Human MicroRNAs Interacting With SARS-CoV-2 RNA Sequences: Computational Analysis and Experimental Target Validation

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel RNA virus affecting humans, causing a form of acute pulmonary respiratory disorder named COVID-19, declared a pandemic by the World Health Organization. MicroRNAs (miRNA) play an emerging and important role in the interplay between viruses and host cells. Although the impact of host miRNAs on SARS-CoV-2 infection has been predicted, experimental data are still missing. This study started by a bioinformatics prediction of cellular miRNAs potentially targeting viral RNAs; then, a number of criteria also based on experimental evidence and virus biology were applied, giving rise to eight promising binding miRNAs. Their interaction with viral sequences was experimentally validated by transfecting luciferase-based reporter plasmids carrying viral target sequences or their inverted sequences into the lung A549 cell line. Transfection of the reporter plasmids resulted in a reduction of luciferase activity for five out of the eight potential binding sites, suggesting responsiveness to endogenously expressed miRNAs. Co-transfection of the reporter plasmids along with miRNA mimics led to a further and strong reduction of luciferase activity, validating the interaction between miR-219a-2-3p, miR-30c-5p, miR-378d, miR-29a-3p, miR-15b-5p, and viral sequences. miR-15b was also able to repress plasmid-driven Spike expression. Intriguingly, the viral target sequences are fully conserved in more recent variants such as United Kingdom variant B.1.1.7 and South Africa 501Y.V2. Overall, this study provides a first experimental evidence of the interaction between specific cellular miRNAs and SARS-CoV-2 sequences, thus contributing to understanding the molecular mechanisms underlying virus infection and pathogenesis to envisage innovative therapeutic interventions and diagnostic tools.

Keywords: COVID-19; RNA virus; SARS-CoV-2; anti-viral; microRNA; non-coding RNA.

FIGURE 1

Prediction of human microRNA targets in the genomic RNA of SARS-CoV-2. Selection was performed with RNAhybrid 2.2 (Krüger and Rehmsmeier, 2006) and MirTarget (Chen and Wang, 2020). Seed regions are marked in bold. Nucleotide numbering refers to isolate Wuhan-Hu-1, complete genome (NC_045512.2). ¹Microarrays data from the Human miRNA Tissue Atlas at https://ccb-web.cs.uni-saarland.de/tissueatlas/ (Ludwig et al., 2016). Readings of chip hybridization signals had been normalized according to Huber et al. (2002).

FIGURE 2

Screening of viral sequences for responsiveness to selected miRNAs in the A549 cell line. SARS-CoV-2 sequences predicted to be targeted by the indicated miRNAs were cloned in the reporter vector psiCheck-2. 48 h after transfection, luciferase activities were recorded; the Renilla luciferase activity (Rl) was normalized to the firefly luciferase activity (Luc), whose gene is also contained in the reporter vector; the values are reported as fold mean value ± s.d. relative to the luciferase activity determined for parental vector psiCheck-2 transfection (V), which was set to 1. p-values at Student’s t-test were *p < 0.05, **p < 0.01, or ***p < 0.001.

FIGURE 3

Silencing effects toward viral sequences are modulated by synthetic miRNA mimics. (A) A549 cells were transfected with the luciferase-based reporter constructs containing the viral target sequence of indicated miRNA (WT-miRNA abbreviation name) or the control plasmids with inverted target sequence (I-miRNA abbreviation name), along with 50 nM (+) of the specific miRNA mimic, or unrelated molecule used as negative control, Ctrl-miRNA. The values are reported as fold mean ± s.d. relative to Rl/Luc recorded for transfections of control molecules of each series (specific I construct and Ctrl-miRNA), which were set to 1. (B) A549 were transfected with Spike-expressing plasmid (pSpike) along with Ctrl-miRNA or miR-15b; 48 h after transfection, Spike expression was quantitated by RT-qPCR. Data are the mean ± s.d. of two independent experiments, each with three data sets. *, **, or ***, p-values as indicated in Figure 2.