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Respiratory syncytial virus non-structural protein 1 facilitates virus replication through miR-29a-mediated inhibition of interferon-α receptor
Biochemical and Biophysical Research Communications xxx (2016) 1e6
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Respiratory syncytial virus non-structural protein 1 facilitates virus replication through miR-29a-mediated inhibition of interferon-a receptor Yao Zhang, Lihua Yang, Hao Wang, Guocheng Zhang*, Xin Sun** Department of Pediatrics, Xijing Hospital of the Fourth Military Medical University, Xi'an 710032, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 August 2016 Accepted 24 August 2016 Available online xxx
Human respiratory syncytial virus (RSV) non-structural protein 1 (NS1) has recently been suggested to inhibit type-I interferon (IFN)-dependent immune responses during RSV infection. However, the precise function of RSV NS1 protein in reducing the antiviral effects of IFNs against RSV is poorly understood. The roles of cellular miRNAs in the defence against RSV infection are not well characterized. In this study, qRT-PCR analysis revealed that miR-29a expression was upregulated in the recombinant wild-type RSV (rRSV-WT) group compared with the control group, but no changes were observed in the recombinant RSV mutant lacking NS1 (rRSV-DNS1) group. Using dual-luciferase reporter assay, we demonstrated that miR-29a could directly target IFNAR1 30 -UTR and downregulate IFNAR1 expression. In addition, RSV NS1 suppressed IFNAR1 expression at both RNA level and protein level in human lung adenocarcinoma cell line A549. RSV plaque assays showed that the number of RSV plaques in miR-29a mimics group was significantly higher than that in miR-29a inhibitor group or miRNA scramble control group. HA-NS1 overexpression increased the numbers of RSV plaque, but the promotive effect on virus replication was attenuated in cells transfected with miR-29a inhibitors. These results suggest that miR-29a, upregulated during RSV infection, is a negative regulator of IFNAR1 and is critical for RSV NS1-induced virus replication. © 2016 Elsevier Inc. All rights reserved.
Keywords: Respiratory syncytial virus Non-structural protein 1 miR-29a Interferon-a receptor Replication
1. Introduction Human respiratory syncytial virus (RSV) is a member of the family Paramyxoviridae, which is the leading cause of severe bronchiolitis or pneumonia among infants and the aged around the world [1]. RSV could infect airway epithelium, which further aggravates inflammation and accelerates the development or progression of airway diseases [2]. There is increasing evidence that RSV proteins, especially the surface proteins on the virion, play crucial roles in promoting RSV infection and replication [3]. Nonstructural (NS) proteins NS1 and NS2 are encoded by RSV genome. NS1 and NS2 have been proved to regulate viral replication and to suppress type I interferon (IFN) production, with NS1 exerting a greater inhibition [4].
* Corresponding author. Department of Pediatrics, Xijing Hospital of the Fourth Military Medical University, 127 Changle West Road, Xi'an 710032, China. ** Corresponding author. Department of Pediatrics, Xijing Hospital of the Fourth Military Medical University, 127 Changle West Road, Xi'an 710032, China. E-mail addresses: [email protected] (G. Zhang), [email protected] (X. Sun).
Type I IFNs, a family of cytokines, function as modulators of immune responses during viral infections directly and/or indirectly by regulating other mediators [5]. In humans and mice, type I IFNs consist of IFN-a and IFN-b which are located on the surface of IFNproducing cells. Type I IFNs exert their antiviral effects through binding to the IFN-a receptor (IFNAR1) and activating receptorassociated tyrosine kinases Jak1 and Tyk2 [6]. IFN-a leads to the activation of signal transducer and activator of transcription 1 (STAT1) and STAT2 through phosphorylating tyrosines in their C-terminal domain [7]. Prestwood et al. have demonstrated that IFN-g receptor was able to reduce systemic levels of Dengue virus. They found that IFN-g receptor exhibited the antiviral action by inhibiting early viral replication in the spleen and bone marrow and thereby restricted dengue virus diffusion [8]. Type I IFNs were implicated in the process of inflammatory injury. IFNAR was required for interleukin (IL)-15 induction in response to vesicular stomatitis virus infection [9]. By using mice with IFNAR knockout or with Treg cell-specific IFNAR deletion, Metidji and colleagues discovered that IFNAR signaling contributes to regulatory T cell development in thymus and improves their function. Moreover, IFNAR deletion in regulatory T cells resulted in an upregulation of
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the pro-apoptotic gene Bim and active caspase [10]. MicroRNAs (miRNAs) are small non-protein-coding RNAs of approximately 20e22 nucleotides that exert regulatory effects in diverse cellular biological processes such as cell cycle, development, differentiation, proliferation, apoptosis, invasion and metastasis, inflammation and immune response [11]. miR-199a-3p and miR-210 have been demonstrated to be upregulated in HepG2 2.2.15 cells in comparison with those in HepG2 cells. Further experiments have confirmed that both of miR-199a-3p and miR-210 suppress HBV replication and controlled the production of virion via directly targeting the transcripts of critical HBV genes [12]. miR221 inhibits RSV replication, prevents the infection and promotes the apoptosis rate in RSV-infected human bronchial epithelium by downregulating NGF expression at transcriptional level [13]. Human RSV NS1 has been proved to inhibit miR-24 expression by enhancing the expression of Kruppel-like factor 6, a transcription factor, which modulates the transforming growth factor b signaling pathway [14]. A microarray-based analysis revealed that the expression level of miR-29a was 1.5-fold higher in RSV-infected A549 cells as compared with the control group, suggesting that miR-29a may be involved in the process of viral replication [15]. Therefore, we speculated that miR-29a-mediated suppression of the IFNAR may be a cause of RSV replication. In the present study, we identified a novel mechanism underlying NS1-induced inhibition of IFNAR synthesis. Our findings revealed a previously unexplained role for miR-29a as a negative regulator of IFNAR expression and elucidated the mechanism by which NS1 contributed to RSV replication. 2. Materials and methods 2.1. Cell maintenance and virus infection The human lung adenocarcinoma cell line A549 and the human laryngeal carcinoma cell line HEp-2 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with GlutaMAX (Gibco), 100 U/mL penicillin (Sigma-Aldrich, Louis, MO, USA), 100 mg/L streptomycin (Sigma-Aldrich) and 10% (vol/vol) heat-inactivated fetal bovine serum (Gibco) at 37 C in a humid 5% CO2 atmosphere. RSV strain A2 (ATCC) was propagated in HEp-2 cells. For virus titration, HEp-2 cells were seeded in 96-well flat-bottom plates (104 cells/well) one day before infection. The infected cells and supernatants were determined by plaque assay [16]. For virus infection, A549 cells (1 105 cells/mL) were seeded in 25 cm2 flask 1 day before infection. Cells were infected with RSV at a multiplicity of infection (MOI) of 0.1, 1 and 5 for 2 h, extensively washed with phosphate-buffered saline (PBS), and then cultured in complete DMEM at 37 C for the indicated times.
IFNAR1 30 -UTR were purchased from Genepharma (GenePharma, Shanghai, China). A549 cells were planted into 6-well flasks 24 h before transfection. The next day, A549 cells were co-transfected with WT or mutant IFNAR1 30 -UTR luciferase reporter plasmid and miR-29a mimics or miRNA scramble control (miR-NC) using Lipofectamine 2000 (Invitrogen). Twenty-four h after transfection, cells were lysed with Passive Lysis Buffer (Promega). Luciferase Assay Buffer was added into a 96-well plate. The activities of luciferases in cell lysates were measured using a dual-luciferase reporter system (Promega) according to the manufacturer's instructions. 2.4. Western blot A549 cells were transfected with NS1 plasmids using Lipofectmin 2000 (Invitrogen) according to the instruction and harvested 48 h after transfection. Cells were gently washed twice with ice-cold PBS and cell pellets were then lysed with ice-cold RIPA lysis buffer (0.5 M TriseHCl, 1.5 M NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.004% sodium azide, pH 7.4) containing a 1 mM of protease inhibitor (Sigma-Aldrich). Equal mass of protein was separated by a SDS-PAGE gel. Total proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) and then blocked with 5% non-fat milk for 1 h at room temprature. The membrane was incubated with a anti-NS1 antibody (Abcam, Cambridge, MA, USA) anti-IFNAR1 antibody (Abcam) or a anti-HA antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 12 h at 4 C. After washing with TBST buffer, blots were probed with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma-Aldrich) for 1 h at room temprature. The HRP labelled antibodies were visualized with an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) and exposed to an X-ray film in a dark room. 2.5. RNA isolation and quantitive RT-PCR Total RNA was isolated from A549 cells using a miRVANA miRNA isolation kit (Ambion, Austin, TX, USA) in accordance with the manufacturer's instructions. Equal amounts of RNA were reversely transcripted into cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacture's protocols. The qRT-PCR assay was performed with Power SYBR Green PCR Master Mix (Applied Biosystems). U6 small nuclear RNA was used as a reference control for miRNA expression. qRT-PCR reactions were conducted using a Real-Time PCR System (ABI 7500, Applied Biosystems). The relative expression of miR-29a was calculated and estimated from triplicate qRTPCR reactions using 2DDCt method. 2.6. Plaque assay
To obtain plasmids pcDNA3-NS1 with HA-tagged, RSV NS1 genes were constructed by amplifying cDNA clones of RSV by PCR using specific primers. The PCR fragments were digested with EcoRI and NotI restriction enzymes (Promega, Madison, WI, USA) and then ligated into the same sites of pcDNA3 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). Plasmids pcDNA3-NS1 were amplified in Escherichia coli TOP10 and then isolated using commercially available kits (Qiagen, Valencia, CA, USA).
RSV titration was measured with plaque formation assay as described in detail elsewhere [17]. In brief, transfected cells were infected with RSV at an MOI of 5 for 2 h. After incubation, cells were overlaid with 1% (wt/vol) methylcellulose containing 50% (vol/vol) 2 DMEM and 2% (vol/vol) FBS and cultured for another 5 days before the overlay medium was removed, and cells were fixed and stained with 2% (wt/vol) crystal violet in 20% (vol/vol) ethanol. Wells containing of 30e100 plaques were counted and the viral titre was calculated using the following formula: virus titre (pfu/ mL) ¼ Plaques dilution 5.
2.3. Dual-luciferase reporter assay
2.7. Statistical analysis
2.2. Construction of plasmids
Luciferase reporter plasmids with wild-type (WT) or mutant
All experiments were repeated at least three times. The SPSS
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17.0 software (SPSS, Inc., Chicago, IL, USA) was used to process experimental data. Statistical comparisons were conducted using a two-tailed Student's t-test or one-way analysis of variance (ANOVA). A P value of less than 0.05 was considered statistically significant. 3. Results 3.1. RSV NS1 enhances miR-29a expression An microarray data analysis has suggested that miR-29a is induced 1.5-fold in RSV-infected A549 cells [15]. To confirm the result, qRT-PCR was performed to measure the expression level of miR-29a in RSV-infected A549 cells. After the infection of RSV for 36 h, miR-29a level was significantly higher in the group at an MOI of 5 compared with the group at an MOI of 1 (Fig. 1A). To identify whether RSV NS1 protein affected miR-29a expression, A549 cells were infected with recombinant RSV (rRSV-WT) or recombinant RSV mutant lacking NS1 (rRSV-DNS1) at an MOI of 5. The expression level of miR-29a was evaluated at 36 h after infection. As shown in Fig. 1B, expression of miR-29a was significantly upregulated in rRSV-WT-infected cells, but in the absence of the NS1 protein gene, expression of miR-29a was unchanged. These results demonstrated that RSV NS1 protein enhanced miR-29a expression. 3.2. IFNAR1 is a direct target gene of miR-29a miR-29a was predicted to have multiple interactions with IFNAR1 mRNA [18]. To conform that miR-29a indeed targeted the 30 -untranslated region (UTR) of IFNAR1 as predicted, a luciferase reporter gene containing IFNAR1 30 -UTR or a control plasmid containing mutant 30 -UTR of IFNAR1 was cotransfected with miR-29a mimics or miRNA scramble control (miR-NC) into A549 cells. The results showed that luciferase activity was significantly decreased in the miR-29a mimics group when compared with the miR-NC group, but no reduction was observed in the mutant 30 -UTR groups (Fig. 2A). To further determine whether IFNAR1 expression was modified by miR-29a, the expression level of IFNAR1 protein was measured by western blot. The expression of miR-29a was significantly increased in the miR-29a mimics group and was significantly decreased in the miR-29a inhibitor group, compared with the miR-NC group (data not shown). We found that miR-29 mimic transfection resulted in a reduction of IFNAR1 protein expression and downregulation of miR-29a elevated the levels of IFNAR1 in A549 cells (Fig. 2B). Therefore, miR-29a could directly target IFNAR1 30 -UTR and downregulate IFNAR1 at protein expression level in human A549 cells.
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3.3. RSV NS1 reduces IFNAR1 expression To confirm the inhibitory role of NS1 in IFNAR1 production, A549 cells were transfected with HA-NS1-expressing plasmid or empty vector control plasmid, or cotransfected with HA-NS1expressing plasmid and miR-29a inhibitor. qRT-PCR was performed to determine the mRNA expression level of IFNAR1. As predicted, there was a significant reduction of IFNAR1 mRNA in the HA-NS1 group when compared with the empty vector controls, but the IFNAR1 mRNA levels in the HA-NS1 þ miR-29a inhibitor group was increased compared with the HA-NS1 group (Fig. 3A). Western blot analysis was done to measure the protein expression level of NS1 and IFNAR1 (Fig. 3B). The results showed that NS1 was overexpressed after transfection with HA-NS1-expressing plasmid or cotransfection with HA-NS1-expressing plasmid and miR-29a inhibitor (Fig. 3C). The protein levels of IFNAR1 in the HA-NS1 group were significantly decreased compared with empty vector group, but the IFNAR1 levels in the HA-NS1 þ miR-29a inhibitor group were significantly increased compared with the HA-NS1 group (Fig. 3D). 3.4. miR-29a promotes virus replication To assess the effect of miR-29a on virus replication, A549 cells were transfected with miR-29a mimics, inhibitor, or miRNA scramble control (miR-NC), followed by infection with RSV at an MOI of 5. qRTPCR was performed to confirm the expression of miR-29a (Fig. 4A). RSV plaque assays showed that the number of RSV plaques in miR-29a mimics group was significantly increased when compared with miR29a inhibitor group or miRNA scramble control group (Fig. 4B). These results indicated that miR-29a contributes to RSV replication. 3.5. Inhibition of miR-29a attenuates the promotive effects of NS1 on RSV replication In order to further elucidate the precise mechanism that NS1 promotes virus replication, A549 cells were cotransfected with empty vector or plasmids expressing HA-NS1 and miR-29a inhibitor or miR-29a inhibitor NC, and then infected (MOI ¼ 5) with WT RSV or recombinant RSV lacking NS1 (DNS1). At 36 h after infection, the viral titers were determined by plaque assay. The RSV plaque assays showed that HA-NS1 overexpression increased the numbers of RSV plaque, but the promotive effect on virus replication was attenuated in presence of miR-29a inhibitor (Fig. 4C). 4. Discussion In recent years, a large amount of studies have been conducted
Fig. 1. RSV infection upregulates the expression of miR-29a in A549 cells. (A) A549 cells were infected with or without RSV at an MOI of 0.1, 1 or 5. qRT-PCR assay was performed to determined the expression levels of miR-29a 6, 12, 18, 24, 36 and 48 h after infection respectively. U6 RNA was used as the internal control in each sample. Data are expressed as the mean ± SD (n ¼ 3). **P < 0.01.
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Fig. 2. IFNAR1 is a target of miR-29a. (A) A549 cells were cotransfected with plasmids containing the WT or mutant IFNAR1 30 -UTR and miR-29a mimics or miRNA scramble control (miR-NC). After culture for 24 h, cells were harvested and then the relative luciferase activity was measured. (B) Western blot was performed to detect the expression level of IFNAR1 in A549 cells transfected with miR-29a mimics, inhibitor, or miRNA scramble control (miR-NC). Data are expressed as the mean ± SD (n ¼ 3). NS, not significant, **P < 0.01, ***P < 0.001.
to explore the mechanism underlying the inhibitory effect of NS1 on host immune responses, particularly the type I IFN synthesis. NS1 protein has also been shown to counteract the IFN-mediated inhibition of virus replication [19]. However, the exact molecular mechanism by which NS1 facilitated RSV replication is not yet elucidated. These experiments found that miR-29a was upregulated in A549 cells infected with RSV. In addition, overexpression of RSV NS1 protein enhanced the expression of miR-29a in A549 cells. We discovered that miR-29a acted as an important regulator of immune response through inhibiting IFNAR1 production and thereby promoting RSV replication. IFN-a plays a vital role in regulating immune responses, angiogenesis, and cell proliferation via directly interacting with IFNAR, its
high-affinity membrane receptor, which ultimately activates STATs [20]. It has been identified that RSV inhibits IFN-a/b signaling pathway by expression of viral NS1 and NS2 in epithelial host cells during infection [21]. An experiment discovered that RSV NS1 colocalized with mitochondrial antiviral signaling protein (MAVS) in A549 human epithelial cells infected with RSV. On the other hand, their study showed that NS1 interacted to MAVS and disrupted the MAVS interaction with retinoic acid inducible gene and thereby indirectly affected IFN production [22]. During virus infections, IFN-a and tumor necrosis factor-alpha (TNF-a) are produced mainly by macrophages and dendritic cells. In order to replicate in the host cells, human RSV NS1 and NS2 attenuate their immune defences by decreasing the production of innate defensive
Fig. 3. Effect of NS1 protein on IFNAR1 expression. A549 cells were transfected with HA-NS1 expressing plasmids or empty vectors, or cotransfected with HA-NS1-expressing plasmid and miR-29a inhibitor. Cells were collected at 48 h after transfection, qRT-PCR (A) and western blot (B) were done to measure the IFNAR1 expression at mRNA level and protein level. (C, D) The blots were quantified by NIH image software and normalized with b-actin. Data are expressed as the mean ± SD (n ¼ 3). NS, not significant, **P < 0.01, ***P < 0.001.
Please cite this article in press as: Y. Zhang, et al., Respiratory syncytial virus non-structural protein 1 facilitates virus replication through miR29a-mediated inhibition of interferon-a receptor, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.08.142
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Fig. 4. Effect of miR-29a on RSV replication. A549 cells were transfected with miR-29a mimics, miR-29a inhibitor or miRNA scramble control (miR-NC), and then infected with RSV at an MOI of 5 for 2 h qRT-PCR was conducted to determine the expression level of miR-29a (A). Three days after infection, cells and supernatants were collected and the number of RSV p.f.u. was calculated using a plaque assay (B). A549 cells were cotransfected with empty vector or plasmids expressing HA-NS1 and miR-29a inhibitor or miR-NC, followed by infection with rRSV-DNS1 or WT RSV at an MOI of 5 for 2 h. After incubation for 3 days, cell-associated RSV and supernatant RSV were collected and the viral titers were determined (C). Data are expressed as the mean ± SD (n ¼ 3). NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.
cytokines including IFN-a/b, IFN-g, nuclear factor-kappa B, TNF-a, IL-1a, and IL-6 [23]. Both NS1a and NS1b, the two proteolytic fragments of NS1, were able to inhibit the activity of TNF-a promoter by abrogating the Sp1 transactivation [24]. NS1 and NS2 of bovine RSV reduce the activity of interferon regulatory factor 3. NS protein-mediated inhibition of IRF-3 and IFN secretion may play vital roles in the pathogenesis and immunogenicity of bovine RSV infection [25]. H5N1 NS1 decreased the phosphorylation levels of STAT1, STAT2 and STAT3 in HeLa cells treated with IFN-b, which is a result of NS1-mediated inhibition of IFNAR1 expression. Moreover, NS1 directly disrupt the IFN signaling pathway to promote viral replication [26]. Our data showed that NS1 overexpression upregulated miR-29a expression level and resulted in a significant reduction of IFNAR1 at both mRNA level and protein level. A large amount of studies have found that miRNAs play a part in antiviral immune responses via direct degradation or translational inhibition of their target mRNAs [27]. miR-29a inhibited protein expression of tristetraprolin, which was implicated in the degradation of mRNAs, and affected oncogenic Ras signaling to result in epithelial-to-mesenchymal transition and metastasis of breast cancer [28]. A study found that human miRNA miR-29a was expressed in human peripheral blood mononuclear cells. miR-29a suppresses the protein expression of Nef by directly targeting its 30 -UTR, thereby interfering with HIV-1 replication [29]. The expression level of IFNAR1 in epithelium lacking miR-29a was approximately 8-fold higher than that in WT epithelium [30]. Here, we demonstrated that IFNAR1 was a target of miR-29a using the dual-luciferase reporter assay system. By using plaque-forming assay, we found that miR-29a was able to suppress RSV replication. To further assess the function of miR-29a in the RSV replication, A549 cells were cotransfected with HA-NS1 overexpressing plasmids or empty vector and miR-29a inhibitor or miR-NC, and then infected with WT RSV or rRSV-DNS1. We found that miR-29a downregulation efficiently interfered with NS1-induced RSV replication, suggesting that NS1 may inhibit RSV replication in a miR-29a-dependent manner. In conclusion, our evidence suggested that RSV NS1 induced upregulation of miR-29a. In addition, miR-29a suppressed the expression of IFNAR1, which is essential for the function of Type I IFNs. The promotive effect of NS1 on RSV replication was attenuated by inhibition of miR-29a expression. These findings demonstrated a new mechanism underlying the inhibitory role of NS1
against RSV-induced IFNAR1 expression. Further studies should be performed to clarify the specific mechanism of NS1-induced inhibition of IFNAR1 during RSV infection. Conflict of interest No conflicts of interest were declared. Acknowledgements This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.08.142. References [1] P.L. Collins, Respiratory syncytial virus and metapneumovirus, in: B.N. Fields, D.M. Knipe, P.M. Howley (Eds.), Fields Virology, Lippincott Williams & Wilkins, Philadelphia, 2007, pp. 1601e1646. [2] R.F. Foronjy, A.J. Dabo, C.C. Taggart, S. Weldon, P. Geraghty, Respiratory syncytial virus infections enhance cigarette smoke induced COPD in mice, PloS One 9 (2014) e90567. [3] E.C. Moore, J. Barber, R.A. Tripp, Respiratory syncytial virus (RSV) attachment and nonstructural proteins modify the type I interferon response associated with suppressor of cytokine signaling (SOCS) proteins and IFN-stimulated gene-15 (ISG15), Virol. J. 5 (2008) 116. [4] K.M. Spann, K.C. Tran, B. Chi, R.L. Rabin, P.L. Collins, Suppression of the induction of alpha, beta, and gamma interferons by the NS1 and NS2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages, J. Virol. 78 (2004) 4363e4369. [5] F. McNab, K. Mayer-Barber, A. Sher, A. Wack, A. O'Garra, Type I interferons in infectious disease, Nat. Rev. Immunol. 15 (2015) 87e103. [6] L.C. Platanias, Mechanisms of type-I-and type-II-interferon-mediated signalling, Nat. Rev. Immunol. 5 (2005) 375e386. [7] M.T. Dill, Z. Makowska, G. Trincucci, A.J. Gruber, J.E. Vogt, M. Filipowicz, D. Calabrese, I. Krol, D.T. Lau, L. Terracciano, Pegylated IFN-a regulates hepatic gene expression through transient Jak/STAT activation, J. Clin. Investig. 124 (2014) 1568e1581. [8] T.R. Prestwood, M.M. Morar, R.M. Zellweger, R. Miller, M.M. May, L.E. Yauch, S.M. Lada, S. Shresta, Gamma interferon (IFN-g) receptor restricts systemic dengue virus replication and prevents paralysis in IFN-a/b receptor-deficient mice, J. Virol. 86 (2012) 12561e12570. [9] S.L. Colpitts, T.A. Stoklasek, C.R. Plumlee, J.J. Obar, C. Guo, L. Lefrançois, Cutting edge: the role of IFN-a receptor and MyD88 signaling in induction of IL-15
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Respiratory syncytial virus non-structural protein 1 facilitates virus replication through miR-29a-mediated inhibition of interferon-a receptor Yao Zhang, Lihua Yang, Hao Wang, Guocheng Zhang*, Xin Sun** Department of Pediatrics, Xijing Hospital of the Fourth Military Medical University, Xi'an 710032, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 August 2016 Accepted 24 August 2016 Available online xxx
Human respiratory syncytial virus (RSV) non-structural protein 1 (NS1) has recently been suggested to inhibit type-I interferon (IFN)-dependent immune responses during RSV infection. However, the precise function of RSV NS1 protein in reducing the antiviral effects of IFNs against RSV is poorly understood. The roles of cellular miRNAs in the defence against RSV infection are not well characterized. In this study, qRT-PCR analysis revealed that miR-29a expression was upregulated in the recombinant wild-type RSV (rRSV-WT) group compared with the control group, but no changes were observed in the recombinant RSV mutant lacking NS1 (rRSV-DNS1) group. Using dual-luciferase reporter assay, we demonstrated that miR-29a could directly target IFNAR1 30 -UTR and downregulate IFNAR1 expression. In addition, RSV NS1 suppressed IFNAR1 expression at both RNA level and protein level in human lung adenocarcinoma cell line A549. RSV plaque assays showed that the number of RSV plaques in miR-29a mimics group was significantly higher than that in miR-29a inhibitor group or miRNA scramble control group. HA-NS1 overexpression increased the numbers of RSV plaque, but the promotive effect on virus replication was attenuated in cells transfected with miR-29a inhibitors. These results suggest that miR-29a, upregulated during RSV infection, is a negative regulator of IFNAR1 and is critical for RSV NS1-induced virus replication. © 2016 Elsevier Inc. All rights reserved.
Keywords: Respiratory syncytial virus Non-structural protein 1 miR-29a Interferon-a receptor Replication
1. Introduction Human respiratory syncytial virus (RSV) is a member of the family Paramyxoviridae, which is the leading cause of severe bronchiolitis or pneumonia among infants and the aged around the world [1]. RSV could infect airway epithelium, which further aggravates inflammation and accelerates the development or progression of airway diseases [2]. There is increasing evidence that RSV proteins, especially the surface proteins on the virion, play crucial roles in promoting RSV infection and replication [3]. Nonstructural (NS) proteins NS1 and NS2 are encoded by RSV genome. NS1 and NS2 have been proved to regulate viral replication and to suppress type I interferon (IFN) production, with NS1 exerting a greater inhibition [4].
* Corresponding author. Department of Pediatrics, Xijing Hospital of the Fourth Military Medical University, 127 Changle West Road, Xi'an 710032, China. ** Corresponding author. Department of Pediatrics, Xijing Hospital of the Fourth Military Medical University, 127 Changle West Road, Xi'an 710032, China. E-mail addresses: [email protected] (G. Zhang), [email protected] (X. Sun).
Type I IFNs, a family of cytokines, function as modulators of immune responses during viral infections directly and/or indirectly by regulating other mediators [5]. In humans and mice, type I IFNs consist of IFN-a and IFN-b which are located on the surface of IFNproducing cells. Type I IFNs exert their antiviral effects through binding to the IFN-a receptor (IFNAR1) and activating receptorassociated tyrosine kinases Jak1 and Tyk2 [6]. IFN-a leads to the activation of signal transducer and activator of transcription 1 (STAT1) and STAT2 through phosphorylating tyrosines in their C-terminal domain [7]. Prestwood et al. have demonstrated that IFN-g receptor was able to reduce systemic levels of Dengue virus. They found that IFN-g receptor exhibited the antiviral action by inhibiting early viral replication in the spleen and bone marrow and thereby restricted dengue virus diffusion [8]. Type I IFNs were implicated in the process of inflammatory injury. IFNAR was required for interleukin (IL)-15 induction in response to vesicular stomatitis virus infection [9]. By using mice with IFNAR knockout or with Treg cell-specific IFNAR deletion, Metidji and colleagues discovered that IFNAR signaling contributes to regulatory T cell development in thymus and improves their function. Moreover, IFNAR deletion in regulatory T cells resulted in an upregulation of
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the pro-apoptotic gene Bim and active caspase [10]. MicroRNAs (miRNAs) are small non-protein-coding RNAs of approximately 20e22 nucleotides that exert regulatory effects in diverse cellular biological processes such as cell cycle, development, differentiation, proliferation, apoptosis, invasion and metastasis, inflammation and immune response [11]. miR-199a-3p and miR-210 have been demonstrated to be upregulated in HepG2 2.2.15 cells in comparison with those in HepG2 cells. Further experiments have confirmed that both of miR-199a-3p and miR-210 suppress HBV replication and controlled the production of virion via directly targeting the transcripts of critical HBV genes [12]. miR221 inhibits RSV replication, prevents the infection and promotes the apoptosis rate in RSV-infected human bronchial epithelium by downregulating NGF expression at transcriptional level [13]. Human RSV NS1 has been proved to inhibit miR-24 expression by enhancing the expression of Kruppel-like factor 6, a transcription factor, which modulates the transforming growth factor b signaling pathway [14]. A microarray-based analysis revealed that the expression level of miR-29a was 1.5-fold higher in RSV-infected A549 cells as compared with the control group, suggesting that miR-29a may be involved in the process of viral replication [15]. Therefore, we speculated that miR-29a-mediated suppression of the IFNAR may be a cause of RSV replication. In the present study, we identified a novel mechanism underlying NS1-induced inhibition of IFNAR synthesis. Our findings revealed a previously unexplained role for miR-29a as a negative regulator of IFNAR expression and elucidated the mechanism by which NS1 contributed to RSV replication. 2. Materials and methods 2.1. Cell maintenance and virus infection The human lung adenocarcinoma cell line A549 and the human laryngeal carcinoma cell line HEp-2 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with GlutaMAX (Gibco), 100 U/mL penicillin (Sigma-Aldrich, Louis, MO, USA), 100 mg/L streptomycin (Sigma-Aldrich) and 10% (vol/vol) heat-inactivated fetal bovine serum (Gibco) at 37 C in a humid 5% CO2 atmosphere. RSV strain A2 (ATCC) was propagated in HEp-2 cells. For virus titration, HEp-2 cells were seeded in 96-well flat-bottom plates (104 cells/well) one day before infection. The infected cells and supernatants were determined by plaque assay [16]. For virus infection, A549 cells (1 105 cells/mL) were seeded in 25 cm2 flask 1 day before infection. Cells were infected with RSV at a multiplicity of infection (MOI) of 0.1, 1 and 5 for 2 h, extensively washed with phosphate-buffered saline (PBS), and then cultured in complete DMEM at 37 C for the indicated times.
IFNAR1 30 -UTR were purchased from Genepharma (GenePharma, Shanghai, China). A549 cells were planted into 6-well flasks 24 h before transfection. The next day, A549 cells were co-transfected with WT or mutant IFNAR1 30 -UTR luciferase reporter plasmid and miR-29a mimics or miRNA scramble control (miR-NC) using Lipofectamine 2000 (Invitrogen). Twenty-four h after transfection, cells were lysed with Passive Lysis Buffer (Promega). Luciferase Assay Buffer was added into a 96-well plate. The activities of luciferases in cell lysates were measured using a dual-luciferase reporter system (Promega) according to the manufacturer's instructions. 2.4. Western blot A549 cells were transfected with NS1 plasmids using Lipofectmin 2000 (Invitrogen) according to the instruction and harvested 48 h after transfection. Cells were gently washed twice with ice-cold PBS and cell pellets were then lysed with ice-cold RIPA lysis buffer (0.5 M TriseHCl, 1.5 M NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.004% sodium azide, pH 7.4) containing a 1 mM of protease inhibitor (Sigma-Aldrich). Equal mass of protein was separated by a SDS-PAGE gel. Total proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) and then blocked with 5% non-fat milk for 1 h at room temprature. The membrane was incubated with a anti-NS1 antibody (Abcam, Cambridge, MA, USA) anti-IFNAR1 antibody (Abcam) or a anti-HA antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 12 h at 4 C. After washing with TBST buffer, blots were probed with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma-Aldrich) for 1 h at room temprature. The HRP labelled antibodies were visualized with an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) and exposed to an X-ray film in a dark room. 2.5. RNA isolation and quantitive RT-PCR Total RNA was isolated from A549 cells using a miRVANA miRNA isolation kit (Ambion, Austin, TX, USA) in accordance with the manufacturer's instructions. Equal amounts of RNA were reversely transcripted into cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacture's protocols. The qRT-PCR assay was performed with Power SYBR Green PCR Master Mix (Applied Biosystems). U6 small nuclear RNA was used as a reference control for miRNA expression. qRT-PCR reactions were conducted using a Real-Time PCR System (ABI 7500, Applied Biosystems). The relative expression of miR-29a was calculated and estimated from triplicate qRTPCR reactions using 2DDCt method. 2.6. Plaque assay
To obtain plasmids pcDNA3-NS1 with HA-tagged, RSV NS1 genes were constructed by amplifying cDNA clones of RSV by PCR using specific primers. The PCR fragments were digested with EcoRI and NotI restriction enzymes (Promega, Madison, WI, USA) and then ligated into the same sites of pcDNA3 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). Plasmids pcDNA3-NS1 were amplified in Escherichia coli TOP10 and then isolated using commercially available kits (Qiagen, Valencia, CA, USA).
RSV titration was measured with plaque formation assay as described in detail elsewhere [17]. In brief, transfected cells were infected with RSV at an MOI of 5 for 2 h. After incubation, cells were overlaid with 1% (wt/vol) methylcellulose containing 50% (vol/vol) 2 DMEM and 2% (vol/vol) FBS and cultured for another 5 days before the overlay medium was removed, and cells were fixed and stained with 2% (wt/vol) crystal violet in 20% (vol/vol) ethanol. Wells containing of 30e100 plaques were counted and the viral titre was calculated using the following formula: virus titre (pfu/ mL) ¼ Plaques dilution 5.
2.3. Dual-luciferase reporter assay
2.7. Statistical analysis
2.2. Construction of plasmids
Luciferase reporter plasmids with wild-type (WT) or mutant
All experiments were repeated at least three times. The SPSS
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17.0 software (SPSS, Inc., Chicago, IL, USA) was used to process experimental data. Statistical comparisons were conducted using a two-tailed Student's t-test or one-way analysis of variance (ANOVA). A P value of less than 0.05 was considered statistically significant. 3. Results 3.1. RSV NS1 enhances miR-29a expression An microarray data analysis has suggested that miR-29a is induced 1.5-fold in RSV-infected A549 cells [15]. To confirm the result, qRT-PCR was performed to measure the expression level of miR-29a in RSV-infected A549 cells. After the infection of RSV for 36 h, miR-29a level was significantly higher in the group at an MOI of 5 compared with the group at an MOI of 1 (Fig. 1A). To identify whether RSV NS1 protein affected miR-29a expression, A549 cells were infected with recombinant RSV (rRSV-WT) or recombinant RSV mutant lacking NS1 (rRSV-DNS1) at an MOI of 5. The expression level of miR-29a was evaluated at 36 h after infection. As shown in Fig. 1B, expression of miR-29a was significantly upregulated in rRSV-WT-infected cells, but in the absence of the NS1 protein gene, expression of miR-29a was unchanged. These results demonstrated that RSV NS1 protein enhanced miR-29a expression. 3.2. IFNAR1 is a direct target gene of miR-29a miR-29a was predicted to have multiple interactions with IFNAR1 mRNA [18]. To conform that miR-29a indeed targeted the 30 -untranslated region (UTR) of IFNAR1 as predicted, a luciferase reporter gene containing IFNAR1 30 -UTR or a control plasmid containing mutant 30 -UTR of IFNAR1 was cotransfected with miR-29a mimics or miRNA scramble control (miR-NC) into A549 cells. The results showed that luciferase activity was significantly decreased in the miR-29a mimics group when compared with the miR-NC group, but no reduction was observed in the mutant 30 -UTR groups (Fig. 2A). To further determine whether IFNAR1 expression was modified by miR-29a, the expression level of IFNAR1 protein was measured by western blot. The expression of miR-29a was significantly increased in the miR-29a mimics group and was significantly decreased in the miR-29a inhibitor group, compared with the miR-NC group (data not shown). We found that miR-29 mimic transfection resulted in a reduction of IFNAR1 protein expression and downregulation of miR-29a elevated the levels of IFNAR1 in A549 cells (Fig. 2B). Therefore, miR-29a could directly target IFNAR1 30 -UTR and downregulate IFNAR1 at protein expression level in human A549 cells.
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3.3. RSV NS1 reduces IFNAR1 expression To confirm the inhibitory role of NS1 in IFNAR1 production, A549 cells were transfected with HA-NS1-expressing plasmid or empty vector control plasmid, or cotransfected with HA-NS1expressing plasmid and miR-29a inhibitor. qRT-PCR was performed to determine the mRNA expression level of IFNAR1. As predicted, there was a significant reduction of IFNAR1 mRNA in the HA-NS1 group when compared with the empty vector controls, but the IFNAR1 mRNA levels in the HA-NS1 þ miR-29a inhibitor group was increased compared with the HA-NS1 group (Fig. 3A). Western blot analysis was done to measure the protein expression level of NS1 and IFNAR1 (Fig. 3B). The results showed that NS1 was overexpressed after transfection with HA-NS1-expressing plasmid or cotransfection with HA-NS1-expressing plasmid and miR-29a inhibitor (Fig. 3C). The protein levels of IFNAR1 in the HA-NS1 group were significantly decreased compared with empty vector group, but the IFNAR1 levels in the HA-NS1 þ miR-29a inhibitor group were significantly increased compared with the HA-NS1 group (Fig. 3D). 3.4. miR-29a promotes virus replication To assess the effect of miR-29a on virus replication, A549 cells were transfected with miR-29a mimics, inhibitor, or miRNA scramble control (miR-NC), followed by infection with RSV at an MOI of 5. qRTPCR was performed to confirm the expression of miR-29a (Fig. 4A). RSV plaque assays showed that the number of RSV plaques in miR-29a mimics group was significantly increased when compared with miR29a inhibitor group or miRNA scramble control group (Fig. 4B). These results indicated that miR-29a contributes to RSV replication. 3.5. Inhibition of miR-29a attenuates the promotive effects of NS1 on RSV replication In order to further elucidate the precise mechanism that NS1 promotes virus replication, A549 cells were cotransfected with empty vector or plasmids expressing HA-NS1 and miR-29a inhibitor or miR-29a inhibitor NC, and then infected (MOI ¼ 5) with WT RSV or recombinant RSV lacking NS1 (DNS1). At 36 h after infection, the viral titers were determined by plaque assay. The RSV plaque assays showed that HA-NS1 overexpression increased the numbers of RSV plaque, but the promotive effect on virus replication was attenuated in presence of miR-29a inhibitor (Fig. 4C). 4. Discussion In recent years, a large amount of studies have been conducted
Fig. 1. RSV infection upregulates the expression of miR-29a in A549 cells. (A) A549 cells were infected with or without RSV at an MOI of 0.1, 1 or 5. qRT-PCR assay was performed to determined the expression levels of miR-29a 6, 12, 18, 24, 36 and 48 h after infection respectively. U6 RNA was used as the internal control in each sample. Data are expressed as the mean ± SD (n ¼ 3). **P < 0.01.
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Fig. 2. IFNAR1 is a target of miR-29a. (A) A549 cells were cotransfected with plasmids containing the WT or mutant IFNAR1 30 -UTR and miR-29a mimics or miRNA scramble control (miR-NC). After culture for 24 h, cells were harvested and then the relative luciferase activity was measured. (B) Western blot was performed to detect the expression level of IFNAR1 in A549 cells transfected with miR-29a mimics, inhibitor, or miRNA scramble control (miR-NC). Data are expressed as the mean ± SD (n ¼ 3). NS, not significant, **P < 0.01, ***P < 0.001.
to explore the mechanism underlying the inhibitory effect of NS1 on host immune responses, particularly the type I IFN synthesis. NS1 protein has also been shown to counteract the IFN-mediated inhibition of virus replication [19]. However, the exact molecular mechanism by which NS1 facilitated RSV replication is not yet elucidated. These experiments found that miR-29a was upregulated in A549 cells infected with RSV. In addition, overexpression of RSV NS1 protein enhanced the expression of miR-29a in A549 cells. We discovered that miR-29a acted as an important regulator of immune response through inhibiting IFNAR1 production and thereby promoting RSV replication. IFN-a plays a vital role in regulating immune responses, angiogenesis, and cell proliferation via directly interacting with IFNAR, its
high-affinity membrane receptor, which ultimately activates STATs [20]. It has been identified that RSV inhibits IFN-a/b signaling pathway by expression of viral NS1 and NS2 in epithelial host cells during infection [21]. An experiment discovered that RSV NS1 colocalized with mitochondrial antiviral signaling protein (MAVS) in A549 human epithelial cells infected with RSV. On the other hand, their study showed that NS1 interacted to MAVS and disrupted the MAVS interaction with retinoic acid inducible gene and thereby indirectly affected IFN production [22]. During virus infections, IFN-a and tumor necrosis factor-alpha (TNF-a) are produced mainly by macrophages and dendritic cells. In order to replicate in the host cells, human RSV NS1 and NS2 attenuate their immune defences by decreasing the production of innate defensive
Fig. 3. Effect of NS1 protein on IFNAR1 expression. A549 cells were transfected with HA-NS1 expressing plasmids or empty vectors, or cotransfected with HA-NS1-expressing plasmid and miR-29a inhibitor. Cells were collected at 48 h after transfection, qRT-PCR (A) and western blot (B) were done to measure the IFNAR1 expression at mRNA level and protein level. (C, D) The blots were quantified by NIH image software and normalized with b-actin. Data are expressed as the mean ± SD (n ¼ 3). NS, not significant, **P < 0.01, ***P < 0.001.
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Fig. 4. Effect of miR-29a on RSV replication. A549 cells were transfected with miR-29a mimics, miR-29a inhibitor or miRNA scramble control (miR-NC), and then infected with RSV at an MOI of 5 for 2 h qRT-PCR was conducted to determine the expression level of miR-29a (A). Three days after infection, cells and supernatants were collected and the number of RSV p.f.u. was calculated using a plaque assay (B). A549 cells were cotransfected with empty vector or plasmids expressing HA-NS1 and miR-29a inhibitor or miR-NC, followed by infection with rRSV-DNS1 or WT RSV at an MOI of 5 for 2 h. After incubation for 3 days, cell-associated RSV and supernatant RSV were collected and the viral titers were determined (C). Data are expressed as the mean ± SD (n ¼ 3). NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.
cytokines including IFN-a/b, IFN-g, nuclear factor-kappa B, TNF-a, IL-1a, and IL-6 [23]. Both NS1a and NS1b, the two proteolytic fragments of NS1, were able to inhibit the activity of TNF-a promoter by abrogating the Sp1 transactivation [24]. NS1 and NS2 of bovine RSV reduce the activity of interferon regulatory factor 3. NS protein-mediated inhibition of IRF-3 and IFN secretion may play vital roles in the pathogenesis and immunogenicity of bovine RSV infection [25]. H5N1 NS1 decreased the phosphorylation levels of STAT1, STAT2 and STAT3 in HeLa cells treated with IFN-b, which is a result of NS1-mediated inhibition of IFNAR1 expression. Moreover, NS1 directly disrupt the IFN signaling pathway to promote viral replication [26]. Our data showed that NS1 overexpression upregulated miR-29a expression level and resulted in a significant reduction of IFNAR1 at both mRNA level and protein level. A large amount of studies have found that miRNAs play a part in antiviral immune responses via direct degradation or translational inhibition of their target mRNAs [27]. miR-29a inhibited protein expression of tristetraprolin, which was implicated in the degradation of mRNAs, and affected oncogenic Ras signaling to result in epithelial-to-mesenchymal transition and metastasis of breast cancer [28]. A study found that human miRNA miR-29a was expressed in human peripheral blood mononuclear cells. miR-29a suppresses the protein expression of Nef by directly targeting its 30 -UTR, thereby interfering with HIV-1 replication [29]. The expression level of IFNAR1 in epithelium lacking miR-29a was approximately 8-fold higher than that in WT epithelium [30]. Here, we demonstrated that IFNAR1 was a target of miR-29a using the dual-luciferase reporter assay system. By using plaque-forming assay, we found that miR-29a was able to suppress RSV replication. To further assess the function of miR-29a in the RSV replication, A549 cells were cotransfected with HA-NS1 overexpressing plasmids or empty vector and miR-29a inhibitor or miR-NC, and then infected with WT RSV or rRSV-DNS1. We found that miR-29a downregulation efficiently interfered with NS1-induced RSV replication, suggesting that NS1 may inhibit RSV replication in a miR-29a-dependent manner. In conclusion, our evidence suggested that RSV NS1 induced upregulation of miR-29a. In addition, miR-29a suppressed the expression of IFNAR1, which is essential for the function of Type I IFNs. The promotive effect of NS1 on RSV replication was attenuated by inhibition of miR-29a expression. These findings demonstrated a new mechanism underlying the inhibitory role of NS1
against RSV-induced IFNAR1 expression. Further studies should be performed to clarify the specific mechanism of NS1-induced inhibition of IFNAR1 during RSV infection. Conflict of interest No conflicts of interest were declared. Acknowledgements This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.08.142. References [1] P.L. Collins, Respiratory syncytial virus and metapneumovirus, in: B.N. Fields, D.M. Knipe, P.M. Howley (Eds.), Fields Virology, Lippincott Williams & Wilkins, Philadelphia, 2007, pp. 1601e1646. [2] R.F. Foronjy, A.J. Dabo, C.C. Taggart, S. Weldon, P. Geraghty, Respiratory syncytial virus infections enhance cigarette smoke induced COPD in mice, PloS One 9 (2014) e90567. [3] E.C. Moore, J. Barber, R.A. Tripp, Respiratory syncytial virus (RSV) attachment and nonstructural proteins modify the type I interferon response associated with suppressor of cytokine signaling (SOCS) proteins and IFN-stimulated gene-15 (ISG15), Virol. J. 5 (2008) 116. [4] K.M. Spann, K.C. Tran, B. Chi, R.L. Rabin, P.L. Collins, Suppression of the induction of alpha, beta, and gamma interferons by the NS1 and NS2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages, J. Virol. 78 (2004) 4363e4369. [5] F. McNab, K. Mayer-Barber, A. Sher, A. Wack, A. O'Garra, Type I interferons in infectious disease, Nat. Rev. Immunol. 15 (2015) 87e103. [6] L.C. Platanias, Mechanisms of type-I-and type-II-interferon-mediated signalling, Nat. Rev. Immunol. 5 (2005) 375e386. [7] M.T. Dill, Z. Makowska, G. Trincucci, A.J. Gruber, J.E. Vogt, M. Filipowicz, D. Calabrese, I. Krol, D.T. Lau, L. Terracciano, Pegylated IFN-a regulates hepatic gene expression through transient Jak/STAT activation, J. Clin. Investig. 124 (2014) 1568e1581. [8] T.R. Prestwood, M.M. Morar, R.M. Zellweger, R. Miller, M.M. May, L.E. Yauch, S.M. Lada, S. Shresta, Gamma interferon (IFN-g) receptor restricts systemic dengue virus replication and prevents paralysis in IFN-a/b receptor-deficient mice, J. Virol. 86 (2012) 12561e12570. [9] S.L. Colpitts, T.A. Stoklasek, C.R. Plumlee, J.J. Obar, C. Guo, L. Lefrançois, Cutting edge: the role of IFN-a receptor and MyD88 signaling in induction of IL-15
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Please cite this article in press as: Y. Zhang, et al., Respiratory syncytial virus non-structural protein 1 facilitates virus replication through miR29a-mediated inhibition of interferon-a receptor, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.08.142