We reported a natural antisense (AS) long non-coding RNA as an important modulator of interferon-Alpha1 (IFNA1) mRNA levels. We showed that IFN-Alpha1 AS promotes IFNA1 mRNA stability by transient duplex formation and inhibition of miR-1270-induced mRNA decay. Here, we performed a proof-of-concept experiment to verify that the AS-mRNA regulatory axis exerts in vivo control of innate immunity. We established a model system for influenza virus infection using guinea pig, which encodes a functional MX1 gene for the type I IFN pathway. This system allowed us to investigate the effects of antisense oligoribonucleotides representing functional domains of guinea pig IFN-Alpha1 AS on gpIFNA1 mRNA levels and, consequently, on viral proliferation in the respiratory tract of influenza virus-infected animals. We demonstrated that pulmonary-administered asORNs inhibited the proliferation of the virus in the animals by modulating IFNA1 mRNA levels. These results indicate that, in light of the proposed actions, asORNs may modulate the level of IFNA1 mRNA in vivo, indicating that IFN-Alpha1 AS plays a pivotal role in determining the outcome of type I IFN responses.
During the last decade, high-throughput technologies, such as next generation sequencing, have shown that the vast majority of a genome is transcribed, allowing the complexity of whole transcriptomes to be fully appreciated. As a result, an overwhelming number of long non-coding RNAs (lncRNAs) has been newly identified, leading to an increased understanding of the complexity of biological processes.
LncRNAs are defined as transcripts of more than 200 nucleotides that are not translated into proteins. They form a heterogeneous class of non-coding transcripts (1), which are subdivided in five categories based on their genomic localization: pRNAs (promoter-associated RNAs), eRNAs (enhancer-associated RNAs), gsRNAs (gene body-associated RNAs), lincRNAs (long intergenic or intervening non-coding RNAs) and NATs (Natural Antisense Transcripts) (for a review see (2) and REFERENCES therein).
It is thought that most lncRNAs originate within a 2 kb region surrounding the Transcription Start Site (TSS) of protein-coding genes (65% of lncRNAs overlap with a promoter, pRNAs), map to enhancer regions (19%; eRNAs), derive from antisense transcripts that overlap with annotated gene bodies (5%, NATs), or are associated with the bodies of protein-coding genes (gsRNA) (3, 4). The remaining lncRNAs (11%) originate from more distal (>2 kb) unannotated regions and are referred to as lincRNAs (5, 6).
Transcriptional regulatory elements, such as enhancers and promoters, can initiate transcription bi-directionally (7, 8); therefore, the majority of lncRNAs are initiated at promoters or enhancers. Many lncRNAs are localized in the nucleus (9), supporting the notion that lncRNAs are key regulators of epigenetic regulation. However, recent reports of local gene regulation by lncRNA loci suggest that in many cases, the act of transcription or DNA elements within the lncRNA locus are more likely to be a source of regulatory activity than the lncRNA itself (for a review see (10) and references therein), indicating more precise roles of lncRNAs as scaffolds for nuclear processes or guides for ribonucleoprotein complexes (2).
Another crucial role of lncRNAs has been highlighted in the field of post-transcriptional gene regulation (11, 12). For this function, lncRNAs leave the site of transcription and operate in trans. Trans-acting lncRNAs may function by modulating the activity or abundance of proteins or RNAs to which they bind (10). For example, the lncRNA, NORAD (noncoding RNA activated by DNA damage), functions as a molecular decoy for the RNA-binding proteins, PUMILIO1 (PUM1) and PUMILIO2 (PUM2) (13, 14), indicating that NORAD functions as a negative regulator of PUM1/PUM2 by limiting the availability of these proteins to interact with mRNA targets (10). Moreover, lncRNAs have the ability to regulate the abundance or activity of other RNAs to which they bind through base-paring interactions. These lncRNAs, termed competing endogenous RNAs or ceRNAs, have been suggested to broadly titrate microRNA (miR) availability (15).
A key feature of these lncRNAs is that they often require stoichiometric interaction with their target molecules to exert measureable regulatory effects. It is, therefore, interesting to note that these lncRNAs often form networks with ceRNA partners to fulfill the gap between the cellular copy numbers of the lncRNA and its target(s) (16) (for a review see also (17)).
Type I interferons (IFNs) and their downstream products 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 IFN regulation studies have focused on IFN-Alpha protein function, particularly on the transcriptional activation of type I IFN genes and the IFN-Alpha signal transduction cascade. However, regulation at the RNA level has received less attention (18).
During our studies on a novel cis-acting element that is responsible for the CRM1-dependent nuclear export of human IFN-Alpha1 (IFNA1) mRNA (19), we observed that deletion of a core structure in a stem-loop region formed by the element selectively impaired the stability of the IFNA1 mRNA. This finding led us to identify IFN-Alpha1 antisense RNA (AS), a natural AS transcribed from the opposite strand of the IFNA1 gene that acts as an important modulator of IFNA1 mRNA levels by promoting IFNA1 mRNA stability (18). This regulatory function was mediated in two ways; by transient duplex formation between IFN-Alpha1 AS and the mRNA, and by inhibiting miR-1270-induced mRNA decay through the shared miR-1270 response elements (MRE-1270s) between IFN-Alpha1 AS and mRNA (16, 18).
In the accompanying report (20) and our previous study (18), we identified guinea pig (Cavia porcellus) IFNA1, whose mRNA was expressed with IFN-Alpha1 AS in a concordant manner, indicating a concordant regulation in the expression of gpIFN-Alpha1 AS/mRNA, similar to the observations in Sendai virus-infected human Namalwa lymphocytes.
In the present study, we performed a proof-of-concept (POC) experiment to confirm that the NAT-mRNA regulatory network exerts control over innate immunity in vivo. For this, we established a unique model system in guinea pigs, which possess a functional Mx (Mx dynamin-like GTPase) gene (21). This enabled us to investigate the IFN response to influenza virus infection (22). We employed poly(D,L-lactide-co- glycolide) (PLGA) nanoparticles as a respiratory drug carrier (23, 24), which allowed us to deliver antisense oligoribonucleotides (asORNs) designed from the functional domains of guinea pig (gp)IFN-Alpha1 AS directly to the sites of virus infection. With this system, we verified that gpIFN-Alpha1 AS functional domains modulated the expression levels of gpIFN-Alpha1 AS/mRNA, resulting in inhibition of viral proliferation in the respiratory tract of influenza A virus-infected animals.
PLGA (lactide:glycolide = 75:25, MW = 20,000) and polyvinylalcohol (PVA; Gohsenol EG05) were purchased from Wako Pure Chemical Industries (Osaka, Japan) and Nippon Synthetic Chemical Industry Co. (Tokyo, Japan), respectively. Chitosan (GH-400EF) was obtained from NOF Corporation (Tokyo, Japan). All other chemicals were obtained commercially and were of analytical grade.
The rodent-adapted PR/8 virus, influenza virus A/Puerto Rico/8/34 (A/PR/8/34, H1N1) (25) was propagated in the allantoic cavity of 10-day-old embryonated hen eggs as previously described (18). The viral titers were measured with a plaque-forming assay using MDCK cells.
To silence the expression of gpIFN-Alpha1 AS, 2.9 µg, (an amount previously optimized for both human and guinea pig cells (18)), of each phosphorothioate-modified sense oligodeoxynucleotide (seODN) (Eurofins genomics, Tokyo, Japan) was added to 104C1 cells and magnet-assisted transfection (MATra; IBA, Goettingen, Germany) was performed as described previously (18). pSV-Beta-galactosidase control vector (Promega, Madison, WI, USA) (0.1 µg) was included in each transfection mixture to normalize transfection efficiencies. After incubation for 6 h at 37°C, transfected cells were infected with the PR/8 virus at a multiplicity of infection of 25.5 as previously described (18). After further incubation at 37°C for 0-48 h, infected cells were harvested to examine gpIFNA1 mRNA/AS as described below or to measure Beta-galactosidase activities according to the manufacturer’s instructions (Beta-Glo assay system; Promega). The expression levels of IFNA1 mRNA/AS steadily increased in both Namalwa lymphocytes and 104C1 cells for the first 24 h after viral infections (18); therefore, 104C1 cells were collected for RNA analysis at 0, 24, 36 and 48 h after viral infection. For the Beta-galactosidase analysis, the cells were collected at 48 h after virus infection. The seODNs were designed according to the gpIFNA1 mRNA sequence and included loops that might cross-hybridize with the loops of the IFN-Alpha1 AS, as previously described (18). The names and sequences of seODNs are listed in Table 2.
asORNs (Gene-Design, Osaka, Japan) were employed to introduce the functional domain-bearing fragment of gpIFN-Alpha1 AS into 104C1 cells as described above or into PR/8 virus-infected guinea pigs airways as described below. RNA samples were collected for subsequent RT-qPCR analysis as described above. The asORNs were designed to avoid Toll-like receptor (TLR)7/8-mediated immune effects by excluding the GU-rich motifs (26) and contained 2′-O- methylation modifications at each nucleotide position (27).
Their sequences and names are as follows (an M indicates 2′-O-methylation and an asterisk indicates a phosphorothioate bond) (Table 1).
Total RNA was isolated from 104C1 cells as described previously (18) or from airway organs as described below. Strand-specific RT-qPCR was performed essentially as described previously (18). The copy numbers of gpIFNA1 mRNA/AS were normalized to gpBeta-actin mRNA and determined by the regression lines from a set of standards as described previously (18). The names and sequences employed for the analysis were essentially the same as those described previously (18).
PLGA-asORN were prepared by the emulsion solvent diffusion method in water as described previously (28). Briefly, PLGA was dissolved in an acetone/ethanol mixture and asORN3 or ncasORN solution was added to the PLGA solution. The resulting organic solutions were poured into an aqueous PVA solution containing chitosan and moderately stirred using a propeller-type agitator. The mixtures were agitated under reduced pressure to evaporate organic solvent, and asORN3 or ncasORN solution was further added to the resulting PLGA-asORN suspension to create PLGA-asORN3 and PLGA–ncasORN, respectively, before freeze-drying.
For PLGA-asORN transfection, 3 or 6 µg of asORN3 or ncasORN, contained in PLGA nanoparticles at the various percentages ranging from 1.46 - 2.26% (w/w), were resuspended in RNase-free water, vortexed, and sonicated at 4°C. The PLGA-asORNs were then added to 104C1 cells, which were then incubated in RPMI-1640 medium without fetal calf serum (FCS) for 4 hours. The culture medium was then diluted twice with 2 × concentrated RPMI-1640 containing 20% FCS, and the transfected cells were incubated at 37°C for an additional 2 hours prior to PR/8 virus infection.
Hartley guinea pigs (female, 4-weeks-old) were purchased from Shimizu Laboratory Supplies (Kyoto, Japan). During challenge studies, animals were housed in the animal facility at the Research Center for Animal Life Science, Shiga University of Medical Science (Shiga, Japan) according to an Institutional Care and Use Committee-approved protocol. Groups of four guinea pigs were anesthetized by intraperitoneal injection of pentobarbital sodium salt (30 mg/kg body weight) (Kyoritsu Seiyaku Corporation, Tokyo, Japan). They were subsequently inoculated intranasally with a non-lethal dose (106 plaque forming units (pfu)) of PR/8 virus (29) by instillation of 100 µl diluted virus into one nostril. For evaluation of PLGA-asORN3 effects on PR/8 virus proliferation, guinea pigs were pretreated with 100 µl PLGA-asORN3 or -ncasORN suspension (3% w/v) as described above at 6 h prior to the PR/8 virus infection. Some animals were given another inoculation of PLGA-asORN3 suspension at 18 h after the virus infection. Nasal and tracheal washes were collected from the anesthetized animals at various intervals after virus infection to assess the effects of PLGA-asORN3 on the PR/8 virus proliferation profile in the upper and lower respiratory pathways. The guinea pigs were monitored at the same intervals for changes in weight and rectal temperature to assess PLGA nanoparticle-induced morbidity. To determine the effects of PLGA-asORN3 on gpIFNA1 mRNA/AS expression levels, animals were humanely euthanized by intraperitoneal injection of pentobarbital sodium salt (200 mg/kg of body weight) and the larynx and trachea tissues were harvested for strand-specific RT-qPCR RNA analyses.
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) and AF508792 (Cavia porcellus ACTB).
We previously reported that IFN-Alpha1 AS, a natural antisense RNA, is involved in determining post-transcriptional IFNA1 expression (18). IFN-Alpha1 AS rapidly and reversibly upregulates IFNA1 mRNA levels upon infection of human Namalwa B lymphocytes with Sendai virus and following infection of guinea pig 104C1 cells with PR/8 virus. The cytoplasmic sense-antisense interaction between the complementary transcripts resulted in stabilization of IFNA1 mRNA. Alternatively, IFN-Alpha1 AS functions as a ceRNA to prevent miR-1270 from acting on IFNA1 mRNA (16). These regulatory mechanisms play a pivotal role in determining the biological outcomes of type I IFN responses and whether or not pathogens are cleared effectively.
In the accompanying report (20), we confirmed identification of gpIFNA1 and characterized its anti-viral function against PR/8 virus or EMCV infection. These results enabled us to investigate the in vivo IFN response against viral infection by employing the guinea pig as a model animal. In the present study, we performed a POC experiment to confirm that the NAT-mRNA regulatory axis exerts control over innate immunity in vivo.
To explore this concept and to provide a possible platform for therapeutic intervention, we first planned to determine the functional domains of gpIFN-Alpha1 AS that are required for stabilizing gpIFNA1 mRNA.
We therefore employed the seODN-mapping strategy (18) to analyze changes in mRNA levels after silencing gpIFN-Alpha1 AS expression. This silencing strategy is based on the sequence-specific binding of a seODN to the target AS RNA. The formation of an RNA-DNA hybrid then leads to destruction of the AS RNA by RNase H (18), indicating the functional domains of gpIFN-Alpha1 AS that recognize gpIFNA1 mRNA and that are necessary for stabilizing the mRNA (18). These data informed the POC experiment using the guinea pig model.
An RNA folding study by energy minimization using nearest neighbor energy parameters (30), indicated that most of the ten most likely theoretical secondary structures of gpIFNA1 mRNA (data not shown) possess a common local domain structure (Figure 1A). We therefore designed seODNs, S2 to S6, whose sequences were taken from the single-stranded ‘bubble’ or ‘loop’ region of the common structure (Figure 1A). The seODNs were then tested for their ability to destroy endogenous gpIFN-Alpha1 AS. We did not include a negative control seODN at this stage, which could be designed from the double-stranded region of the common structure. We learned from the previous study (18) that not all seODNs designed from the single-stranded regions could recognize gpIFN-Alpha1 AS, and can therefore be employed as a negative control seODN.
Silencing of gpIFN-Alpha1 AS by seODNs. (A) Location of seODNs S2-6 (black bold lines) are depicted along the common stem-loop structure present in the ten most predicted secondary structures of the gpIFNA1 mRNA. (B) The effects of S2-6 on the constitutive expression levels of gpIFN-Alpha1 AS RNA were examined by strand-specific RT-PCR (top) and agarose gel electrophoresis (bottom). gpBeta-actin mRNA indicates an internal RNA standard. Values of three independent experiments are presented as the mean ± s.e.m. of three or four samples. Mock denotes mock-transfected. RT-minus shows no contamination of genomic DNA in the total cellular RNA samples examined.
As shown in Figure 1B, S2, S4 and S6 seODNs silenced the constitutive expression of gpIFN-Alpha1 AS, while S3 and S5 failed to silence the AS RNA (Figure 1B, compare S3 and S5 with mock). The silencing effects by S2, 4, and 6 were specific because gpBeta-actin mRNA levels were unaffected. We thus selected S4 to examine the effects of silencing gpIFN-Alpha1 AS on gpIFNA1 mRNA levels. S3 seODN was employed as a tentative negative control for gpIFN-Alpha1 AS silencing.
104C1 cells were then transfected with either S4 or S3. As expected, S4-transfected cells had reduced gpIFN-Alpha1 AS expression at time 0; i.e. S4 reduced the constitutive expression levels of gpIFN-Alpha1 AS (Figure 2A top, compare S4 with S3 or mock gpIFN-Alpha1 AS copy number at 0 h) but neither S3-transfected nor mock-transfected cells showed this effect. Following PR/8 virus infection, expression was restored, reaching maximal levels 36 h after infection with approximately 4-fold higher expression in S4 cells than in S3 or mock cells. While mock cells then largely maintained gpIFN-Alpha1 AS expression levels, S4 cells showed a rapid reduction in AS copy numbers, of 76% at 48 h after PR/8 virus infection. These expression profiles of gpIFN-Alpha1 AS in 104C1 cells were very similar to that of human IFN-Alpha1 AS when its constitutive expression was silenced by a seODN and the transfected Namalwa cells were infected with SeV (18). The S3 seODN, however, unexpectedly increased gpIFN-Alpha1 AS copy number by approximately 3-fold, 48 h after PR/8 virus infection compared with at 36 h.
(A) Effect of silencing gpIFN-Alpha1 AS RNA on gpIFNA1 mRNA expression. S3 (filled circle with broken line), S4 (open box with bold line) seODNs- or mock (open circle with bold line)-transfected 104C1 cells were subjected to PR/8 virus infection as described in the Materials and methods (S3, S4 and mock, respectively). RNA samples were collected at the indicated time points after viral infection and the copy numbers of gpIFN-Alpha1 AS RNA (top) and gpIFNA1 mRNA (bottom) were determined. The results are presented as “Number of gpIFN-Alpha1 AS RNA or mRNA copies/100 ng of total cellular RNA” ± s.e.m. of three or four samples. Representative values of three independent experiments are presented. Error bars cannot be seen if they are smaller than the graph symbols. (B) Effect of asORNs on gpIFN-Alpha1 AS RNA and gpIFNA1 mRNA expression levels. asORN3 (filled circle with broken line), asORN4 (open box with bold line)- or ncasORN (open circle with bold line)-transfected 104C1 cells were subjected to PR/8 virus infection as described in the legend to (A) (asORN3, 4 and ncasORN, respectively). RNA samples were collected at the indicated time points after viral infection and the copy numbers of gpIFN-Alpha1 AS RNA (top) and gpIFNA1 mRNA (bottom) were determined as described in the legend to (A). The results are presented as “Number of gpIFN-Alpha1 AS RNA or mRNA copies/100 ng of total cellular RNA” ± s.e.m. of three or four samples. Representative values of three independent experiments are presented. Error bars cannot be seen if they are smaller than the graph symbols.
The time-course of gpIFNA1 mRNA expression in S4-, S3- and mock-transfected cells was very similar to that of AS RNA (Figure 2A, bottom). While mock cells largely maintained similar mRNA copy numbers after reaching a maximal level at 36 h after virus infection, S4 cells rapidly reduced the expression of IFNA1 mRNA from the peak level at 36 h, by 64% at 48 h after PR/8 virus infection. However, as observed for gpIFN-Alpha1 AS expression, S3 also increased gpIFNA1 mRNA expression by 20% at 48 h after infection.
Neither S4 nor S3 seODNs encompass nonmethylated CpG motifs, the ligand recognized by TLR9 (26, 31), and no increase in gpIFNA1 mRNA levels was observed in S4- or S3-transfected cells at time 0 after infection; i.e. at 6 h after transfection (Figure 1A bottom, compare S4 or S3 with mock). The subsequent increase in expression levels of gpIFNA1 mRNA in the transfected cells precluded the possibility that S4 or S3 induced the gpIFN response by activation of TLR9 but that the response was caused following PR/8 virus infection.
The above results indicated that by analogy with silencing human IFN-Alpha1 AS (18), the S4 seODN could represent a recognition site of gpIFN-Alpha1 AS in the mRNA. We, therefore, further investigated gpIFN-Alpha1 AS function on mRNA expression, by over-expressing the mRNA recognition site of gpIFN-Alpha1 AS. We designed antisense 2′-O-methyl oligoribonucleotide 4 (asORN4) with phosphorothioate bonds from the nucleotide sequence of S4 seODN. AsORN3 was also designed and included in the over-expression experiment because S3 showed a positive effect on gpIFNA1 mRNA expression at the later stage of the silencing experiment.
As a negative control for the experiment, the nucleotide sequence of S3 seODN (gpIFNA1, nt 252 to 270) was reversed to create ncasORN (see Figure 1A). The following BLAT search (http://smithlabdb.usc.edu/cgi-bin/hgBlat?command) of ncasORN showed no off-targets present in the guinea pig genome (data not shown). AsORN4/3, ncasORN and seODNs were designed to exclude GU-rich motifs (26) to avoid TLR7/8-mediated IFN-Alpha and TNF-Alpha production.
104C1 cells were then transfected with asORNs or ncasORN. Six hours after transfection, cells were infected with PR/8 virus and subjected to analysis by strand-specific RT-qPCR. AsORN4 cells showed that gpIFNA1 mRNA levels reached 4.7-fold higher levels compared with those expressed in ncasORN cells at 24 h after PR/8 virus infection (Figure 2B, bottom). As expected from the previous experiments investigating the asORN effects on human IFNA1 mRNA levels in Namalwa B lymphocytes (18), asORN4 transfection did not change the levels of gpIFN-Alpha1 AS compared with those in ncasORN-transfected cells (Figure 2B, top). Neither asORNs nor ncasORN had any effect on gpBeta-actin expression (data not shown).
In contrast, asORN3 exerted unexpected effects on both gpIFN-Alpha1 AS and gpIFNA1 mRNA expression levels. After transfection of asORN3, gpIFN-Alpha1 AS maintained higher expression levels throughout the experiment compared with those induced by asORN4, reaching maximal levels 36 h after infection with approximately 1.7-fold higher expression compared with asORN4 or ncasORN cells (Figure 2B, top).
asORN3 and asORN4 produced similar expression levels of gpIFNA1 mRNA at 24 h after viral infection, but the mRNA levels were further raised by asORN3, reaching maximal levels 36 h after virus infection, similar to gpIFN-Alpha1 AS attaining the maximal level at the same time point. The highest expression levels of gpIFNA1 mRNA in asORN3 cells were approximately 65% higher than those in asORN4 cells (Figure 2B, bottom).
The increase of gpIFNA1 mRNA levels was not caused by aberrant RIG-I (retinoic acid-inducible gene-I) recognition of the asORNs, which are chemically synthesized 5′OH ssRNAs, or 5′OH dsRNAs formed between asORN4 or asORN3 and recognition sites of gpIFN-Alpha1 AS in the mRNA, because these molecules lack the 5′ triphosphate structure, a hallmark of viral RNAs and a prerequisite for RIG-I recognition (18, 32, 33).
The expression levels of both gpIFN-Alpha1 AS and gpIFNA1 mRNA were higher in asORN3-transfected cells than in asORN4-transfected cells; therefore, asORN3 was selected for the following POC experiments using the guinea pig as the animal model.
To investigate whether the in vitro effects of asORN3 on the levels of gpIFN-Alpha1 AS/mRNA expression similarly modulate the AS and mRNA levels in vivo, thereby, inhibiting PR/8 virus proliferation in the guinea pig model, we selected the PLGA nanoparticle, a biodegradable polymeric nanoparticle (34) (uptake of 400 nm diameter PLGA by A549 cells: Vmax and Km values were 39.8.4 µg/mg cell protein and 44 µg/ml, respectively (23)), as a drug carrier for pulmonary administration of asORN (23).
PLGA was successfully employed to transfect pig arteries in vivo with plasmid DNAs (35) and to deliver miRNA mimics in a biomaterial implant model (36). Importantly, the expression of inflammatory and IFN response markers was not increased by PLGA (37).
Prior to the in vivo experiments, we conducted a series of preparatory transfection experiments using various lots of PLGA-asORN3 with PR/8 virus-infected 104C1 cells. PLGA nanoparticles with various surface charges (indicated by zeta-potential) and particle sizes (diameter) were synthesized to contain a range of asORN3 contents. They were then tested to optimize the amount of asORN3 and the time for transfection to maximize the expression levels of gpIFN-Alpha1 AS/mRNA.
Our previous experiments (22, 27) showed that decreasing the diameter to submicron levels and positive zeta potentials based on the chitosan-dependent protonation of the PLGA amino group increased the cellular uptake of PLGA nanoparticles; therefore, the initial experiment was performed using PLGA-asORN3 with a mean diameter of 157 nm and a zeta potential of +41 mV.
104C1 cells were transfected with 3 µg asORN3 by either the PLGA-asORN3 (1.3 w/w %) or the MATra method (see Materials and methods for the transfection control). The expression levels of gpIFNA1 mRNA were then compared between the two methods. As expected from the results shown in Figure 2B, PLGA-asORN3 reproduced a time-dependent increase of gpIFNA1 mRNA levels (Figure 3). The mRNA levels in PLGA-asORN3 cells were approximately 4-fold higher at 36 h after PR/8 virus infection compared with those observed in cells receiving virus infection alone or empty PLGA-transfection followed by virus infection (Figure 3, PR/8 virus and Vehicle).
Preparatory experiments to optimize PLGA-dependent transfection efficiency in 104C1 cells. 104C1 cells were transfected with asORN3 (filled bar), ncasORN (open bar) or vehicle control (PLGA alone; light grey bar) by the PLGA or MATra methods, and the transfected cells were subjected to PR/8 virus infection, as described in the Materials and methods. Cells receiving the virus infection alone are indicated by dark grey bars. Transfections with 2.9 µg asORN3 or ncasORN as well as 0.1 µg pSV-Beta-galactosidase control vector were performed. RNA samples were collected at 24 or 36 h after viral infection and the copy numbers of gpIFNA1 mRNA were determined as described in the Materials and methods. The results are presented as “gpIFNA1 mRNA copies/100 ng of total cellular RNA” ± s.e.m. of three or four samples. Representative values of three independent experiments are presented.
In contrast, the PLGA-ncasORN cells at the same time point showed approximately 70% higher levels of gpIFNA1 mRNA expression compared with those shown by the control cells, which were considerably lower (approximately 60%) than the levels expressed in the PLGA-asORN3 cells. However, the transfection efficiencies of PLGA-asORN3 were still lower than those obtained by the MATra method (Figure 3, compare filled bars at 36 h between PLGA and MATra).
To surpass the levels of gpIFN-Alpha1 AS/mRNA expressed by the MATra method, we tested the following two lots of PLGA-asORN3; lot A: 78 nm, +30 mV and 1.46 w/w % and lot B: 108 nm, +38 mV and 2.10 w/w %. The corresponding PLGA-ncasORN had mean diameters of 87 or 85 nm, zeta potentials of +28 mV each and 1.48 or 2.26 w/w % contents, respectively.
As shown in Figure 4, when 3 µg of asORN3 was transfected using lot A, PLGA-asORN3 generated 150% and 163% higher levels of gpIFN-Alpha1 AS and mRNA, respectively, compared with those obtained by the MATra method. In contrast, the corresponding PLGA-ncasORN showed approximately 46% lower levels of gpIFN-Alpha1 AS and mRNA compared with PLGA-asORN3. Six micrograms of lot A and both 3 and 6 µg of lot B did not surpass the expression levels of gpIFN-Alpha1 AS and mRNA by MATra. Based on these results, we concluded that PLGA-asORN3 of 78 nm, +30 mV and 1.46 w/w% and PLGA-ncasORN of 87 nm, +28 mV and 1.48 w/w% were applicable for the following in vivo POC experiment.
Optimization of PLGA-dependent transfection efficiencies of asORN3 to maximize gpIFN-Alpha1 AS RNA/mRNA expression levels in 104C1 cells. 104C1 cells were transfected with asORN3 (filled bar) or ncasORN (open bar) by the PLGA or MATra method, and the transfected cells were subjected to PR/8 virus infection, as described in the legend to Figure 2A. For PLGA-dependent transfection, 3 or 6 µg asORN3 or ncasORN, contained at various percentages (w/w) in PLGA nanoparticles (as described below the x axis of the figure) were employed, whereas 3 µg was used for MATra-associated transfection. RNA samples were collected at 36 h after viral infection and the copy numbers of gpIFN-Alpha1 AS RNA (top) and gpIFNA1 mRNA (bottom) were determined as described in the legend to Figure 2A. The results are presented as “Number of gpIFN-Alpha1 AS RNA or mRNA copies/100 ng of total cellular RNA” ± s.e.m. of three or four samples. Representative values of three independent experiments are presented. According to the manufacturer’s instructions, the amount of nucleotides for MATra-transfection was limited to 3 µg.
To investigate whether asORN3 raises gpIFN-Alpha1 AS and mRNA levels in the larynx and trachea tissues of influenza virus-infected guinea pigs, thereby inhibiting viral proliferation, guinea pigs received a single inoculation of PLGA-asORN3 (3 µg of asORN3) (Figure 5A, PLGA-asORN3/1). After 6 h the guinea pigs were challenged with a non-lethal dose of PR/8 virus (106 pfu/animal). Half of the guinea pigs received an additional 3 µg of asORN3 18 h after the virus infection (Figure 5A, PLGA-asORN3/1+1).
In vivo evaluation of asORN3 against respiratory PR/8 virus infection. (A) Effect of nasally administered asORN3 on gpIFN-Alpha1 AS RNA and gpIFNA1 mRNA expression levels. PLGA-asORN3 (closed bar) or –ncasORN (open bar) was nasally administered to groups of guinea pigs (four animals per group) at -6 hours prior to PR/8 virus infection. Half of the guinea pigs that received PLGA-asORN3 was further administered PLGA-asORN3 (grey bar) at 18 hours after virus infection. The larynx and trachea tissues were collected at various time points ranging from 0 h to 48 h after virus infection. The tissue samples were subsequently subjected to strand-specific RT-qPCR analyses for gpIFN-Alpha1 AS RNA (top) and gpIFNA1 mRNA (bottom) expression levels. The results are presented as “Mean copy number of gpIFN-Alpha1 AS RNA or mRNA/100 ng of total cellular RNA” ± s.e.m. of four samples. Representative values of three independent experiments are presented. Error bars cannot be seen if they are smaller than the graph symbols. (B) Effect of nasally administered asORN3 on PR/8 virus proliferation profile. PLGA-asORN3 (closed bar or grey bar) or PLGA-ncasORN (open bar) was nasally administered, as described in the Materials and methods. The nasal and tracheal washes were collected at various time points as described in the legend to (A) and were subjected to PR/8 virus titration to monitor the viral proliferation profile in the upper and lower respiratory pathways. The results are presented as “Mean titers (×105 pfu/ml)” ± s.e.m. of four samples. Representative values of three independent experiments are presented.
As expected from the in vitro data (Figure 2B), quantification of gpIFN-Alpha1 AS showed that the nasal administration of asORN3 increased the basal level of AS RNA expressed in both the upper and lower respiratory tracts. While the following viral challenge increased the gpIFN-Alpha1 AS expression in both the asORN3- and ncasORN-treated animals, the AS RNA levels in the asORN3-treated animals were 2.0-fold to 4.9-fold higher between time points 24 and 48 h post-PR/8 virus infection compared with those observed in the negative control, ncasORN-treated animals (Figure 5A top, PLGA-asORN3/1 and PLGA-ncasORN). The additional inoculation of asORN3 at 18 h post-infection further increased the AS RNA levels 3.6-fold to 6.1-fold at the same time points (24 – 48 h) post-infection compared with those obtained by ncasORN (Figure 5A top, PLGA-asORN3/1+1 and PLGA-ncasORN).
The time-course of gpIFNA1 mRNA expression in the asORN3-treated guinea pigs was similar to that of gpIFN-Alpha1 AS. The initial asORN3 inoculation raised the levels of gpIFNA1 mRNA from 4.9-fold to a more modest 4.0-fold at time points between 24 and 48 h post-infection compared with the negative control animals (Figure 5A bottom, PLGA-asORN3/1 and PLGA-ncasORN). Additional asORN3 inoculation further increased gpIFNA1 mRNA levels 8.3-fold to 4.8-fold between 24 and 48 h post-infection compared with levels obtained by ncasORN (Figure 5A bottom, PLGA-asORN3/1+1 and PLGA-ncasORN). These results thus successfully indicated that, as shown for asORN3-transfected 104C1 cells, nasally administered PLGA-asORN3 reproduced the oligoribonucleotide-effects on gpIFN-Alpha1 AS/mRNA expression levels in the respiratory tracts of PR/8 virus-infected guinea pigs.
We then investigated whether or not the increased gpIFNA1 mRNA levels were capable of reducing PR/8 virus titers in the asORN3-treated animals. Although PR/8 virus infection induced basal levels of gpIFNA1 mRNA expression in PLGA-ncasORN-treated animals, this level of innate immune response was not enough to limit viral replication in the upper respiratory tract, resulting in increased viral titers in the respiratory washes up to 36 h post-infection (Figure 5B). In contrast, PLGA-asORN3/1 treatment significantly reduced the viral titers in a time-dependent manner by 12% (24 h) to 53% (48 h; p < 0.01) at the time points after virus infection indicated within the brackets, respectively.
The additional administration of asORN3 at 18 h post-infection caused further reduction of the viral titers by 23% (p < 0.05) at 24 h, 40% (p < 0.01) at 36 h, and 67% (p < 0.01) at 48 h after virus infection (Figure 5B, PLGA-asORN3/1+1). Neither PR/8 virus infection nor nasal administration of PLGA nanoparticles positively or negatively affected the clinical course of the infection as indicated by comparable levels of both rectal temperature and body weight of animals receiving PLGA-asORNs and virus infection (Figure 6).
The nasal inoculation of PLGA nanoparticles produced no differences in rectal temperature or body weight between guinea pigs administered asORN3 (closed circle) or ncasORN (open circle) once and guinea pigs administered asORN3 twice (open box). The rectal temperature (top) and body weight (bottom) were monitored at -6 h prior to PR8 virus infection and at 0, 24, 36 and 48 h after the viral infection.
To demonstrate, in a proof-of-concept experiment, that asORNs designed from gpIFN-Alpha1 AS functional domains for gpIFNA1 mRNA, increase mRNA levels, and thereby, induce anti-viral effects in vivo, we developed an animal model system that allowed us to investigate the effects of asORNs on the levels of gpIFN-Alpha1 AS and gpIFNA1 mRNA in the respiratory tract of PR/8 virus-infected animals.
Standard laboratory mouse strains, such as BALB/c and C57BL/6, lack functional Mx1, the mouse homolog of human MX1 (38, 39); therefore, we selected the guinea pig as a model animal, which encodes this important antiviral effector in the type I IFN pathway (21, 29).
In the accompanying study (20) together with our previous report (18), we identified gpIFNA1 and characterized its anti-viral function. gpIFNA1 mRNA was expressed with gpIFN-Alpha1 AS in a concordant manner upon PR/8 virus infection in 104C1 cells; therefore, these expression profiles suggested that there is concordant regulation in the expression of gpIFN-Alpha1 AS/mRNA, similar to the observations in Sendai virus-infected human Namalwa lymphocytes (18).
We therefore performed an seODN-oriented mapping of the gpIFNA1 mRNA domains that interact with gpIFN-Alpha1 AS. The subsequent transfection of asORN3/4, representing the gpIFN-Alpha1 AS domains that interact with the mRNA, confirmed in vitro their effects on raising the expression levels of gpIFNA1 mRNA/AS. We then selected PLGA nanoparticles as the drug carrier and determined the optimum packaging conditions of asORN3, which enabled the pulmonary administration of the asORN to the guinea pig model animal. PLGA-asORN3 was then employed to investigate whether the in vitro effects of asORNs on the expression levels of gpIFNA1 mRNA/AS were similar in vivo.
Using this experimental strategy, we demonstrated that a functional domain of gpIFN-Alpha1 AS promoted gpIFNA1 mRNA expression in the respiratory tract of PR/8 virus-infected guinea pigs. This POC experiment thus provided evidence that the natural antisense RNA-mRNA regulatory network we reported previously (16, 18) modulated innate immunity, thereby causing inhibition of PR/8 virus proliferation in vivo.
ncRNAs, which do not contain information for the synthesis of specific proteins were predicted in a review in 1965 (40). Today, ncRNAs are gaining increasing recognition because of the development of comprehensive methods that can rapidly analyze entire transcriptomes (10). The lncRNAs are a group of these ncRNAs and 120,353 are currently annotated in the human genome (LNCipedia.org v 5.0.; https://lncipedia.org) (41). Most of them are only predicted by computational analysis, and for the majority of them no function has been attributed. The challenge is, therefore, not only to confirm putative lncRNAs, but also to unveil their functional roles in vitro and in vivo (42).
We have identified and characterized human IFN-Alpha1 AS, a NAT and a lncRNA, which plays a critical role in the post-transcriptional regulation of IFNA1, and subsequently IFN-Alpha protein production (18). A ~ 4 kb, spliced IFN-Alpha1 AS recognizes a single-stranded region, termed the bulged-stem loop (BSL) within a conserved secondary structure element formed in the IFNA1 mRNA-coding region. Upon recognition of the BSL region, the cytoplasmic sense-antisense interaction results in stabilization of the IFNΑ1 mRNA. This effect was verified by transfection of an asORN, which was designed from the IFN-Alpha 1 AS domain that interacts with the BSL of IFNA1 mRNA. The asORN raised the levels of IFNA1 mRNA expression 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 (18).
Furthermore, IFN-Alpha1 AS has another synergistic role in the post-transcriptional regulation of human IFNA1 mRNA stability. IFN-Alpha1 AS shares the MRE-1270s with IFNA1 mRNA. Interactions between IFN-Alpha1 AS and miR-1270 through the response element titrated away the microRNA from IFNA1 mRNA, indicating that the AS acts as an endogenous decoy or ceRNA for IFNA1 mRNA. It followed that an asORN that shares the MRE-1270 sequence, silenced endogenous miR-1270, causing de-repression of not only IFN-Alpha1 AS expression but also IFNA1 mRNA expression (16).
Similar to the human asORN bearing MRE-1270, guinea pig asORN3 also raised the levels of both gpIFN-Alpha1 AS/mRNA expression, suggesting the possibility that the IFN-Alpha1 AS could promote gpIFNA1 mRNA expression by inhibiting miR-induced mRNA decay. We, therefore, examined the possibility that asORN3 may act as a decoy to sponge a guinea pig miR (s), leading to the increase of both IFN-Alpha1 AS and mRNA expression levels.
There is no miR database available for Cavia porcellus; therefore, we searched for any possible miRs from Cricetulus griseus, Mus musculus and Rattus norvegicus (downloaded from miRBase; http:www.mirbase.org/cgi-bin/browse.pl) that might cross-target asORN3 as a MRE. Following bioinformatic analyses of the miR databases (RegRNA2.0.; http://regrna2.mbe.nctu.edu.tw/; (43), RNA hybrid in BiBiserv; http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/; (44)), the predicted mmu-miR-5617-3p could recognize the asORN3 sequence as a MRE (Table 3).
A subsequent homology search of the guinea pig genome by BLAST (https://www.ensembl.org/index.html) revealed that scaffold_99: 2408710-2408695 and scaffold_25: 9297802-92978171 shared the seed sequence of mmu-miR-56171-3p, which could target the asORN3 sequence region of gpIFN-Alpha1 AS (Table 4) as well as a region of gpIFNA1 mRNA (Table 5).
asORN3 encodes a probable 6-mer MRE site (45) (see also Table 4); therefore, increased levels of gpIFN-Alpha1 AS and mRNA expression in the asORN3 cells strongly suggested that transfection of asORN3 resulted in potent antagonism of the predicted miRs, leading to the specific de-repression of both gpIFN-Alpha1 AS and gpIFNA1 mRNA expression.
Furthermore, increased expression levels of gpIFNA1 mRNA but not gpIFN-Alpha1 AS by asORN4, indicated that gpIFN-Alpha1 AS might raise the levels of gpIFNA1 mRNA expression by transient duplex formation between the corresponding domain of the AS and the targeted single-stranded region of gpIFNA1 mRNA in a similar manner to that of human IFN-Alpha1 AS. These findings thus provide the molecular mechanisms of action for guinea pig and human IFN-Alpha1 AS, and indicate the possibility that asORNs from the IFN-Alpha1 AS functional domains could be employed for prophylactic and/or therapeutic use to treat the influenza virus infections by modulating IFNA1 mRNA expression levels, resulting in a type I IFN response against viral infection.
As recently reported by Coch and colleagues (46), type I IFNs are required and sufficient for the treatment of influenza virus infection. The IFNs are expressed and secreted following the detection of viral components within infected cells. Subsequent binding to cognate receptors on cell surfaces leads to the up-regulation of anti-viral genes or IFN-stimulated genes (ISGs). Several ISGs have direct anti-influenza virus activity, including the Mx family of GTPases, which inhibit nuclear import and/or proliferation of viral nucleocapsids, and the IFN-induced trans-membrane (IFITM) family, (especially IFITM3), which interfere with fusion between viral and endosomal membranes, thereby limiting viral entry (for a review see (47) and references therein).
This highlights the fundamental role of IFNs in efficiently limiting further proliferation and spread of the influenza virus. Because inhibition of viral proliferation by type I IFN occurs in infected cells and in surrounding uninfected cells, the induction of IFN-induced anti-viral activities provides a strong rationale for the use of IFN-Alpha1 AS asORNs, not only for prophylaxis, but also to ameliorate the clinical signs of ongoing influenza virus infection by inhibiting both viral entry and viral proliferation.
Our results support, at least in part, the rationale that the initial administration of asORN3 to guinea pigs prior to PR/8 virus infection elevated both IFN-Alpha1 AS and IFNA1 mRNA expression in the larynx and trachea tissues to higher levels than the control infection even at the time of virus administration and up to 48 h after infection.
The increased RNA levels were associated with significant reductions of viral titers in the upper and lower respiratory tracts in the infected animals up to 48 h after infection. Moreover, the same dose of asORN3 ameliorated the expression of IFN-Alpha1 AS/mRNA and further reduced PR/8 virus titers to a significant degree if administered in the course of the ongoing influenza A virus infection. These results thus indicate that modulation of type I IFN expression could serve as a promising strategy to treat influenza virus infection in both therapeutic and prophylactic settings (48).
In conclusion, we performed a POC experiment to provide evidence that the natural antisense RNA-mRNA regulatory circuitry controls the expression of IFNA1 mRNA, and subsequently IFN-Alpha production, thereby restricting the proliferation of influenza A virus in vivo. Optimization of asORN sequences and administration protocols will help the development of new nucleic acid therapeutics for both prevention and treatment of human influenza virus infection.
Ryou Sakamoto and Shiwen Jiang 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. All applicable international, national, and institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors. We are grateful to Tomoaki Nakano, a former member of the Kimura laboratory, who participated at early stages of the project, Shin-Ichiro Nakamura for providing technical advice for the guinea pig experiments, and to Mikio Nishizawa for critical reading of the manuscript and 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.