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

Pablo Armas,Ernesto J. Piga,Georgina Bezzi,Andrés Binolfi

doi:10.3390/ijms22052614

Abstract

The Coronavirus Disease 2019 (COVID-19) pandemic has become a global health emergency with no effective medical treatment and with incipient vaccines. It is caused by a new positive-sense RNA virus called severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). G-quadruplexes (G4s) are nucleic acid secondary structures involved in the control of a variety of biological processes including viral replication. Using several G4 prediction tools, we identified highly putative G4 sequences (PQSs) within the positive-sense (+gRNA) and negative-sense (−gRNA) RNA strands of SARS-CoV-2 conserved in related betacoronaviruses. By using multiple biophysical techniques, we confirmed the formation of two G4s in the +gRNA and provide the first evidence of G4 formation by two PQSs in the −gRNA of SARS-CoV-2. Finally, biophysical and molecular approaches were used to demonstrate for the first time that CNBP, the main human cellular protein bound to SARS-CoV-2 RNA genome, binds and promotes the unfolding of G4s formed by both strands of SARS-CoV-2 RNA genome. Our results suggest that G4s found in SARS-CoV-2 RNA genome and its negative-sense replicative intermediates, as well as the cellular proteins that interact with them, are relevant factors for viral genes expression and replication cycle, and may constitute interesting targets for antiviral drugs development.

Highlights

By the end of 2019, an unexpected outbreak of a new severe acute respiratory syndrome (SARS) termed by the World Health Organization (WHO) as Coronavirus Disease2019 (COVID-19) emerged in Wuhan (China) [1]
−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
These processes are mediated by the replicase-transcriptase complex (RTC) formed by several non-structural proteins [9,10]

Summary

Introduction

By the end of 2019, an unexpected outbreak of a new severe acute respiratory syndrome (SARS) termed by the World Health Organization (WHO) as Coronavirus Disease. The organization of the coding regions for the main viral proteins arefor represented for each location of the selected PQSs in the analyzed genomes. We have used a novel G4 prediction pipeline for the identification of the PQSs with high probability of G4 formation within the +gRNA and −gRNA of SARS-CoV-2 and related betacoronaviruses. We performed biophysical and molecular approaches to demonstrate for the first time that cellular nucleic acid binding protein (CNBP), the main cellular protein bound to SARS-CoV-2 RNA genome in infected human cells [19], binds and promotes the unfolding of G4s formed by both +gRNA and −gRNA of SARS-CoV-2. G4s found in SARS-CoV-2 +gRNA and −gRNA, and the cellular proteins that interact with them, are important elements for viral replication cycle and may be novel targets for developing antiviral drugs against COVID-19

G4 Prediction and Selection in the SARS-CoV-2 Positive and Negative Genome

Information of selected

Confirmation That the Selected PQSs Fold In Vitro as G4

CNBP binds and unfolds the

Integration of Results with Putative Functions of G4 and CNBP in SARS-CoV-2

Possible

Bioinformatics

Oligonucleotides

ThT Fluorescence Assays

CNBP Expression and Purification