Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication (preprint)

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their mRNAs, protect them from degradation by cellular 5- exoribonucleases, and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bi-functional replicase subunit harboring an N-terminal 3-to-5; exoribonuclease (ExoN) domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14s enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome-CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

A dual mechanism of action of AT-527 against SARS-CoV-2 polymerase (preprint)

A worldwide effort is ongoing to discover drugs against the Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2), which has so far caused >3.5 million fatalities (https://covid19.who.int/). The virus essential RNA-dependent RNA polymerase complex is targeted by several nucleoside/tide analogues whose mechanisms of action and clinical potential are currently evaluated. The guanosine analogue AT-527, a double prodrug of its 5'-triphosphate AT-9010, is currently in phase III clinical trials as a COVID19 treatment. Here we report the cryo-EM structure at 2.98 Å resolution of the SARS-CoV-2 nsp12-nsp7-(nsp8)2 complex with RNA showing AT-9010 bound at three sites of nsp12. At the RdRp active-site, one AT-9010 is incorporated into the RNA product. Its 2'-methyl group prevents correct alignment of a second AT-9010 occupying the incoming NTP pocket. The 2'-F, 2'-methyl 3'-OH ribose scaffold explains the non-obligate RNA chain-termination potency of this NA series for both HCV NS5 and SARS-CoV RTCs. A third AT-9010 molecule 5'-diphosphate binds to a coronavirus-specific pocket in the nsp12 N-terminus NiRAN domain, a SelO pseudo-kinase structural and functional homologue. This unique binding mode impedes NiRAN-mediated UMPylation of SARS-CoV-2 nsp8 and nsp9 proteins. Our results suggest a mechanism of action for AT-527 in line with a therapeutic use for COVID19.

Implications of Spike-glycoprotein processing at S1/S2 by Furin, at S2' by Furin and/or TMPRSS2 and shedding of ACE2: cell-to-cell fusion, cell entry and infectivity of SARS-CoV-2 (preprint)

The Spike (S)-protein of SARS-CoV-2 binds host-cell receptor ACE2 and requires proteolytic "priming" at PRRAR685{downarrow} into S1 and S2 (cleavage at S1/S2), and "fusion-activation" at KPSKR815{downarrow} (cleavage at S2') for viral entry. Both cleavages occur at Furin-like motifs suggesting that proprotein convertases might promote virus entry. In vitro Furin cleaved peptides mimicking the S1/S2 cleavage site more efficiently than S2', whereas TMPRSS2 cleaved at both sites. In HeLa cells endogenous Furin-like enzymes cleave mainly at S1/S2 during intracellular protein trafficking, as confirmed by mutagenesis. We also mapped the S2' cleavage site by proteomics and further showed that S2'-processing by Furin, while limited, was strongly enhanced in the presence of ACE2. In contrast, the S2' KRRKR815{downarrow} mutant (S2') was considerably better cleaved by Furin, whereas individual/double KR815AA mutants are retained in the endoplasmic reticulum (ER). Pharmacological inhibitors of convertases (Boston Pharmaceuticals - BOS-inhibitors) effectively blocked endogenous S-protein processing in HeLa cells. However, under co-expression the S-protein was prematurely cleaved by TMPRSS2 into ER-retained, non-O-glycosylated S2 and S2' products. Quantitative analysis of cell-to-cell fusion and Spike processing using Hela cells revealed the key importance of the Furin sites for syncytia formation and unveiled the enhanced fusogenic potential of the - and {delta}-variants of the S-protein of SARS-CoV-2. Our fusion assay indicated that TMPRSS2 enhances S2' formation, especially in the absence of Furin cleavage, as well as ACE2 shedding. Furthermore, we provide evidence using pseudoparticles that while entry by a "pH-dependent" endocytosis pathway in HEK293 cells did not require Furin processing at S1/S2, a "pH-independent" viral entry in lung-derived Calu-3 cells was sensitive to inhibitors of Furin and TMPRSS2. Consistently, in Calu-3 cells BOS-inhibitors or Camostat potently reduce infectious viral titer and cytopathic effects and this outcome was enhanced when both compounds were combined. Overall, our results show that Furin and TMPRSS2 play synergistic roles in generating fusion-competent S-protein, and promote viral entry, supporting the combination of Furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2.

An appeal for an open scientific debate about the proximal origin of SARS-CoV-2 (preprint)

One year after the onset of the COVID-19 pandemic, the origin of SARS-CoV-2 still eludes humanity. Early publications firmly stated that the virus was of natural origin, and the possibility that the virus might have escaped from a lab was discarded in most subsequent publications. However, based on a re-analysis of the initial arguments, highlighted by the current knowledge about the virus, we show that the natural origin is not supported by conclusive arguments, and that a lab origin cannot be formally discarded. We call for an opening of peer-reviewed journals to a rational, evidence-based and prejudice-free evaluation of all the reasonable hypotheses about the virus' origin. We advocate that this debate should take place in the columns of renowned scientific journals, rather than being left to social media and newspapers.

A high-throughput fluorescence polarization assay to discover inhibitors of arenavirus and coronavirus exoribonucleases (preprint)

Viral exoribonucleases are uncommon in the world of RNA viruses. To date, this activity has been identified only in the Arenaviridae and the Coronaviridae families. These exoribonucleases play important but different roles in both families: for mammarenaviruses the exoribonuclease is involved in the suppression of the host immune response whereas for coronaviruses, exoribonuclease is both involved in a proofreading mechanism ensuring the genetic stability of viral genomes and participating to evasion of the host innate immunity. Because of their key roles, they constitute attractive targets for drug development. Here we present a high-throughput assay using fluorescence polarization to assess the viral exoribonuclease activity and its inhibition. We validate the assay using three different viral enzymes from SARS-CoV-2, lymphocytic choriomeningitis and Machupo viruses. The method is sensitive, robust, amenable to miniaturization (384 well plates) and allowed us to validate the proof-of-concept of the assay by screening a small focused compounds library (23 metal chelators). We also determined the IC50 of one inhibitor common to the three viruses.

Protein-primed RNA synthesis in SARS-CoVs and structural basis for inhibition by AT-527 (preprint)

How viruses from the Coronaviridae family initiate viral RNA synthesis is unknown. Here we show that the SARS-CoV-1 and -2 Nidovirus RdRp-Associated Nucleotidyltransferase (NiRAN) domain on nsp12 uridylates the viral cofactor nsp8, forming a UMP-Nsp8 covalent intermediate that subsequently primes RNA synthesis from a poly(A) template; a protein-priming mechanism reminiscent of Picornaviridae enzymes. In parallel, the RdRp active site of nsp12 synthesizes a pppGpU primer, which primes (-)ssRNA synthesis at the precise genome-poly(A) junction. The guanosine analogue 5'-triphosphate AT-9010 (prodrug: AT-527) tightly binds to the NiRAN and inhibits both nsp8-labeling and the initiation of RNA synthesis. A 2.98 A resolution Cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-(nsp8)2 /RNA/NTP quaternary complex shows AT-9010 simultaneously binds to both NiRAN and RdRp active site of nsp12, blocking their respective activities. AT-527 is currently in phase II clinical trials, and is a potent inhibitor of SARS-CoV-1 and -2, representing a promising drug for COVID-19 treatment.

Furin cleaves SARS-CoV-2 spike-glycoprotein at S1/S2 and S2'for viral fusion/entry: indirect role for TMPRSS2 (preprint)

The Spike (S)-protein of SARS-CoV-2 binds host-cell receptor ACE2 and requires proteolytic 'priming' (S1/S2) and 'fusion-activation' (S2') for viral entry. The S-protein furin-like motifs PRRAR685{downarrow} and KPSKR815{downarrow} indicated that proprotein convertases promote virus entry. We demonstrate that furin and PC5A induce cleavage at both sites, ACE2 enhances S2' processing, and their pharmacological inhibition (BOS-inhibitors) block endogenous cleavages. S1/S2-mutations (S1/S2) limit S-protein-mediated cell-to-cell fusion, similarly to BOS-inhibitors. Unexpectedly, TMPRSS2 does not cleave at S1/S2 or S2', but it can: (i) cleave/inactivate S-protein into S2a/S2b; (ii) shed ACE2; (iii) cleave S1-subunit into secreted S1', activities inhibited by Camostat. In lung-derived Calu-3 cells, BOS-inhibitors and S1/S2 severely curtail 'pH-independent' viral entry, and BOS-inhibitors alone/with Camostat potently reduce infectious viral titer and cytopathic effects. Overall, our results show that: furin plays a critical role in generating fusion-competent S-protein, and indirectly, TMPRSS2 promotes viral entry, supporting furin and TMPRSS2 inhibitors as potential antivirals against SARS-CoV-2

SARS-CoV-2 protein ORF3a is pathogenic in Drosophila and causes phenotypes associated with COVID-19 post-viral syndrome (preprint)

The Coronavirus Disease 2019 (COVID-19) pandemic has caused millions of deaths and will continue to exact incalculable tolls worldwide. While great strides have been made toward understanding and combating the mechanisms of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection, relatively little is known about the individual SARS-CoV-2 proteins that contribute to pathogenicity during infection and that cause neurological sequela after viral clearance. We used Drosophila to develop an in vivo model that characterizes mechanisms of SARS-CoV-2 pathogenicity, and found ORF3a adversely affects longevity and motor function by inducing apoptosis and inflammation in the nervous system. Chloroquine alleviated ORF3a induced phenotypes in the CNS, arguing our Drosophila model is amenable to high throughput drug screening. Our work provides novel insights into the pathogenic nature of SARS-CoV-2 in the nervous system that can be used to develop new treatment strategies for post-viral syndrome.

Tracing the origins of SARS-CoV-2 in coronavirus phylogenies (preprint)

SARS-CoV-2 is a new human coronavirus (CoV), which emerged in China in late 2019 and is responsible for the global COVID-19 pandemic that caused more than 59 million infections and 1.4 million deaths in 11 months. Understanding the origin of this virus is an important issue and it is necessary to determine the mechanisms of its dissemination in order to contain future epidemics. Based on phylogenetic inferences, sequence analysis and structure-function relationships of coronavirus proteins, informed by the knowledge currently available on the virus, we discuss the different scenarios evoked to account for the origin - natural or synthetic - of the virus. The data currently available is not sufficient to firmly assert whether SARS-CoV2 results from a zoonotic emergence or from an accidental escape of a laboratory strain. This question needs to be solved because it has important consequences on the evaluation of risk/benefit balance of our interaction with ecosystems, the intensive breeding of wild and domestic animals, as well as some lab practices and on scientific policy and biosafety regulations. Regardless of its origin, studying the evolution of the molecular mechanisms involved in the emergence of pandemic viruses is essential to develop therapeutic and vaccine strategies and to prevent future zoonoses. This article is a translation and update of a French article published in M{\'e}decine/Sciences, Aug/Sept 2020 (http://doi.org/10.1051/medsci/2020123).

Favipiravir strikes the SARS-CoV-2 at its Achilles heel, the RNA polymerase (preprint)

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. Here we show that Favipiravir exerts an antiviral effect as a nucleotide analogue through a combination of chain termination, slowed RNA synthesis and 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 high-error 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.

Targeting furin activity through in silico and in vitro drug repurposing strategy for SARS-CoV-2 spike glycoprotein cleavage repression (preprint)

In December 2019, a new coronavirus was identified in the Hubei province of central china and named SRAS-CoV-2. This new virus induces COVID-19, a severe respiratory disease with high death rate. The spike protein (S) of SARS-CoV-2 contains furin-like cleavage sites absent the other SARS-like viruses. The viral infection requires the priming or cleavage of the S protein and such processing seems essential for virus entry into the host cells. Furin is highly expressed in the lung tissue and the expression is further increased in lung cancer, suggesting the exploitation of this mechanism by the virus to mediate enhanced virulence as shown by the higher risk of COVID-19 in these patients. In this study, we used structure- based virtual screening and a collection of about 8,000 unique approved and investigational drugs suitable for docking to search for molecules that could inhibits furin activity. Sulconazole, a broad-spectrum anti-fungal agent, was found to be of potential interest. Using Western blot analysis, Sulconazole was found to inhibit the cleavage of the cell surface furin substrate MT1-MMP that contains two furin cleavage sites similar to those of the SARS- CoV-2 spike protein. Sulconazole and analogs could be interesting for repurposing studies and to probe the yet not fully understood molecular mechanisms involved in cell entry.

In vitro screening of a FDA approved chemical library reveals potential inhibitors of SARS-CoV-2 replication (preprint)

A novel coronavirus, named SARS-CoV-2, emerged in 2019 from Hubei region in China and rapidly spread worldwide. As no approved therapeutics exists to treat Covid-19, the disease associated to SARS-Cov-2, there is an urgent need to propose molecules that could quickly enter into clinics. Repurposing of approved drugs is a strategy that can bypass the time consuming stages of drug development. In this study, we screened the Prestwick Chemical Library(R) composed of 1,520 approved drugs in an infected cell-based assay. 90 compounds were identified. The robustness of the screen was assessed by the identification of drugs, such as Chloroquine derivatives and protease inhibitors, already in clinical trials. The hits were sorted according to their chemical composition and their known therapeutic effect, then EC50 and CC50 were determined for a subset of compounds. Several drugs, such as Azithromycine, Opipramol, Quinidine or Omeprazol present antiviral potency with 2