Secondary Structure of Subgenomic RNA M of SARS-CoV-2

SARS-CoV-2 belongs to the Coronavirinae family. Like other coronaviruses, SARS-CoV-2 is enveloped and possesses a positive-sense, single-stranded RNA genome of ~30 kb. Genomic RNA is used as the template for replication and transcription. During these processes, positive-sense genomic RNA (gRNA) and subgenomic RNAs (sgRNAs) are created. Several studies presented the importance of the genomic RNA secondary structure in SARS-CoV-2 replication. However, the structure of sgRNAs has remained largely unsolved so far. In this study, we probed the sgRNA M model of SARS-CoV-2 in vitro. The presented model molecule includes 5′UTR and a coding sequence of gene M. This is the first experimentally informed secondary structure model of sgRNA M, which presents features likely to be important in sgRNA M function. The knowledge of sgRNA M structure provides insights to better understand virus biology and could be used for designing new therapeutics

Structural and Functional RNA Motifs of SARS-CoV-2 and Influenza A Virus as a Target of Viral Inhibitors

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic, whereas the influenza A virus (IAV) causes seasonal epidemics and occasional pandemics. Both viruses lead to widespread infection and death. SARS-CoV-2 and the influenza virus are RNA viruses. The SARS-CoV-2 genome is an approximately 30 kb, positive sense, 5′ capped single-stranded RNA molecule. The influenza A virus genome possesses eight single-stranded negative-sense segments. The RNA secondary structure in the untranslated and coding regions is crucial in the viral replication cycle. The secondary structure within the RNA of SARS-CoV-2 and the influenza virus has been intensively studied. Because the whole of the SARS-CoV-2 and influenza virus replication cycles are dependent on RNA with no DNA intermediate, the RNA is a natural and promising target for the development of inhibitors. There are a lot of RNA-targeting strategies for regulating pathogenic RNA, such as small interfering RNA for RNA interference, antisense oligonucleotides, catalytic nucleic acids, and small molecules. In this review, we summarized the knowledge about the inhibition of SARS-CoV-2 and influenza A virus propagation by targeting their RNA secondary structure

A Conserved Secondary Structural Element in the Coding Region of the Influenza A Virus Nucleoprotein (NP) mRNA Is Important for the Regulation of Viral Proliferation - Fig 6

<p><b>A.</b> (+) RNA5 motif with marked complementary region to anisense oligonucleotides. In green were marked differences in sequence between A/California/04_NYICE_E3/2009 strain and consensus sequence of M121. <b>B.</b> Effect of antisense oligonucleotides targeting M121 motif of scIAV A/California/04_NYICE_E3/2009 in cell line MDCK-HA. C—control; L—control with lipofectamine, R—positive control with ribavirin; N—negative control with oligonucleotide N.</p

Predicted structure probability of M121 by RNAstructure 5.5 program.

<p>Predicted structure probability of M121 by RNAstructure 5.5 program.</p

Secondary structure of M121 predicted by RNAalifold and bioinformatics calculations for possibility of each base pair.

<p><b>A.</b> Base pairs in secondary structure are colored according to % of canonical base pairs calculated for type A influenza (last column of table): red >98%; 95 ≤ orange < 98%; 90 ≤ green < 95%; 85 ≤ blue < 90%. <b>B.</b> In table are marked preserving mutations: indicated by green shadow are evidence of compensatory mutation.</p

Primers for PCR and transcription of M121 and (+)RNA5.

<p>Primers for PCR and transcription of M121 and (+)RNA5.</p

Secondary structure of influenza RNA motif.

<p><b>A.</b> M121, isolated RNA with consensus sequence; secondary structure was predicted by RNAstructure 5.5 using chemical mapping results from SHAPE. <b>B.</b> Chemical mapping results of M121 in entire segment 5 (+)RNA (A/VietNam/1203/2004 (H5N1)); in blue is marked difference in sequence comparing to M121 on panel A.</p

Lead ion cleavage of M121.

<p>RNA was incubated with 1 mM Pb(OAc)₂, 100 mM KCl and 5 mM MgCl₂, 10 mM Tris-HCl pH 7 in time course: lanes 1–6 - 0, 1, 5, 15, 30 and 60 min, respectively. Lane 7—control reaction: M121 incubated in 100 mM KCl and 5 mM MgCl₂, 10 mM Tris-HCl pH 7 for 60 min. Lane 8—RNase T1 ladder. Lane 9—formamide ladder.</p

Enzymes mapping of M121.

<p>All reactions were conducted in 23°C for 30 min. Lane 1 –control reaction: M121 incubated in 100 mM KCl and 5 mM MgCl₂, 10 mM Tris-HCl, pH 7, for 30 min in 23°C. Lanes 2–4—RNase V1 cuts in increasing concentration of enzyme: 0.5x10^-3 U/μl, 1x10^-3 U/μl and 3x10^-3 U/μl, respectively. Lanes 5–7—RNase T1 cuts in increasing concentration of enzyme: 0.15 U/μl, 0.25 U/μl and 0.75 U/μl, respectively. Lanes 8–10—RNase S1 cuts in increasing concentration of enzyme: 0.05 U/μl, 0.3 U/μl, 1 U/μl, respectively, Lane 11—formamide ladder, Lane 12—RNase T1 ladder.</p