The emergence of the current coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen, warrants the need for collaborative research in finding therapeutic targets.
Developing drugs for severe COVID-19 can help patients to recover faster and can reduce mortality in those that become critically unwell. Apart from viral proteins, exploring the structured ribonucleic acid (RNA) elements of SARS-CoV-2 – its genetic code – provides a promising alternative as antiviral drug targets.
An international team of researchers have observed the RNA folding structures of the SARS-CoV-2 genome that aids the infection process. These structures are akin to various other betacoronaviruses, which means that the discovery could help find drugs not only for COVID-19 but also for new coronaviruses that may emerge in the future.
SARS-CoV-2's genetic code
The SARS-CoV-2 virus has a genetic code of 29,902 characters, connected through a long RNA molecule. It comprises the information for producing 27 proteins, which is significantly less than the 40,000 types of proteins that a human cell can produce. The virus hijacks its host cell's metabolic processes to instigate viral replication. Viruses can accurately control the synthesis of this process.
The novel coronavirus utilizes the spatial folding of its RNA hereditary molecule to control protein production, especially in areas that do not code for the viral proteins. To date, evidence for this has been based on computer analysis.
Characterizing the structure of regulatory elements
In the study, which was published in the journal Nucleic Acids Research, the researchers systematically characterized the secondary structures of all conserved cis-acting RNA elements of SARS-CoV-2 by nuclear magnetic resonance (NMR) spectroscopy. This procedure exposes the atoms of the RNA to a strong magnetic field and provides high-resolution experimental secondary structure models.
The team analyzed nine RNA constructs representing the eight system-loop (SL) domains present at the genomic 5’-end, two RNA constructs that correspond to the cis-acting elements from the open reading frame 1a (ORF1a), and four RNA constructs that represent the functional SLs within the viral 3′-UTR.
The researchers then characterized the structure of a total of 15 regulatory elements. They compared the findings of the procedure with the findings from a chemical process involving dimethyl sulfate footprint, allowing RNA single strand regions to be differentiated from the RNA double strand regions.
"Our findings have laid a broad foundation for future understanding of how exactly SARS-CoV2 controls the infection process. Scientifically, this was a huge, very labor-intensive effort which we were only able to accomplish because of the extraordinary commitment of the teams here in Frankfurt and Darmstadt together with our partners in the COVID-19-NMR consortium," Professor Harald Schwalbe from the Center for Biomolecular Magnetic Resonance at Goethe University Frankfurt said.
"But the work goes on: together with our partners, we are currently investigating which viral proteins and which proteins of the human host cells interact with the folded regulatory regions of the RNA and whether this may result in therapeutic approaches," he added.
The team also noted that apart from the analysis of individual cis-elements, experimental data on the full-length RNA will guide further detailed structural studies. For instance, DMS footprinting suggested an additional helical part for 5_SL4, facilitating both RNA translation and replication.
An alternative model for SARS-CoV-2 is proposed by computational predictions, wherein a small SL forms directly downstream of SL4. For this type of investigation, NMR is the method of choice to delineate which of the two secondary structures form in an RNA construct that covers the complete sequence.
"In general, we provide a thorough experimental validation of phylogenetic-based in silico models for the RNA elements characterized in this study," the researchers wrote in the study.
"Differences found between prediction and experimental data show a trend toward fewer stable base pairs in the experimentally determined structures," they added.
The researchers explained that it is reasonable to expect very similar functional properties for cis-acting RNAs in SARS-CoV-2, and the severe acute respiratory syndrome coronavirus (SARS-CoV), the pathogen responsible for the SARS epidemic in 2002.
This study has provided an extensive set of chemical shift data, which will become a reliable basis for further research on the SARS-CoV-2 RNA genome. It opens up new avenues for investigation into alternate RNA-led therapies and could facilitate major progress toward fighting COVID-19.