In vivo secondary structural analysis of Influenza A virus genomic RNA.

Influenza A virus (IAV) is a respiratory virus that causes epidemics and pandemics. Knowledge of IAV RNA secondary structure in vivo is crucial for a better understanding of virus biology. Moreover, it is a fundament for the development of new RNA-targeting antivirals. Chemical RNA mapping using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) coupled with Mutational Profiling (MaP) allows for the thorough examination of secondary structures in low-abundance RNAs in their biological context. So far, the method has been used for analyzing the RNA secondary structures of several viruses including SARS-CoV-2 in virio and in cellulo. Here, we used SHAPE-MaP and dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) for genome-wide secondary structure analysis of viral RNA (vRNA) of the pandemic influenza A/California/04/2009 (H1N1) strain in both in virio and in cellulo environments. Experimental data allowed the prediction of the secondary structures of all eight vRNA segments in virio and, for the first time, the structures of vRNA5, 7, and 8 in cellulo. We conducted a comprehensive structural analysis of the proposed vRNA structures to reveal the motifs predicted with the highest accuracy. We also performed a base-pairs conservation analysis of the predicted vRNA structures and revealed many highly conserved vRNA motifs among the IAVs. The structural motifs presented herein are potential candidates for new IAV antiviral strategies.

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.