SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction

Abstract

The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds.

Pressure Perturbation Studies of Noncanonical Viral Nucleic Acid Structures

“…Recently, potentially GQ-forming sequences were found in several viral genomes, and it opens the possibility to target viral GQs using molecules developed for cancer research. Additionally, an interaction between viral proteins and GQs was also reported recently, which underlines the importance of GQs in viral research [9]. In this paper we report the evaluation of three GQ-forming sequence of the hepatitis B virus (HBV).…”

G-Quadruplex Targeting in the Fight against Viruses: An Update

“…Primary structure alignment of SARS-CoV and SARS-CoV-2 SUD proteins revealed high similarity of the corresponding macrodomains, coupled with high conservation of the amino acids necessary for G4 binding and viral replication. Thus, disruption of the SUD/G4 interaction could constitute an alternative antiviral approach [104]. To this end, some G4 ligands were recently employed in the in vitro investigation of SARS-CoV-2 SUD interaction with G4-forming sequences.…”

“…With a Homogeneous Time-Resolved Fluorescence (HTRF) assay using a cellular RNA G4 sequence (TRF2), Lavigne et al showed that all tested molecules inhibited SUD/G4 interaction, with IC 50 values between 15 and 50 nM. The three most stable PQSs predicted in SARS-CoV-2 genome were also tested, but none of them interacted with the SUD domain, suggesting a preferential interaction with host cell DNA or RNA, rather than viral RNA [104].…”

Characterization of G-Quadruplexes Folding/Unfolding Dynamics and Interactions with Proteins from Single-Molecule Force Spectroscopy

“…G4 formation may obstruct DNA replication; thus, G4 formation regulated by proteins such as RPA and FANCJ helicase at the replication fork has an important role in maintaining genome stability [125]. Intriguingly, a SARS unique macrodomain (SUD) within the Nsp3 protein of both SARS-CoV and SARS-CoV-2 also can bind G4 structures, thus suggesting the potential roles of G4s in viral replication and transcription [126,127]. The role of G4s as regulatory elements in telomere, promoter, and RNA regions requires that their folding and unfolding (unwinding) is temporally controlled [128].…”