Fast identification and Analysis of SARS-CoV-2 variants using Viral Instant Mutation Viewer (VIMVer)

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), responsible for the COVID-19 pandemic, has become one of the most studied viruses since its emergence. Two years after its outbreak, SARSCoV- 2 still represents a major public health priority as it keeps spreading at an alarming rate through the rise of many pathogenic variants. These latter can be a serious threat the more their genomic sequence diverge from the original strain, the less efficient the vaccine will remain. Therefore, it is critical for medical services to be able to determine straight out the strain they are dealing with, also as the location of the occurred genomic mutations and identify which viral protein has evolved. In this context, our main goal is to understand the nuances behind the mutations observed simultaneously in the genome and the proteome. To do so, we developed a user-friendly web-service software (Viral Instant Mutation Viewer or VIMVer) which allows an instant identification of new mutations and displayed them in both the nucleotide and protein sequences in comparison to a reference sequence (wuhan-1). Given a SARS-CoV-2 nucleotide sequence (as a newly sequenced genome), our software will instantly extract, analyse and visualize mutations on the genome and the proteome with the proper numbering and positioning. Additionally, the output is linked to Phylogenetic Assignment of Named Global Outbreak LINeages (Pangolin COVID-19) (2), which will thus allow an automatic identification of the Lineage or its position in relation to known lineage. We believe this tool will help many in their daily process to analyse their data. The source code is released under public licence and can be adapted for further development.

Identification and functional-structural characterization of the enzymes linked with stable genome size increase of (+) RNA viruses

Nidovirales is an extraordinary order of complex positive-stranded RNA viruses including some of the largest RNA genomes (12-41 kb) among which notable human health burdens: SARS-CoV-1, SARS-CoV-2, MERS-CoV, etc. Recent advance in genome sequencing is slowly filling the gaps between and beyond the classified nidoviral families. Still, the research is lagging behind to understand the evolution of RNA genomes. For example, how are these large genome RNA viruses able to bypass the length and stability constraints of an RNA molecule? Is there any link between increasing length and gaining a functional domain or a special structural feature? To answer these questions, we started with database mining to extract novel nidoviral genomes and annotated different domains in polyproteins of classified and unclassified nidoviruses using HHpred and HHblits tools (Zimmermann L, et al. 2018). We observed a significant variation across the order regarding presence/absence, fold/structure type, co-factor (or enhancer) presence/absence, presence of one motif or the other and genome location of enzymes: Exonuclease (ExoN), N-7 Methyltransferase (MTase), 2'-O-MTase and RNA dependent RNA polymerase (RdRp). A trend seen with this bioinformatic analysis directly implies that stable RNA genome increase as well as maintenance is driven by the synergy of modifying enzymes: MTases, RNA proofreading by ExoNs and fast & processive RdRps (Ferron F, et al. 2021). Next, after their identification, we are trying to characterize these large RNA genome genetic markers: MTase(s) & ExoN, to have a comprehensive understanding of nidoviruses evolution. We have identified, expressed and purified a new nidoviral MTase from a Tobaniviridae family member, White Bream Virus (WBV). This enzyme is unique in terms of its location in ORF1a and not in ORF1b (Ferron F, et al. 2019). Functional and mutational studies show this new MTase to contain N-7 guanine specific, S-adenosyl-methionine (SAM) dependent capping activity (cap-0). Aligning with our predictions, structural characterization confirms that it has a Rossmann fold (RF) SAMdependent RNA-cap N7-guanine MTase. This study answers the missing link of capping activity in these members, which is somewhat only established for coronaviruses in this large genome order. Evaluating such enzymes is a step forward in the direction of fundamental understanding of how these RNA viruses are successfully expanding and maintaining their large genomes as well as coping up to fight against the host innate immunity.

Macro domain F156 residue: a hallmark of SARS-CoV-2 de-MARylation specificity

The Coronavirus disease 2019 (Covid-2019) pandemic currently provokes a global health and economic crisis due to the generalized spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a large, enveloped and positive sense single stranded RNA virus. The SARS-CoV-2 genome encodes 16 non-structural proteins (nsp 1-16), forming a large membrane bound replication complex. The largest protein of this complex is nsp3, a multi-domain protein that contain a well conserved Macro domain (also called X domain or ADP-ribose phosphatase domain). The Macro domain can bind to mono-ADP-ribose (MAR) and poly-ADP-ribose (PAR) in their free form or conjugated to a protein or RNA substrates. Macro domains also carry hydrolase activities including de-MARylation and de-PARylation implicated in the inflammation process and the regulation of innate immunity. Herein, we report a mutagenesis study focusing on SARS-CoV-2 F156 and SARSCoV N157 residues, stipulated important for ADP-ribose orientation within the binding clef. Our data suggest that the exchange of these residues or their substitution to alanine slightly influence ADP-ribose binding, but drastically impact Macro domain de-MARylation activity.

Identification of potent inhibitors of SARS-CoV-2 exoribonuclease by fluorescence polarization assay

The emergence of SARS-CoV-2 has triggered a pandemic with devastating consequences to the world. One of the proteins essential to the virus life cycle is nsp14, which is a bifunctional protein that encodes a 3'to 5' exoribonuclease activity in its N-terminus, and a methyl transferase activity in its C-terminus. Nsp14 in complex with the accessory protein nsp10 is involved in a proofreading mechanism that ensures the genetic stability of its massive viral genome, and is associated to the resistance against nucleotide analogs targeting the polymerase nsp12. Because of its key role, nsp14-nsp10 complex constitutes an attractive target for antiviral development. Here we present a fluorescence polarization (FP) assay development to measure the exoribonuclease activity and its inhibition in vitro. The FP method is sensitive, robust, amenable to miniaturization and offers confirmation by visualizing the degradation of the fluorescent RNA in acrylamide gels. We performed a screening of a focused library of 113 metal chelators at 20 and 5 μM compound concentration and IC50 measurement of 9 hits showing efficiency at micromolar level. We also tested the focused library in SARS-CoV-2 infected Vero cells and we confirmed 3 hits previously detected in the in vitro screening out of 6 promising inhibitors. In conclusion the FP method proposed is a reliable tool to discover inhibitors of the SARS-CoV-2 exoribonuclease activity and will help to find new antivirals to be used in combination with nucleoside analogs.

Development of a High throughput screening method based on fluorescence to discover inhibitors of viral exoribonucleases

The exoribonuclease activity (ExoN) is quite bizarre in the world of RNA viruses, as it is present uniquely in the Arenaviridae and the Coronaviridae families. ExoN plays important but different roles in both families : for arenaviruses the ExoN is involved in the suppression of the host immune response whereas for coronaviruses, ExoN is likely involved in the proofreading mechanism for the viral genome's replication. Because of their key roles, they are attractive targets for drug development, however, the most common current available technique to measure the ExoN activity and inhibition is the use of radiolabeled gel assays, which is not suitable for the screening of compounds libraries. Here we developed a method using fluorescence polarization to assess the ExoN activity and inhibition and we validated the method on three different viral enzymes (SARS-CoV-2, lymphocytic choriomeningitis virus and Machupo Virus. The method is very sensitive, robust, amenable to miniaturization (384 well plates) and allow us to screen a small compounds library (24). We are confident that this method is a method of choice for screening large libraries and will become a commonly used HTS screening method.

Rapid incorporation of Favipiravir by the fast and permissive viral RNA polymerase complex results in SARS-CoV-2 lethal mutagenesis

The ongoing Corona Virus Disease 2019 (Covid-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. We demonstrate here that Favipiravir predominantly exerts an antiviral effect through lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and higherror rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of Covid-19.

The SARS-CoV ORF1b enzymes : Fast, furious, and unique

With the current Covid-19 ongoing outbreak originating inWuhan, human pathogenic coronaviruses demonstrate their ability to emerge abruptly and spread serious pulmonary disease. The Orf1b enzymes promote replication and transcription within a complex with unique enzyme having outstanding properties. The RdRp core sequence of the Covid-19 isolate published in january 2020 shows a high (>95% aa) sequence homology to the SARSCoV emerged in 2003. Analysis of polymorphisms show that aa changes are mostly located at the protein surface, unlikely to affect any basic function of the RdRp.We have reconstituted a highly active SARS-CoV RdRp complex made of nsp7, nsp8, and nsp12, and studied its polymerization activity on a variety of RNA templates using steady-state and pre-steady state kinetics. The RdRp is able to incorporate single NTPs at the astonishing rate of >500 s-1, about 10-fold faster than any known viral RdRp. Fast synthesis occurs at the expense of fidelity, which is at least 10-fold lower than that of Dengue virus NS5 or Coxsackie virus RdRps. Such low fidelity must be corrected by the nsp14 ExoN subdomain -able to remove 3'-terminal mismatches to match genome stability observed in infected cells, and account for the large size of the Coronavirus RNA genome. The nsp14 enzyme is a bi-functional enzyme made of an Exonuclease domain, activated through binding to nsp10, and an RNA methyltransferase able to execute N7-guanine methylation of RNA caps. It is the only example of RNA cap MTase which does not have a Rossmann fold, which poses interesting questions given the overwhelming success of the Rossmann fold through evolution. Nsp13 is a type 1 helicase, whose role is unclear. The nsp15 RNA endonuclease is a RNase A type endonuclease specific for Uracile, inhibited by 2'-O methylation of RNA, while nsp16 is a 2'-O methyltransferase also activated by nsp10. Our work provides a structural and functional view of this sophisticated replication complex, with highly active enzyme preparations suitable for robotized high-throughput inhibition assays aiming at the discovery of pan-coronavirus inhibitors Orf1b enzymes.