Repurposed Transcriptomic Data Reveal Small Viral RNA Produced by Influenza Virus during Infection in Mice

<div><p>Influenza virus, a highly infectious ssRNA virus, replicates in the nucleus of host cells. This unusual feature brings the possibility that the virus may hijack host small noncoding RNA metabolism. Influenza small viral RNA production has been examined <i>in vitro</i> but has not yet been studied in an <i>in vivo</i> setting. We assessed small RNA species from influenza virus during mouse infection by mining publicly available mouse small RNA transcriptome data. We uncovered 26 nt reads corresponding to svRNA, a small viral RNA previously detected <i>in vitro</i> that regulates the transition from transcription to replication during infection, and found a strong positive correlation between svRNA production and host susceptibility to influenza virus infection. We also detected significant overrepresentation of a non-coding 23 nt sequence that we speculate may behave like a miRNA and work with influenza protein NS1 to prevent the transcription and maturation of interferon-stimulated mRNAs.</p></div

The eight viral segments of the influenza A genome.

<p>The eight viral segments of the influenza A genome.</p

Relationship between host immunity and svRNA abundance.

<p>For each infected mouse, the susceptibility of the mouse strain to influenza virus was plotted against the abundance of svRNA in the PR8 reads for that sample. Lower values indicate that the mouse strain is more susceptible to PR8 (high viral titer, inflammation and weight loss); higher values indicate that the mouse strain is more resistant to PR8 (low viral titer, little inflammation, and weight loss). Pearson’s linear correlation was applied to the dataset to quantify the relationship between the variables, and the line connecting the data points represents the linear regression of the data.</p

Evaluation of total and PR8-specific reads in infected and mock-infected mice.

<p>(A) Total small RNA reads detected in mock-infected and infected mouse lungs. Lung samples were taken at two days post infection with influenza A virus PR8. Small RNAs were purified and sequenced on an Illumina Genome Analyzer IIx system. The infected mouse group contained two replicates from each mouse strain (129, CAST, PWK, WSB) and the mock-infected group contained a single sample from each mouse strain. Groups were compared with a Mann-Whitney test. (B) Percentage of influenza virus-specific reads in each mouse group. Adaptor-trimmed reads mapping uniquely to the PR8 genome are shown as a percentage of total RNA reads. Mock-infected and infected groups were compared with a Mann-Whitney test. The data are presented in a box-and-whiskers plot that uses the centerline to indicate median, the box to indicate quartiles, and the whiskers to indicate range.</p

Size and segment distributions of svRNA reads.

<p>(A) Percent of total svRNA attributed to each read length. (B) Percent of total svRNA attributed to each segment. Segments were sorted from most represented to least represented in the svRNA and segment percentages were compared with unpaired t-tests.</p

Segment-specific genome coverage of reads mapped to PR8.

<p>Influenza virus-specific reads were sorted by genomic coordinate. Strand-specific coverage of each base in the reference sequence was quantified and base coverage values for the seven infected mouse samples were normalized for the total number of PR8-specific reads in the respective sample and displayed as base coverage per thousand reads. For each position in the PR8 genome, the mean and 95% CI of the normalized coverage of the vRNA were calculated and are displayed as black and red, respectively. Nucleotide position is counted from the 5’ end of the vRNA.</p

Interferon responses to Pam2-ODN.

<p>Transcriptional responses of interferon and known interferon-sensitive antiviral genes 4 h after treatment of MLE-15 cells <i>in vitro</i> or mouse lungs <i>in vivo</i> with Pam2-ODN.</p><p>Fold change compares Pam2-ODN-treated samples to PBS-treated samples. ↔ indicates no significant change in gene expression between PBS treated and Pam2-ODN treated samples, ↑ indicates induction of transcription by Pam2-ODN, ↓ indicates repression of transcription by Pam2-ODN.</p

Pam2 treatment synergizes with all classes of TLR9-stimulating CpG oligodeoxynucleotides.

<p>Mice were challenged with influenza 1 day after a single inhaled treatment with the described treatments. Shown are survival (<b>A</b>) and body weight (<b>B</b>) of mice through 22 days after challenge (mean ± s.d.). (n = 20 mice/group; * p<0.00001 vs. PBS treated, ** p = 0.0004 vs. PBS treated, † p = 0.01 vs. Pam2+ODN 2006 treated, † p = 0.1 vs Pam2+ODN 2006 treated).</p

Inflammatory cytokine responses to Pam2-ODN.

<p>Transcriptional responses of interferon-independent inflammatory cytokines and chemokines 4 h after treatment of MLE-15 cells <i>in vitro</i> or mouse lungs <i>in vivo</i> with Pam2-ODN.</p><p>Fold change compares Pam2-ODN-treated samples to PBS-treated samples. ↔ indicates no significant change in gene expression between PBS treated and Pam2-ODN treated samples, ↑ indicates induction of transcription by Pam2-ODN, ↓ indicates repression of transcription by Pam2-ODN.</p

Synergistic TLR2/6 and TLR9 protects against influenza with or without TLR3 stimulation.

<p>Mice were challenged with influenza 1 day after a single inhaled treatment with the described treatments. Shown are survival (<b>A</b>) and body weight (<b>B</b>) of mice through 22 days after challenge (mean ± s.d.). “2x” indicates doubling of the concentration of all TLR ligand components in a corresponding “1x” treatment. (n = 20 mice/group; * p<0.00001 vs. PBS treated, ** p<0.02 vs. PBS treated, † p<0.0001 vs. poly(I∶C) treated, †† p = 0.002 vs. poly(I∶C) treated, # p = 0.004 vs. poly(I∶C) treated).</p