MicroRNA and cellular targets profiling reveal miR-217 and miR-576-3p as proviral factors during Oropouche infection

<div><p>Oropouche Virus is the etiological agent of an arbovirus febrile disease that affects thousands of people and is widespread throughout Central and South American countries. Although isolated in 1950’s, still there is scarce information regarding the virus biology and its prevalence is likely underestimated. In order to identify and elucidate interactions with host cells factors and increase the understanding about the Oropouche Virus biology, we performed microRNA (miRNA) and target genes screening in human hepatocarcinoma cell line HuH-7. Cellular miRNAs are short non-coding RNAs that regulates gene expression post-transcriptionally and play key roles in several steps of viral infections. The large scale RT-qPCR based screening found 13 differentially expressed miRNAs in Oropouche infected cells. Further validation confirmed that miR-217 and miR-576-3p were 5.5 fold up-regulated at early stages of virus infection (6 hours post-infection). Using bioinformatics and pathway enrichment analysis, we predicted the cellular targets genes for miR-217 and miR-576-3p. Differential expression analysis of RNA from 95 selected targets revealed genes involved in innate immunity modulation, viral release and neurological disorder outcomes. Further analysis revealed the gene of decapping protein 2 (DCP2), a previous known restriction factor for bunyaviruses transcription, as a miR-217 candidate target that is progressively down-regulated during Oropouche infection. Our analysis also showed that activators genes involved in innate immune response through IFN-β pathway, as STING (Stimulator of Interferon Genes) and TRAF3 (TNF-Receptor Associated Factor 3), were down-regulated as the infection progress. Inhibition of miR-217 or miR-576-3p restricts OROV replication, decreasing viral RNA (up to 8.3 fold) and virus titer (3 fold). Finally, we showed that virus escape IFN-β mediated immune response increasing the levels of cellular miR-576-3p resulting in a decreasing of its partners STING and TRAF3. We concluded stating that the present study, the first for a <i>Peribunyaviridae</i> member, gives insights in its prospective pathways that could help to understand virus biology, interactions with host cells and pathogenesis, suggesting that the virus escapes the antiviral cellular pathways increasing the expression of cognates miRNAs.</p></div

Interaction network between miRNAs and predicted target genes modulated by OROV infection.

<p>Network depicting the relation among miR-217, miR-576-3p and 92 selected target mRNAs found in at least 3 out 6 databases. Green rectangles displays the two selected up-regulated miRNAs; red ellipses represent predicted down-regulated mRNAs; gray ellipse represents mRNA predicted to be regulated by both miRNAs.</p

Relative expression levels of selected target mRNAs predicted for miR-217 and miR-576-3p.

<p>HuH-7 cells were infected with OROV at MOI 1 and RT-qPCR was done 12 h post infection (<b>A</b>) or 24 h post infection (<b>B</b>). (<b>A</b>) Data denotes mean fold change (y-axis) of infected cells relative to uninfected cells for 18 deregulated mRNA out of 95 selected targets. Gene expression was normalized by endogenous 18S, GAPDH, HPRT1 and GUSB levels. Deregulated target genes are depicted in x-axis. Black columns represent predicted targets for miR-576-3p and gray columns represent predicted targets for miR-217, respectively. Error bars represent Standard Error Mean (SEM) for 6 independent samples. ns = non-significant (0.05 ≤ p ≤ 0.1); * = p ≤ 0.05; ** = p ≤ 0.01. (<b>B</b>) Data denotes mean fold change (y-axis) of infected cells relative to uninfected cells for 6 selected targets 24 h post infection. Gene expression was normalized by endogenous GAPDH levels. Black columns represent predicted targets for miR-576-3p and gray columns represent predicted targets for miR-217, respectively. Error bars represent SD for four independent samples. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001.</p

Mean relative expression levels of the 13 differentially expressed miRNAs.

<p>Mean relative expression levels of the 13 differentially expressed miRNAs.</p

Validation of individual miRNAs expression.

<p>RT-qPCR with specific primers for individual miRNAs. Mean fold change of expression levels in OROV infected cells relative to uninfected cells for (<b>A</b>) miR-217 and (<b>B</b>) miR-576-3p. RT-qPCR was performed at 3, 6, 12 h post-infection. (<b>C</b>) RT-qPCR for miR-26a-1-3p, miR-26a-2-3p and miR-92a-1-5p was performed only at 12 h post-infection time point. MicroRNAs expression was normalized by U6 RNA endogenous levels. Error bars represent SD of triplicates of three independent experiments. Asterisks represent significant values compared to non-infected cells. * = p ≤ 0.05; ** = p ≤ 0.01.</p

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IFN-β antiviral response is attenuated during infection.

<p>HuH-7 cells were infected with OROV at MOI 1 and RT-qPCR was done at indicated post-infection time points. (<b>A</b>) Normalized expression of IFN-β mRNA during infection. HuH-7 cells were infected with OROV at MOI 1 and IFN-β levels were measured at indicated post-infection time points. Error bars represent SD for four independent infections. Data denotes mean fold change (y-axis) of infected cells relative to uninfected cells for (<b>B</b>) STING and (<b>C</b>) TRAF3 RNA levels. Gene expression was normalized by endogenous GAPDH levels. Error bars represent SD for four independent samples. ns = non-significant (p ≤ 0.1); * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001. (<b>D</b>) Schematic representation of IFN-β and miR-576-3p interplay in antiviral response during OROV infection. Values are relative to uninfected cells. miR-576-3p, IFN-β, STING and TRAF3 transcripts levels are represented by green, blue, red and yellow lines, respectively.</p

Enrichment pathway analysis of predicted targets for miR-217 and miR-576-3p.

<p>Gene set enrichment analysis (GSEA) was performed for all predicted targets using gene ontology available in GO, KEGG and REACTOME databases. Over-represented pathways are depicted in the y-axis. Quantity of genes associated to a specific pathway is represented in the x-axis. Black columns represent the quantity of genes expected to be associated to a determined pathway and gray columns represent the observed quantity of genes associated to the same pathway, respectively. Significance of overlapping is represented by the negative log of P-value for each pathway.</p

Inhibition of miR-217 and miR-576-3p affects OROV replication.

<p>HuH-7 cells were transfected with 75 nM negative control inhibitor, miR-217 inhibitor, miR-576-3p inhibitor or both. 3 h post transfection, cells were infected with OROV at MOI 1 and RT-qPCR was performed to measure miRNA, target genes and OROV RNA levels. (<b>A</b>) Fold change of miRNA expression levels 6 h post-infection. MiRNA levels were normalized by U6 levels and infected cells were compared to uninfected cells, both pre-transfected with the respective inhibitor for each condition. Cells transfected with negative inhibitor followed by OROV infection were compared to uninfected cells transfected with the same inhibitor and considered as positive control (set as 1, y-axis) for comparison. Black columns represent miR-217 expression levels and gray columns represent miR-576-3p levels. NQ–not quantified. (<b>B</b>) Fold change of target genes RNA levels 18 h post infection. Target genes expression was normalized by GAPDH RNA levels and deregulation was measured relative to the same target in uninfected cells. Black columns represent DCP2 RNA levels and gray columns represent STING RNA levels. (<b>C</b>) Intracellular OROV segment S RNA levels 18 h post-infection. Positive control (cells transfected with negative inhibitor and infected with OROV) levels were set as 1 (y-axis) for comparison. (<b>D</b>) Viral titration of virus supernatants from experiments performed in <b>C</b>. Viruses in the supernatant were quantified by plaque assay 18 h post-infection and plotted as pfu/ml x 10⁶ (y-axis). Error bars represent SD of triplicates for two independent experiments. NS = non-significant; * = p ≤ 0.05; ** = p ≤ 0.01.</p

Oropouche infection in blood and hepatocyte cell lines.

<p>Jurkat, THP-1 and HuH-7 cells were infected with OROV at MOI 1. At 12 h post infection, infectivity was assessed by flow cytometry using polyclonal anti-OROV antibody. (<b>A</b>) Infection in T lymphocyte (Jurkat), monocyte (THP-1) and hepatocyte (HuH-7) cell lines (x-axis) was performed as described in materials and methods section. Error bars represents standard deviation (SD) of four independent experiments. THP-1 NA = THP-1 not activated; THP-1 (24) = activated for 24 h in PMA; THP-1 (8 d) = activated for 3 days in PMA followed by 5 days without PMA. (<b>B</b>) OROV infection in Huh-7 cells at different MOIs (x-axis). Infectivity was assessed by flow cytometry. Error bars represents SD. (<b>C</b>) Relative cell viability of HuH-7 cells infected with OROV (MOI 1) at different time points. Cell viability was assessed by fluorimetric assay. Absolute values of uninfected cells were set as 1 (left y-axis). Black columns represent uninfected cells and gray columns represent OROV infected cells (MOI 1). Error bars represents SD for five replicates of two independent experiments. Gray line represents mean virus titer in supernatant measured by plaque assay at indicated time points (right y-axis). ** = <i>p</i> ≤ 0.01.</p