confirm these findings in vivo, we aimed to demonstrate, in a proof-of-concept experiment that asORNs designed from a model animal-derived IFN-Alpha1 AS functional domain that recognizes the IFNA1 mRNA, increase mRNA levels, and thereby, induce antiviral effects in vivo. Standard laboratory mouse strains, such as BALB/c and C57BL/6, lack functional M × 1, the mouse homolog of human MX1 (4, 13); therefore, we selected the guinea pig as the model animal, which encodes this important anti-viral effector in the type I interferon pathway (6, 14).
In this work, we identified cpIFNA1 and characterized its anti-viral function against PR/8 virus or EMCV infection. This has enabled us to set up a guinea pig model system to allow investigation of how asORNs regulate gpIFNA1 mRNA levels, and the subsequent effects on viral titers in PR/8 virus-infected animals. In the accompanying manuscript, we report the results from the proof-of-concept experiment, showing that pulmonary-administered asORNs raise the in vivo levels of gpIFNA1 mRNA, leading to inhibition of influenza virus proliferation in the animals (15).
Shiwen Jiang and Ryou Sakamoto contributed equally to this article. The authors declare no competing conflicts of interest with respect to the research, authorship and/or publication of this article. We are grateful to Mikio Nishizawa for critical reading of the manuscript and for stimulating discussions. We also thank all the present members of the Kimura laboratory for stimulating discussions. We thank Jeremy Allen, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. This work was supported by Japan Science and Technology Agency (JST) Grant No. AS2511389Q758 and by the Japan Society for the Promotion of Sciences (JSPS) KAKENHI Grant No. 15K06926 to TK.