GUANINE QUADRUPLEXES AS POTENTIAL REGULATORY ELEMENTS OF THE SARS-COV-2 VIRUS

ARMAS, P.; PIGA, E.J.; BEZZI, G.

online

Congreso; LVI SAIB Meeting - XV SAMIGE Meeting - SAIB-SAMIGE Joint Meeting 2020 on line; 2020

SAIB-SAMIGE

G-quadruplexes (G4) are non-canonical secondary structures formed by nucleic acids (DNA or RNA). These secondary structures have been proposed as new transcriptional and translational regulatory elements that were originally and mainly described in oncogenes. Bioinformatics predictions using a specific search algorithm for the consensus sequence have revealed the widespread presence of putative G4 forming sequences (PG4s) in a large number of species belonging to all kingdoms of life, as well as in various RNA and DNA viruses. The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has quickly turned into a global public health emergency. Understanding the underlying mechanisms and developing innovative treatments is extremely urgent. SARS-CoV-2 is a positive-sense, single-stranded RNA virus that employs a variety of unusual strategies involving different RNA molecules to accomplish its replication. Translation of the positive-strand genomic RNA (+gRNA) gives rise to a large polyprotein that undergoes proteolytic processing to generate an RNA-dependent RNA polymerase (RdRp). RdRp uses +gRNA as template to synthesize a full-length antisense negative-strand RNA (-gRNA) and shorter negative subgenomic RNAs (-sgRNAs). Then, -gRNA and -sgRNAs are used as templates by RdRp to synthesize the +gRNA for encapsidation and positive subgenomic RNAs (+sgRNAs) for translation of structural viral proteins. In this work, we characterized the PG4 on both the + and -gRNA using five G4folding predictors. Although no PG4s of three tetrads were found, we could identify PG4s of two tetrads with the consensus sequence G2N1-15G2N1-15G2N1-15G2 (37 PG4 in the +gRNA and 19 in the ?gRNA). Moreover, we evaluated the conservation of these PG4s in 4 members of the Coronaviridae family evolutionarily related to SARS-CoV-2. Five PG4s (3 in +gRNA and 2 in -gRNA) that presented the highest scores in the G4 predictors (indicating a high probability of G4 formation) and were highly conserved among other betacoronaviruses were selected for further analyses. PG4s in the +gRNA are located within the ORF1ab and nucleocapsid (N) genes. We analyzed in vitro the formation, topology of structure and stability using multiple spectroscopic, biochemical and biophysical techniques. We confirmed that four of the best ranked PG4s (at positions 644 and 3467 of the +gRNA and position -23877 and -13963 in the ?gRNA) effectively fold as G4. We also evaluated the action of the nucleic acids chaperone protein CNBP on SARS-CoV-2 G4s. CNBP was reported to unfold G4 and has been recently informed as the main cellular protein that directly binds SARS-CoV-2 +gRNA and is induced upon infection. Our results provide novel nucleic acids structural elements probably involved in SARS-CoV-2 replicative cycle and suggest that G4s should be considered as suitable targets for antiviral therapeutic strategies against SARS-CoV-2 and treatment of COVID-19.