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. 2014;11(1):66-75.
doi: 10.4161/rna.27504. Epub 2013 Dec 20.

Evidence for a crucial role of a host non-coding RNA in influenza A virus replication

Carla Winterling  1 , Manuel Koch  1 , Max Koeppel  1 , Fernando Garcia-Alcalde  1 , Alexander Karlas  1 , Thomas F Meyer  1
Affiliations

Evidence for a crucial role of a host non-coding RNA in influenza A virus replication

Carla Winterling et al. RNA Biol. 2014.

Abstract

A growing body of evidence suggests the non-protein coding human genome is of vital importance for human cell function. Besides small RNAs, the diverse class of long non-coding RNAs (lncRNAs) recently came into focus. However, their relevance for infection, a major evolutionary driving force, remains elusive. Using two commercially available microarray systems, namely NCode™ and Sureprint™ G3, we identified differential expression of 42 ncRNAs during influenza A virus (IAV) infection in human lung epithelial cells. This included several classes of lncRNAs, including large intergenic ncRNAs (lincRNAs). As analyzed by qRT-PCR, expression of one lincRNA, which we termed virus inducible lincRNA (VIN), is induced by several IAV strains (H1N1, H3N2, H7N7) as well as vesicular stomatitis virus. However, we did not observe an induction of VIN by influenza B virus, treatment with RNA mimics, or IFNβ. Thus, VIN expression seems to be a specific response to certain viral infections. RNA fractionation and RNA-FISH experiments revealed that VIN is localized to the host cell nucleus. Most importantly, we show that abolition of VIN by RNA interference restricts IAV replication and viral protein synthesis, highlighting the relevance of this lincRNA for productive IAV infection. Our observations suggest that viral pathogens interfere with the non-coding portion of the human genome, thereby guaranteeing their successful propagation, and that the expression of VIN correlates with their virulence. Consequently, our study provides a novel approach for understanding virus pathogenesis in greater detail, which will enable future design of new antiviral strategies targeting the host's non-protein coding genome.

Keywords: IAV; VIN; host factor; lincRNA; non-protein coding genome.

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Figures

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Figure 1. Host cell lncRNAs are differentially expressed during influenza A/WSN/33 (H1N1) infection. (A) The contributions of different ncRNA classes on NCode™ and Sureprint™ G3 microarrays alone and common to both are shown. Re-annotation of microarrays was performed according to Ensembl human genome annotation (Release 68). (B) A549 cells were infected with influenza A/WSN/33 (H1N1) virus (MOI 1) for 8 or 24 h. Columns 1–4: log2-fold expression change of 17 lincRNAs (labeled by Ensembl transcript ID) that were differentially regulated at least 2-fold between uninfected and infected samples at both time points using NCode™ and Sureprint™ G3 microarrays. Columns 5–6: regulation of lincRNA expression by UV-treated, infected cell supernatants.
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Figure 2. Infection-induced expression of a novel lincRNA is not specific to A/WSN/33 (H1N1) virus. (A) A549 cells were infected with three IAV strains, IBV, and VSV with MOIs 1 for 6 h. qRT-PCR data are presented as mean fold-changes in VIN expression (+/− SD) compared with non-infected (NI) reference. Data from three independent experiments were analyzed using one-sample t test (* P < 0.01; ** P < 0.005). (B) A549 cells were transfected with poly I:C and RNA was isolated 24 h post-transfection. qRT-PCR was performed for VIN and IFNβ. (C) A549 cells were treated with IFNβ and RNA isolated 8 h later. qRT-PCR was performed for VIN and MX1. Data in (B and C) are presented as fold-changes of expression compared with mock control (means of three independent experiments (+/− SD).
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Figure 3. In silico characterization of VIN. (A) Genomic context of VIN. Shown is the annotation by UCSC genome browser [http://genome.ucsc.edu, Human Feb. 2009 (GRCh37/hg19) Assembly], depicting the position of LOC440900 (VIN, red arrow), an uncharacterized transcript LOC100499194, H3K27ac, and H3K4me1 marks, location of a CpG island and transcription factor binding site clusters. (B) RNA secondary structure prediction of VIN (RNAfold web server, University of Vienna). Shown is a minimal free energy structure (MFE = -396.90 kcal/mol). Base pairing probabilities have been color coded using a scale from 0 (blue) to 1 (red). (C) RNase A stability of VIN. Nuclear RNA extracts of A549 cells were treated with RNase A followed by purification of RNA. qRT-PCR was performed for GAPDH mRNA and VIN. Data from three independent experiments (mean +/− SD) are depicted as VIN transcripts per GAPDH transcript (1/2^delta CT).
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Figure 4. VIN is localized to host cell nuclei. (A) Nuclear and cytoplasmic RNA fractions were prepared from A/WSN/33 (H1N1) virus-infected A549 cells (MOI 5, 8 h). qRT-PCR was performed for GAPDH, RNU1-1 and VIN. Data are presented as mean ∆CT values (cytoplasm-nucleus) +/− SD of three independent experiments. (B) DIG-labeled probes were hybridized to A549 cells mock-infected or infected with A/WSN/33 (H1N1) virus (MOI 5, 6 h p.i.) followed by immunofluorescence staining of DIG (green) and viral NP protein (red) (RNA-FISH). Nuclei were visualized using Draq5 (blue). Images shown are representatives from four independent experiments.
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Figure 5. VIN is essential for productive A/WSN/33 (H1N1) virus infection in human lung epithelial cells. (A) Three unique siRNAs designed to target VIN were used individually (1, 2, and 3) and collectively (P) to transfect A549 cells. Images show NP immunofluorescent staining in MDCK cells infected with supernatants from VIN knockdown cells (1, 2, 3, and P) compared with Allstars control. Immunofluorescence images shown are representatives from at least three independent experiments. (B) NP-positive MDCK cells from infection experiments were quantified using ScanR software and viral titers calculated. Data are presented as mean A/WSN/33 (H1N1) viral titers (plaque forming units (PFU)/ml) +/− SD from at least three independent experiments). Mann Whitney U tests were used for statistical analysis, * P < 0.01; ** P < 0.005; *** P < 0.001. (C) Western blot analysis of IAV protein expression 48 h p.i. in VIN A549 siRNA knockdown cells (1, 2, and 3, and P) compared with siRNA Allstars control. β-Actin expression is shown as a loading control. Blot is a representative of two independent experiments (HA, Hemagglutinin; NP, Nucleoprotein; NS1, non-structural protein 1; M2, Matrix protein 2).
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