A set-up to identify new drugs to inhibit SARS-CoV-2 replication

CELAYES, MARÍA EMILIA; DELGUI, LAURA RUTH; MAYORGA, LUIS; POLO, MARIANO

Virtual

Encuentro; XXXIX Reunión Científica Anual de la Sociedad De Biología De Cuyo; 2021

Severe Acute Respiratory Syndrome-CoronaVirus-2 (SARS-CoV-2) is the causative agent of the atypical pneumonia disease(COVID-19). This previously unknown virus was identified in China and has spread worldwide, causing millions of deathsglobally (https://covid19.who.int/). To date, no specific antiviral therapies against the new SARS-CoV-2 are available.SARS-CoV-2 belongs to the Coronaviridae family, with a single-stranded positive-sense RNA genome. SARS-CoVs genomicsequences include the large protein named non-structural protein 3 (NSP3). That protein contains seven domains which areessential components for viral replication and transcription process. One of those domains is unique for SARS-CoVs (SARSuniquedomain, SUD). That exclusiveness suggests that SUD can be a critical factor involved in the severe pathogenicitycaused by these viruses. Two important features have been previously described regarding SARS-CoVs SUD. On the onehand, it was shown that SUD interacts with oligonucleotides known to form G-quadruplexes (G4), which are self-assembledsecondary structures of nucleic acids composed of tetrads of poly-guanine sequences. On the other hand, the residues mediatingSUD-G4 interaction are essential for SARS-CoV infection.It was recently published that SARS-CoV-2 genomes contain sequences that can form G4s. It has also been demonstrated thatSUD can interact with cellular G4 RNAs, and that this interaction is critical in determining the course of the viral infection.Thus, we hypothesise that drugs hampering SUD-G4 interaction would be viable therapeutic options for COVID-19. Here, weshow a set-up system that allows us to find drugs impeding SUD-G4 interaction to happen, so viral replication is suppressed.To do that, we first verified that SUD is present in the SARS-CoV-2 strain, and it is highly conserved compared to SARSCoVSUD. After analysing genome sequences from over 7.5 thousand recently reported SARS-CoV-2 isolates, we observedvery low variation frequencies of SUD residues. Later we cloned the domain and generated a mutant that is unable to bindDNA as a control. Then, we expressed those constructions using E. coli and purified the proteins by affinity chromatography.We finally generated G4-DNA structures using custom oligonucleotides that we use to measure SUD-G4 interaction byElectrophoretic Mobility Shift Assay (EMSA).We propose to use this set-up to identify drugs interfering with SUD-G4 interactions. Those can be natural products, new orrepurposed drugs. And