In silico identification of conserved cis-acting RNA elements in the SARS-CoV-2 genome

Abstract

Aim: The aim of this study was to computationally predict conserved RNA sequences and structures known as cis-acting RNA elements (CREs) in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome.

Materials & methods: Bioinformatics tools were used to analyze and predict CREs by obtaining viral sequences from available databases.

Results: Computational analysis revealed the presence of RNA stem-loop structures within the 3' end of the ORF1ab region analogous to previously identified SARS-CoV genomic packaging signals. Alignment-based RNA secondary structure predictions of the 5' end of the SARS-CoV-2 genome also identified conserved CREs.

Conclusion: These CREs may be potential vaccine and/or antiviral therapeutic targets; however, further studies are warranted to confirm their roles in the SARS-CoV-2 life cycle.

Keywords: RNA structure; RNA virus; SARS-CoV-2; cis-acting RNA elements.

Figure 1.. Prediction of minimum free energy structures within the severe acute respiratory syndrome coronavirus 2 ORF1b region.

The SARS-CoV-2 genome is about 30 kb in length and organized in 13 ORFs. The viral genome is flanked by the 5′ and 3′ UTRs. The ORF1b region is highlighted in red (upper panel) and a mounting plot represents the prediction of the MFE structures spanning from nt position 15,000–21,541 of the SARS-CoV-2 genome sequences (NC_045512.2) is shown with the highest peak of MFE prediction located between 19,000 and about 20,300 nt of the SARS-CoV-2 ORF1b region (lower panel). Note that the mountain plot indicates secondary structure in a plot of height versus sequence position, where the peaks represent the probabilities of RNA base pairing. MFE: Minimum free energy; nt: Nucleotide; ORF: Open reading frame; SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2; UTR: Untranslated region.

Figure 2.. Prediction of ORF1b RNA secondary structures.

RNA secondary structures located at the 3′ end of the SARS-CoV-2 ORF1b region (nucleotide positions: 19,712–20,294) were predicted using the Mfold web server (A) and compared with RNA secondary structures at the same position for SARS-CoV (NC_004718.3) (B) and bat SARS-like CoV (MG772933) (C). The two identical RNA stem-loops (SL1 and SL2) are indicated in a rectangle among the three viruses. SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2.

Figure 3.. Comparison of the predicted RNA secondary structures among Severe Acute Respiratory Syndrome Coronavirus 2.

The viral RNA sequences in the 3′ end of the ORF1b region from 19,900 to 20,019 nucleotide for both SARS-CoV-2 and bat SARS-like CoV and in the region of 19,830–19,949 nucleotide for SARS-CoV, were folded using the Mfold web server and drawn using the VARNA web applet to compare predicted RNA stem-loops. The similarities of the RNA structures of the predicted SL1 and SL2 among the three viruses are highlighted in green and blue, respectively. In addition, the sequence variation in these two stem-loops are indicated in black. These two RNA stem-loops are part of the previously identified SARS-CoV genomic PS, with SL2 being identified as the PS_core [21,22]. SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2.

Figure 4.. Computational prediction of RNA secondary structures model at the severe acute respiratory syndrome coronavirus 2 5′-terminal region.

Schematic representation of the RNA secondary structures at the 5′ region of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome (nucleotide positions: 1–474) was generated using the Mfold web server and drawn using the VARNA web applet (ΔG = -155.50 kcal/mol). The eight RNA stem-loops (SL1–SL8) are indicated and the conserved core leader transcriptional regulatory sequence (TRS) region located within the SL3 is highlighted in gray. In addition, the conserved 5′-UUUCGU-3′ motifs found within SL5A and SL5B are indicated in boxes and the AUG start codon of ORF1ab is indicated in red. The SARS-CoV-2 reference genome sequence obtained from GenBank (NC_045512.2) was used.

Figure 5.. Alignment-based prediction of the RNA structures at the 5′-terminal regions of severe acute respiratory syndrome coronavirus 2 genomes.

The first 474 nucleotides of SARS-CoV-2, bat SARS-like CoV and SARS-CoV genomes were aligned using LocARNA to predict conserved RNA stem-loops at the 5′ ends. Colors indicate the conservation of base pair types in which red represents conservation of one base pair and yellow and green indicate conservation of two and three base pairs, respectively. Dark color represents that all three viruses contained this base pair, whereas light color shows that one or two of the sequences did not contain the base pairs. SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2.