Whole Genome Identification of Potential G-Quadruplexes and Analysis of the G-Quadruplex Binding Domain for SARS-CoV-2

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has become a global public health emergency. G-quadruplex, one of the non-canonical secondary structures, has shown potential antiviral values. However, little is known about the G-quadruplexes of the emerging SARS-CoV-2. Herein, we characterized the potential G-quadruplexes in both positive and negative-sense viral strands. The identified potential G-quadruplexes exhibited similar features to the G-quadruplexes detected in the human transcriptome. Within some bat- and pangolin-related betacoronaviruses, the G-tracts rather than the loops were under heightened selective constraints. We also found that the amino acid sequence similar to SUD (SARS-unique domain) was retained in SARS-CoV-2 but depleted in some other coronaviruses that can infect humans. Further analysis revealed that the amino acid residues related to the binding affinity of G-quadruplexes were conserved among 16,466 SARS-CoV-2 samples. Moreover, the dimer of the SUD-homology structure in SARS-CoV-2 displayed similar electrostatic potential patterns to the SUD dimer from SARS. Considering the potential value of G-quadruplexes to serve as targets in antiviral strategy, our fundamental research could provide new insights for the SARS-CoV-2 drug discovery.

CNBP Binds and Unfolds In Vitro G-Quadruplexes Formed in the SARS-CoV-2 Positive and Negative Genome Strands

“…During the COVID-19 pandemic, mainly along the last half of 2020, several works analyzed the SARS-CoV-2 RNA genome to seek PQSs. All of them analyzed the +gRNA [ 30 , 31 , 32 , 33 , 34 , 35 , 36 ], while a few made a superficial overview of PQSs on the −gRNA [ 32 , 33 , 36 ]. Although −gRNA and −sgRNAs are minority in respect of their positive-sense RNA counterparts and represent only about 1% of viral RNA [ 9 , 10 ], negative-sense RNAs are key intermediates functioning as templates for +gRNA replication and +sgRNAs transcription.…”

“…Consequently, −gRNA and −sgRNAs may contain G4s with putative regulative functions on these processes. In addition, most of the previous PQSs analyses on SARS-CoV-2 genome have used different bioinformatics prediction tools, some of them designed for DNA-G4, and a variety of criteria for selecting the best PQS candidates, mainly including higher prediction scores [ 30 , 31 , 32 , 33 , 34 , 35 , 36 ] combined with lower potential thermodynamic stability of secondary structures competitive with G4s [ 32 ], uniqueness in SARS-CoV-2 and conservation among variants of SARS-CoV-2 [ 34 ], and conservation among human coronaviruses [ 36 ]. Although there is divergence in G4 prediction tools and selection criteria, none of the predictions have found PQSs with four tracts of three consecutive guanines (with the potential of forming three-tetrads G4s), and have only found PQSs with four tracts of two consecutive guanines (with the potential of forming two-tetrads G4s).…”

“…SARS-CoV SUD binding to viral and/or cellular RNAs with G4s could affect their stability and translation, thus controlling the host cell’s response to the viral infection [ 69 ]. Nsp3 of SARS-CoV-2 contains a similar SUD predicted to conserve critical amino acids for G4 binding [ 32 , 33 , 36 , 78 ], thus probably sharing with SARS-CoV the viral pathogenic mechanism dependent on nsp3-SUD interaction with G4 structures. Another viral protein, the nsp13 with RNA helicase activity, was also informed as able to bind and probably unfold viral RNA G4s, thus favoring viral translation, transcription, and replication processes [ 30 ].…”

“…Critical roles for viral G4s have been described in human viruses, including immunodeficiency virus (HIV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), human papillomavirus (HPV), hepatitis B virus (HBV), Nipah virus, hepatitis C virus (HCV), Zika virus, and Ebola virus [ 27 , 28 , 29 ], and some G4-specific compounds have shown powerful antiviral activity by targeting G4 structures [ 28 , 29 ]. Very recent reports have initiated the search for G4s in the genomes of human coronaviruses, including SARS-CoV-2 [ 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. Beyond the prediction of PQSs, some works have demonstrated the formation of a few G4s in vitro by PQSs found in the +gRNA [ 30 , 34 , 35 , 36 ] and one of them was demonstrated to be formed within cultured human cells and controls the translation efficiency of the nucleocapsid N protein [ 35 , 36 ].…”

Structure and regulation of coronavirus genomes: state-of-the-art and novel insights from SARS-CoV-2 studies

“…Several studies have further started investigating the presence of RNA G-quadruplexes (G4s) in the SARS-CoV-2 genome [73][74][75][76]. Of the many predicted G4s, one G4 located within ORF1a ( position 13385) was shown to bind and to be stabilized by BRACO-19 and TMPyP4, two known G4 binders, suggesting that these compounds might represent good scaffolds for the development of RNA-targeted small-molecule drugs [74].…”