Human respiratory syncytial virus non-structural protein NS1 modifies miR-24 expression via transforming growth factor-β

Abstract

Human respiratory syncytial virus (RSV) is a major health challenge in the young and elderly owing to the lack of a safe and effective vaccine and proven antiviral drugs. Understanding the mechanisms by which viral genes and proteins modulate the host response to infection is critical for identifying novel disease intervention strategies. In this study, the RSV non-structural protein NS1 was shown to suppress miR-24 expression during infection. Lack of NS1 was linked to increased expression of miR-24, whilst NS1 overexpression suppressed miR-24 expression. NS1 was found to induce Kruppel-like factor 6 (KLF6), a transcription factor that positively regulates the transforming growth factor (TGF)-b pathway to induce cell cycle arrest. Silencing of KLF6 led to increased miR-24 expression via downregulation of TGF-β. Treatment with exogenous TGF-β suppressed miR-24 expression and induced KLF6. Confocal microscopy showed co-localization of KLF6 and RSV NS1. These findings indicated that RSV NS1 interacts with KLF6 and modulates miR-24 expression and TGF-β, which facilitates RSV replication.

Fig. 1.

RSV NS1 modulates miR-24 expression. (a) Lack of RSV NS1 induces miR-24 expression. A549 cells were infected with sucrose-purified (sp) RSV A2 (A2sp), ΔNS1 or ΔNS2 virus (m.o.i. 1.0) for 6 or 24 h followed by RNA isolation and quantitative real-time (qRT)-PCR for miR-24 relative to A549 cells treated with 30 % sucrose as mock and 18S rRNA as a housekeeping control. (b) RSV NS1 overexpression suppresses miR-24. miR-24 expression was measured relative to 18S rRNA in A549 cells transfected with pEGFPC1-NS1 or pEGFPC1-NS2 (50 ng each) plasmids for varying times. Data shown are representative of three independent experiments. (c) WT and ΔNS1/ΔNS2 viruses grow at equivalent titres at early time points. Viral titres of RSV A2 (•), ΔNS1 (▪) or ΔNS2 (▴) viruses were determined by plating serial 10-fold dilutions of infected A549 lysates on Vero E6 cells as per the standard protocol and visualized by staining for RSV F protein using mAb-131-2A. Data represent mean ± sem of four biological replicates. ^** P < 0.01.

Fig. 2.

RSV NS1 and IFN-λ induce TGF-β expression via KLF6. (a) Overexpression of RSV NS1 induces TGF-β. A549 cells were transfected with pEGFPC1-NS1 or pEGFPC1 alone for varying times and TGF-β expression was determined by qRT-PCR at different time points following NS1 overexpression. Data represent mean ± sem of three independent experiments. (b) Secondary triggers of TGF-β expression. NS1-transfected A549 cells were either mock treated, or treated with recombinant IFN-λ (10 ng ml^− 1) or LPS (1 μg ml^− 1), or transfected with high-molecular-mass polyI : C (10 ng ml^− 1) for 24 h. TGF-β expression was determined by qRT-PCR. (c) RSV NS1 induces KLF6 expression. WT RSV A2 but not ΔNS1 infection induces KLF6 expression. A549 cells were infected with sucrose purified (sp) WT RSV A2 or recombinant ΔNS1 virus (m.o.i. 1.0) for 24 h followed by qRT-PCR analysis of KLF6 expression. Data represent two independent experiments. (d) Secondary triggers of KLF6 expression. pEGFPC1-NS1-transfected A549 cells were mock treated, or treated with IFN-λ (10 ng ml^− 1), IFN-β (10 ng ml^− 1) or LPS (1 μg ml), or transfected with high-molecular-mass polyI : C (10 ng ml^− 1) for 24 h in two independent experiments. KLF6 or miR-24 expression was measured relative to 18S rRNA. ^*** P < 0.001; ^**** P < 0.0001.

Fig. 3.

KLF6 suppresses miR-24 via TGF-β. (a) KLF6 silencing induces miR-24, and represses TGF-β expression and viral replication. A549 cells were either mock treated or transfected with 25 nM siRNA SMARTpools against KLF6 in DharmaFECT 1 for 24 h. Plates with similar treatment were infected with RSV A2 (m.o.i. 1.0). Total RNA was isolated 24 h post-transfection using RNAzol RT and used to measure miR-24, RSV M2 and TGF-β expression relative to 18S rRNA. Data represent mean ± sem from two independent experiments. (b) Recombinant TGF-β suppresses miR-24 and induces KLF6. A549 cells were treated with recombinant human TGF-β (10 ng ml^− 1) for 6, 12 and 24 h. Total RNA was isolated at the respective time points using RNAzol RT, and analysed for miR-24 and KLF6 expression relative to 18S rRNA. Data represent mean ± sem from three independent experiments. ^** P < 0.01, ^**** P < 0.0001.

Fig. 4.

miR-24 regulates multiple cellular pathways. (a) Work flow used to shortlist miR-24 target genes. Top target predictions were compared to public microarray data on RSV infection. (b, c) Subsets of genes (b) identified were validated using miR-24 inhibition experiments (c). A549 cells (2 × 10⁵) were transfected with miR-24 inhibitor (25 nM) or mock or non-targeting control (NTC) for 24 h followed by analysis of gene expression using target-specific primers. Expression was calculated relative to 18S rRNA and non-targeting control from two or three independent experiments.

Fig. 5.

RSV NS1 interacts with KLF6. A549 cells were transfected with 1.5 μg pEGFPC1-NS1 plasmid for 24 h in a 24-well plate and then transferred to chamber slides. Cells were allowed to adhere overnight, fixed, and stained for GFP expression using an anti-GFP antibody (green) and KLF6 (red). Nuclei were stained with DAPI (1 μg ml^− 1). Confocal images were captured using a Zeiss LSM 710 inverted confocal microscope using an oil immersion lens at × 63. Images are representative of three fields. (a) DAPI alone, (b) GFP alone, (c) KLF6 alone, (d) merged, and (e) orthogonal projection of the optical section showing co-localization of NS1 and KLF6. Bar, 10 μm.