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, 8 (1), 16815

Sendai Virus Infection Induces Expression of Novel RNAs in Human Cells

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Sendai Virus Infection Induces Expression of Novel RNAs in Human Cells

Roli Mandhana et al. Sci Rep.

Abstract

Innate antiviral immune responses are driven by virus-induced changes in host gene expression. While much research on antiviral effectors has focused on virus-inducible mRNAs, recent genome-wide analyses have identified hundreds of novel target sites for virus-inducible transcription factors and RNA polymerase. These sites are beyond the known antiviral gene repertoire and their contribution to innate immune responses is largely unknown. In this study, RNA-sequencing of mock-infected and Sendai virus-infected cells was performed to characterize the virus-inducible transcriptome and identify novel virus-inducible RNAs (nviRNAs). Virus-inducible transcription was observed throughout the genome resulting in expression of 1755 previously RefSeq-annotated RNAs and 1545 nviRNAs. The previously-annotated RNAs primarily consist of protein-coding mRNAs, including several well-known antiviral mRNAs that had low sequence conservation but were highly virus-inducible. The previously-unannotated nviRNAs were mostly noncoding RNAs with poor sequence conservation. Independent analyses of nviRNAs based on infection with Sendai virus, influenza virus, and herpes simplex virus 1, or direct stimulation with IFNα revealed a range of expression patterns in various human cell lines. These phylogenetic and expression analyses suggest that many of the nviRNAs share the high inducibility and low sequence conservation characteristic of well-known primary antiviral effectors and may represent dynamically evolving antiviral factors.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Validation of differential gene expression induced by Sendai virus infection. Namalwa cells were infected with Sendai virus for 6 hours and total RNA was analyzed by RNA-sequencing or by RT-qPCR in independent samples. For each indicated RNA, RNA-sequencing counts are plotted on the graph on the left and RNA abundance from RT-qPCR on the right. Expression of virus-induced (a) previously-annotated genes and (b) previously-unannotated RNAs and (c) virus-suppressed genes was validated. RNA abundance data is representative of ≥2 replicate experiments and is shown normalized to GAPDH expression. Bars indicate average values of technical replicates (n = 3) with error bars representing standard deviation. Statistical analysis was done using a two-tailed Student’s t-test for RT-qPCR measurements (*p-value < 0.05, **p-value < 0.005).
Figure 2
Figure 2
Graphical representation of functional enrichment analysis of Sendai virus-induced RNAs. Sendai virus-induced previously-annotated RNAs were analysed using DAVID to determine enriched (a) GO biological processes and (b) KEGG pathways. The top 10 most significant terms in each analysis are shown here in rank order by p-value. Bars represent number of virus-induced RNAs mapping to each term.
Figure 3
Figure 3
Comparison of genomic distribution of Sendai virus-induced previously-annotated RNAs and nviRNAs. (a) Bar graphs represent the number of virus-induced previously-annotated RNAs (left) and nviRNAs (right) identified on each chromosome. (b) Pie charts illustrate genomic feature distribution of previously RefSeq-annotated RNAs (left) and nviRNAs (right). RNAs are mapped to one of six annotation categories: promoters, transcriptional termination sites (TTS), exons, intergenic regions, introns, and untranslated regions (UTRs; includes 5′ and 3′ UTRs), with the percentage of sites corresponding to each category displayed in parentheses near the label.
Figure 4
Figure 4
Comparison of protein coding potential and vertebrate sequence conservation of Sendai virus-induced previously-annotated RNAs and nviRNAs. (a) The cumulative distribution frequency of PhyloCSF scores for previously-annotated RNAs (blue) and nviRNAs (red). (b) The cumulative distribution frequency of PhyloCSF scores for RNAs mapping to the different genomic feature annotations for previously RefSeq-annotated RNAs and (c) nviRNAs. The different genomic feature annotations include: coding exons (orange), intergenic regions (blue), introns (green), and promoters, transcriptional termination sites, and UTRs (all grouped together; red). (d) The cumulative distribution frequency of the mean PhastCons score for the previously-annotated RNAs (blue) and nviRNAs (red). The mean PhastCons score for each RNA is plotted against the log2 fold change in expression after Sendai virus infection for (e) previously-annotated RNAs and (f) nviRNAs. The 10 most highly-induced previously-annotated RNAs are labeled in blue in (e).
Figure 5
Figure 5
Classification of nviRNA expression in Namalwa cells from various stimuli. Total RNA from Namalwa cells infected with 5 pfu/cell for 10 hours of Sendai virus (SeV), influenza A virus, or HSV-1 or directly treated with 1000 U/mL of IFNα for 6 hours was analyzed by RT-qPCR. Heat map indicates expression of each RNA after infection or treatment with IFNα. Average values (n = 3) of fold change are reported normalized to GAPDH expression.
Figure 6
Figure 6
nviRNA expression in 2fTGH cells. Total RNA from 2fTGH cells infected with 5 pfu/cell Sendai virus (4 hours) or influenza A virus (IAV;10 hours), or transfected with synthetic dsRNA polyI:C (pI:C; 6 hours), or directly treated with IFNα (6 hours) was analyzed by RT-qPCR. Gene-specific primers were used for (a) control genes, (b) nviRNAs inducible by viruses and IFNα in Namalwa cells and (c) nviRNAs only inducible by viruses in Namalwa cells. Data is representative of ≥2 replicate experiments and is shown normalized to GAPDH expression. Bars indicate average values of technical replicates (n = 3) with error bars representing standard deviation. Statistical analysis was done using a two-tailed Student’s t-test (*p-value < 0.05, **p-value < 0.005).
Figure 7
Figure 7
nviRNA expression in THP-1 cells. Total RNA from THP-1 cells infected with 5 pfu/cell Sendai virus (4 hours), influenza A virus (IAV; 10 hours) or HSV-1 (10 hours) or directly treated with IFNα (6 hours) was analyzed by RT-qPCR. Gene-specific primers were used for (a) control genes, (b) nviRNAs inducible by viruses and IFNα in Namalwa cells and (c) nviRNAs only inducible by viruses in Namalwa cells. Data is representative of ≥2 replicate experiments and is shown normalized to GAPDH expression. Bars indicate average values of technical replicates (n = 3) with error bars representing standard deviation. Statistical analysis was done using a two-tailed Student’s t-test (*p-value < 0.05, **p-value < 0.005).

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