An interferon-stimulated long non-coding RNA USP30-AS1 as an immune modulator in influenza A virus infection

Abstract

Long non-coding RNAs (lncRNAs) are essential components of innate immunity, maintaining the functionality of immune systems that control virus infection. However, how lncRNAs engage immune responses during influenza A virus (IAV) infection remains unclear. Here, we show that lncRNA USP30-AS1 is up-regulated by infection of multiple different IAV subtypes and is required for tuning inflammatory and antiviral response in IAV infection. Genetically inactivation of USP30-AS1 enhances viral protein synthesis and viral growth. USP30-AS1 is an interferon-stimulated gene, and the induction of USP30-AS1 can be achieved by JAK-STAT mediated signaling activation. The immune regulation of USP30-AS1 is independent of its proximal protein-coding gene USP30. In IAV infection, deletion of USP30-AS1 unleashes high systemic inflammatory responses involving a broad range of pro-inflammatory factors, suggesting USP30-AS1 as a critical modulator of immune responses in IAV infection. Furthermore, we established a database providing well-annotated host gene expression profiles IAV infection or immune stimulation.

Fig 1. Most universally differential expressed lncRNAs across infection of different IAV subtypes.

A) Upset plot showing the distribution of lncRNAs that were differentially expressed in infection of at least 5 different IAV subtypes in included datasets in the study. The main bar and above number represent the sum of individual different lncRNAs that differentially expressed in black dot indicated infection datasets. The side bar and annotated number indicate the total number of differentially expressed lncRNAs in each infection dataset. B) The expression fold change of USP30-AS1 in A/Hong Kong/483/97 (H5N1), A/Guangzhou/39715/2014 (H5N6), A/Shanghai/2/2013 (H7N9), A/Hong Kong/4550/2016 (H3N2) or A/Oklahoma/370/2005 (H3N2) infected human primary alveolar epithelial cells (with M.O.I. of 2) compared to mock infected control at 24 hours post infection (h.p.i.). Two independent experiments were conducted, a representative experiment was shown. Student’s t-test was used to test the difference between infection and mock. The bar height represents mean and error bar represents standard deviation. C) Expression fold change of USP30-AS1 expression in A/Hong Kong/1/68 (H3N2) (M.O.I. of 0.1) infected A549 or CALU-3 versus PBS treated A549 or CALU-3 at 0 and 24 hours post-infection (h.p.i.). Experiment was conducted in triplicates. Student’s t-test was used to test the difference between infection and mock group. Significant exact two-sided P-value was reported. The bar height represents mean and error bar represents standard deviation.

Fig 2. The expression of USP30-AS1 and different loci of USP30 in response to interferon stimulation.

A) The expression fold change of USP30-AS1 and different regions of USP30, as well as MX1 and ISG15 as positive controls, in response to different dose of interferon α stimulation compared to PBS-based mock treatment at 6 hours post-treatment. B) The expression fold change of USP30-AS1 and different regions of USP30, as well as MX1 and ISG15 as positive controls, in response to different dose of interferon β stimulation compared to PBS-based mock treatment at 6 hours post-treatment. C) The expression fold change of USP30-AS1 and different regions of USP30, as well as MX1 and ISG15 as positive controls, in response to different dose of interferon γ stimulation compared to PBS-based mock treatment at 6 hours post-treatment. USP30 ALLSUS: USP30 CDS region that are consensus across all different USP30 transcripts; USP30 ASOVLP: USP30 genomic region that overlaps with USP30-AS1; USP30 ASUP: USP30 genomic region located in the upstream of USP30-AS1, overlapping with CDS of some transcripts of USP30. Experiment was conducted in triplicates. Student’s t-test was used to test the difference between stimulation and mock group. Significant exact two-sided P-value was reported. The bar height represents mean and error bar represents standard deviation.

Fig 3. Deletion of USP30-AS1 strengthens IAV growth and promotes viral protein synthesis.

A) Viral titer in the supernatant of A/California/04/09 (H1N1) or A/Puerto Rico/8/1934 (H1N1) (M.O.I. of 0.1) infected USP30-AS1^-/- A549 cells or WT A549 cells at 24, 48, and 72 hours post-infection (h.p.i.). A representative experiment was shown from two independent experiments performed in triplicates. Student’s t-test was used to test the difference of viral titer in the supernatant between infected USP30-AS1^-/- A549 and WT A549 cells. Significant exact two-sided P-value was reported. Mean and standard deviation were shown in the plot. CI, confidence interval. B) Detection of IAV viral PB2, PB1, PA, NP proteins and cellular ACTB protein expression by immunoblot in single cycle or multiple cycles A/Puerto Rico/8/1934 (H1N1) infected USP30-AS1^-/- A549 cells compared to WT A549 cells.

Fig 4. Major host defense signaling and the expression of USP30-AS1.

A) Expression fold change of USP30-AS1 between 15 nM JAK Inhibitor-I and DMSO pre-treated (for 24 hours) A549 cells in response to either 100 U/ml interferon β or 100 ng/ml interferon γ stimulation at 6 hours. B) Expression fold change of USP30-AS1 between 5 μM Fludarabine and DMSO pre-treated (for 24 hours) A549 cells in response to either 100 U/ml interferon β or 100 ng/ml stimulation γ at 6 hours C) Expression fold change of USP30-AS1 between 20 μM Stattic and DMSO pre-treated (for 24 hours) A549 cells in response to either 100 U/ml interferon β or 100 ng/ml interferon γ stimulation at 6 hours. D) Expression fold change of USP30-AS1 between 15 nM JAK Inhibitor-I and DMSO pre-treated (for 24 hours) A549 cells infected with either A/California/04/09 (H1N1) (with M.O.I. of 1) or A/Puerto Rico/8/1934 (H1N1) (with M.O.I. of 1) at 24 hour post infection (h.p.i.). E) Expression fold change of USP30-AS1 between either low concentration of 20 nM IKKε/TBK1 inhibitor II and DMSO, or high concentration of 10 μM IKKε/TBK1 inhibitor-II and DMSO pre-treated (for 24 hours) A549 cells in response to either 100 U/ml interferon β or 100 ng/ml interferon γ stimulation at 6 hours. F) Expression fold change of USP30-AS1 between either low concentration of 20 nM IKKε/TBK1 inhibitor II and DMSO, or high concentration of 10 μM IKKε/TBK1 inhibitor-II and DMSO pre-treated (for 24 hours) A549 cells infected with A/California/04/09 (H1N1) (with M.O.I. of 5) at 6 hours post-infection (h.p.i.). G) Expression of USP30-AS1 (normalized counts) in A549 cells in response to 1000 U/mL interferon β stimulation at 12 hours post stimulation after cells treated with either 1 μg/mL R848 or mock treatment (from publicly available datasets, BioProject PRJNA481248). Experiment was conducted in triplicates. Student’s t-test was used to test the difference between two groups. Significant exact two-sided P-value was reported. The bar height represents mean and error bar represents standard deviation.

Fig 5. Loss of USP30-AS1 induces high systemic inflammatory response.

A) MA plot showing up-regulated genes (FC>1.5, adjusted P-value < 0.05) and down-regulated genes (FC<-1.5, adjusted P-value < 0.05) in bulk RNA-seq generated from either A/California/04/09 (H1N1) (with M.O.I. of 1) infected or mock treated USP30-AS1^-/- A549 cells versus either infected or mock treated WT A549 cells. Differential expression calculation was performed under multi-factors adjustment. Bulk RNA-seq was performed in triplicates. B) Heatmap showing the K-mean clustered gene modules across cell genotype (USP30-AS1^-/- or WT) and treatment (A/California/04/09 (H1N1) infection or mock infection) in the bulk RNA-seq data. Type I and Type II interferon stimulated signature genes, as well as up-regulated and down-regulated signature genes of LPS stimulation was also shown for comparison. Each clustered module was annotated with enriched biological processes in Gene Ontology (GO) analysis. C) Volcano plot showing the expression fold change of core members in families of major pro-inflammatory factors in A/California/04/09 (H1N1) infected USP30-AS1^-/- A549 cells versus A/California/04/09 (H1N1) infected WT A549 cells after multi-factors adjustment from bulk RNA-seq data. D) Expression fold change (USP30-AS1^-/- versus WT) of major pro-inflammatory factors observed in RNA-seq analysis in A/California/04/09 (H1N1) (with M.O.I. of 1) infected USP30-AS1^-/- A549 cells compared to infected WT cells at 24 hours post-infection (h.p.i.), same condition performed as bulk RNA-seq. Experiment was conducted in triplicates. Student’s t-test was used to test the difference between USP30-AS1^-/- and WT during infection. Significant exact two-sided P-value was reported. Bar height represents mean and error bar represents standard deviation. E) Protein expression (pg/ml) of major pro-inflammatory mediators in supernatant from same experiment showed in D), by beads-based immunoassay. Experiment was conducted in triplicates. Student’s t-test was used to test the difference between USP30-AS1^-/- and WT group infection. Significant exact two-sided P-value was reported. Bar height represents mean and error bar represents standard deviation. F) The expression fold change of major pro-inflammatory molecules between USP30-AS1^-/- A549 cells and WT cells in response to 100 ng/ml interferon γ stimulation at 6 hours post-treatment. Experiment was conducted in triplicates. Student’s t-test was used to test the fold change difference between USP30-AS1^-/- and WT group infection. Significant exact two-sided P-value was reported. Bar height represents mean and error bar represents standard deviation.