Guanine quadruplexes as regulatory elements of the SARS-COV-2 virus

DIEDRICH, L.; BEZZI, G.; BINOLFI, A.; ARMAS, P.

Rosario

Congreso; LIX Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB); 2023

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The coronavirus disease 2019 (COVID-19) pandemic was triggered by the RNA virus that causes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The replication cycle of this virus depends on different RNA molecules that act not only as viral genome but also as intermediates and templates for several processes such as viral protein translation, RNA dependent RNA synthesis for replication and transcription. Guanine quadruplexes (G4s) are non-canonical secondary structures formed by nucleic acids (DNA or RNA), that function as transcriptional and translational regulatory elements originally and mainly described in human oncogenes. Additionally, G4s have been implicated in the control of a variety of biological processes, including viral replication. Using various G4 computational prediction tools, we found putative G4-forming sequences (PQS) within the SARS-CoV-2 RNA genome (positive sense genomic RNA or +gRNA) and the negative sense genomic RNA (-gRNA) that acts as an intermediate RNA during viral replication. Some of the identified PQS are conserved in various betacoronaviruses related to SARS-CoV-2 while others are exclusive to SARS-CoV-2. Using multiple spectroscopic and biophysical techniques, we confirmed the formation of three G4s in the +gRNA and five G4s in the -gRNA of SARSCoV-2, several of which had not been previously experimentally characterized. We performed CD melting assays in the presence and absence of pyridostatin (PDS), a specific ligand that binds and stabilizes G4s, and demonstrated that the ligand stabilizes most assayed G4s in both +gRNA and -gRNA. On the other hand, spectroscopic and biochemical approaches were used to demonstrate that CNBP, the major human cellular protein bound to the SARS-CoV-2 RNA genome, binds to and promotes the unfolding of most of the G4s formed both in the +gRNA as well as in the -gRNA of SARS-CoV-2. Employing molecular and cellular biology techniques, we assayed the function of the G4s formed by three PQSs of +gRNA on translation in a cellular context. In addition, we studied the effect of one G4 formed by a PQS of the +gRNA on the programmed ribosomal frameshifting, an essential mechanism for viral gene expression. Finally, a computational search for sequence variations on the studied SARS-CoV-2 PQSs allowed us to identify two high-frequency variations that could affect G4s formation in viral variants of interest or concern, encouraging further study on the effects of these variations. Our results suggest that the G4s found in the SARS-CoV-2 RNA genome and their negative-sense replicative intermediates, as well as the cellular proteins and/or ligands that interact with them, are relevant factors for the regulation of viral gene expression and/or for the control of the viral replication cycle, and may constitute interesting targets for the development of antiviral drugs.