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. 2021;13(3):179-193.
doi: 10.1159/000513606. Epub 2021 Feb 24.

Positive Feedback Loop of Long Noncoding RNA OASL-IT1 and Innate Immune Response Restricts the Replication of Zika Virus in Epithelial A549 Cells

Yi Wang  1   2 , Zhiting Huo  1   2 , Quanshi Lin  1   2 , Yuxia Lin  1   2 , Cancan Chen  3 , Yanxia Huang  1   2 , Changbai Huang  1   2 , Junsong Zhang  4 , Junfang He  1   2 , Chao Liu  1   5 , Ping Zhang  6   7
Affiliations

Positive Feedback Loop of Long Noncoding RNA OASL-IT1 and Innate Immune Response Restricts the Replication of Zika Virus in Epithelial A549 Cells

Yi Wang et al. J Innate Immun. 2021.

Abstract

Expression of host noncoding RNAs and coding mRNAs is significantly altered by viral infection. In the current study, we screened the transcriptional profile of human lung epithelial A549 cells infected with Zika virus (ZIKV) by microarray assay. Seventy-nine long noncoding RNAs (lncRNAs) and 140 mRNAs were differentially expressed (DE). The bioinformatics analysis revealed that the mRNAs adjacent to the DE lncRNAs were closely related to the host responses to viral infection. We selected 7 lncRNAs from the top 50 hits for validation. The quantitative real-time PCR data confirmed that expression of selected lncRNAs was induced by ZIKV infection. Moreover, the expression of 7 lncRNAs was induced by infection of dengue virus, Japanese encephalitis virus, or vesicular stomatitis virus, or by treatment of poly(I:C) and IFN-β. Furthermore, loss of innate immune adaptor IPS-1 or receptor IFNAR1 resulted in lower induction levels of several lncRNAs by ZIKV. Overexpression of 3 lncRNAs (RPL27-OT1, OASL-IT1, and REC8-OT3) reduced the virus yields of ZIKV. Knockout of OASL-IT1 significantly enhanced ZIKV replication. In OASL-IT1 knockout cells, the levels of interferons (IFNs) and the activation of 3 innate immune signaling pathways triggered by ZIKV were dramatically reduced. Collectively, our work found a positive feedback loop in the IFN system, in which IFNs and OASL-IT1 regulate each other, thereby promoting establishment of antiviral defense.

Keywords: Innate immunity; Interferon; Interferon-stimulated gene; Long noncoding RNA; Zika virus.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1
Bioinformatics analysis of lncRNAs and mRNAs. a Scatter plot to show variation in lncRNA and mRNA expression. The values of x and y axes were the mean normalized signal values in each group (log2-scaled). The red plots indicate the upregulated genes, and the blue plots indicate the downregulated genes in scatter plots. b Heat maps to show differentially expressed lncRNAs and mRNAs between mock- and ZIKV-infected groups with fold change ≥2.0 and p value ≤0.05. The red color indicates relatively higher expression, and the green color indicates relatively lower expression. c Chromosome distribution of differentially expressed lncRNAs and mRNAs. d Classification of differentially expressed lncRNAs. GO (e, f) and KEGG pathway enrichment analysis (e) of protein-coding genes close to the differently expressed lncRNAs (f). lncRNAs, long noncoding RNAs; ZIKV, Zika virus; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Fig. 2
Fig. 2
Expression levels of mRNAs and lncRNAs. a, b A549 cells were infected with ZIKV for 24 h at MOI 8. The expression levels of 7 mRNAs (a) and 7 lncRNAs (b) were measured by qRT-PCR. c LN229 and MDMs were infected with ZIKV at MOI 8. d A549 cells were infected with DENV2, JEV, or VSV at MOI 8. Mock- and virus-infected cells were collected at 24 h p.i. except VSV-infected cells at 12 h p.i. Total RNAs were extracted for qRT-PCR to detect the levels of 7 lncRNAs. U6 level was set as an internal control. The data were presented as mean ± SD of at least 3 independent experiments. +p < 0.05; ++p < 0.01; +++p < 0.001, unpaired, two-tailed Student's t test. ns, no statistical significance; lncRNAs, long noncoding RNAs; ZIKV, Zika virus; MDMs, monocyte-differentiated macrophages; DENV2, dengue virus; JEV, Japanese encephalitis virus; VSV, vesicular stomatitis virus; p.i., postinfection.
Fig. 3
Fig. 3
Innate immune response was involved in the ZIKV-mediated induction of lncRNAs. a–c qRT-PCR to measure the levels of lncRNAs. A549 cells were transfected with poly(I:C) for 12 h (a) or treated with 100 units of IFN-β for 20 h (b). c Control, IPS-1KO, and IFNAR1KO cells were infected with mock or ZIKV. Cells were harvested for RNA extraction, and the expression of 7 lncRNAs was quantified by qRT-PCR. U6 level was set as an internal control. The data were presented as mean ± SD of at least 3 independent experiments. +p < 0.05; ++p < 0.01; +++p < 0.001, unpaired, two-tailed Student's t test. ns, no statistical significance; lncRNAs, long noncoding RNAs; ZIKV, Zika virus; IFN-β, interferon-beta; IPS-1, interferon promoter stimulator 1; IFNAR1, interferon α/β receptor.
Fig. 4
Fig. 4
Three lncRNAs restricted the ZIKV replication. a Expression levels of lncRNAs expressing A549 cells validated by qRT-PCR. b, c Replication levels of ZIKV. Control and lncRNA-expressing cells were infected with ZIKV at MOI 5. Cells or supernatants were harvested at 24 h p.i. b The viral E protein levels were tested by Western blot. c The viral titers were determined by plaque assay. Data were shown as mean ± SD of at least 3 independent experiments. +p < 0.05; ++p < 0.01; +++p < 0.001, unpaired, two-tailed Student's t test. ns, no statistical significance; lncRNAs, long noncoding RNAs; ZIKV, Zika virus; p.i., postinfection.
Fig. 5
Fig. 5
Characterization of OASL-IT1. a RACE assay to determine the size of full-length OASL-IT1. b–e Prediction of coding ability of OASL-IT1. ORF Finder (b), PhyloCSF (c), CPAT (d), and CPC (e) were employed to predict the coding probability of OASL-IT1. ACTB and GAPDH served as control coding genes, and Lsm3b and NEAT1 served as control noncoding genes. f Western blot. 293 cells were transfected with pcDNA3.1 vector, pcDNA3.1-OASL-IT1-HA, or pcDNA3.1-GFP-HA plasmid. At 24 h posttransfection, cells were harvested for Western blot using anti-HA antibody. Blots were representative of at 3 independent experiments. CPAT, Coding Potential Assessment Tool; CPC, Coding Potential Calculator.
Fig. 6
Fig. 6
OASL-IT1 played an antiviral role. a Subcellular distribution of OASL-IT1. A549 cells were infected with ZIKV at MOI 5 and harvested at 0, 6, 12, and 24 h p.i. for subcellular fractionation and RNA extraction. qRT-PCR was performed to measure the levels of OASL-IT1, U6, and actin, respectively. b Schematic diagram of CRISPR/Cas9 knockout strategy. The positions of OASL-IT1, sgRNA1, sgRNA2, and PCR primers used to amplify the OASL-IT1 fragment were indicated. c DNA gel to show the PCR fragments amplified from the genomic DNA of WT and 2 OASL-IT1KO cells. d, e mRNA levels of OASL-IT1 and OASL. Control and OASL-IT1KO cells were treated with IFN-β for 20 h. Total RNA was extracted for qRT-PCR to measure the levels of OASL-IT1 and OASL.f Plaque assay. Control and OASL-IT1KO cells were infected with ZIKV at MOI 5. At 24 h p.i., supernatants were harvested for plaque assay. g, h Impact of OASL-IT1 knockdown on the viral replication. Control and OASL-IT1KD cells were infected with ZIKV at MOI 5. Total RNAs were prepared for qRT-PCR (g) to measure the levels of OASL-IT1. U6 was set as an internal control. h Supernatants were harvested at 24 h p.i. for plaque assay. Data were shown as mean ± SD of at least 3 independent experiments. +p < 0.05; ++p < 0.01; +++p < 0.001, unpaired, two-tailed Student's t test. ns, no statistical significance; ZIKV, Zika virus; p.i., post infection; WT, wild type.
Fig. 7
Fig. 7
OASL-IT1 promoted the innate immune response. a qRT-PCR to detect the levels of IFN-β, MX1, and IFITM1. The control, OASL-IT1KO, and OASL-IT1KO cells were infected with ZIKV at MOI 5. At indicated time points, total RNAs were prepared for qRT-PCR. GAPDH level was set as an internal control. Data were shown as mean ± SD of at least 3 independent experiments. +++p < 0.001, unpaired, two-tailed Student's t test. b, c Activation levels of 3 innate signaling pathways. Control, OASL-IT1KO, and OASL-IT1KD cells were infected with ZIKV at MOI 5. At 24 h p.i., cells were collected for Western blot (b) or IFM (c). b Whole cell extracts were prepared and subjected to SDS-PAGE and Western blot using indicated antibodies. GAPDH was loaded as an internal control. In the IFM assay, cells were incubated with anti-IRF3 antibody (red) and DAPI (blue). Blots and cell images were representative of at 3 independent experiments. ns, no statistical significance; ZIKV, Zika virus; p.i., postinfection; IFM, immunofluorescence microscopy.
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