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Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance., Author summary The emergence of SARS-CoV-2 has led to a worldwide pandemic with significant morbidity and mortality. Remdesivir is the only antiviral with FDA approval for treatment. Antivirals use comes at a risk, as viruses may acquire mutations to overcome the inhibition. We identified a mutation in the virus polymerase responsible for decreased sensitivity to Remdesivir. A change at this conserved site was not predicted, and the mutation did not cause a replication advantage or change in sensitivity to another antiviral drug. Importantly, this change occurred at very low frequency globally. Unexpectedly, passage of SARS-CoV-2 led to an accumulation of mutations in spike. A number occurred at the same sites but to different residues as those in emerging variants of concern indicating they arise in the absence of immune pressure. Our data indicates low-level Remdesivir resistance in SARS-CoV-2 is different to other RNA viruses and monitoring changes in vitro provides insight into general virus adaptation of newly emerging viruses.

Remdesivir (RDV) is used widely for COVID-19 patients despite varying results in recent clinical trials. Here, we show how serially passaging SARS-CoV-2 in vitro in the presence of RDV selected for drug-resistant viral populations. We determined that the E802D mutation in the RNA-dependent RNA polymerase was sufficient to confer decreased RDV sensitivity without affecting viral fitness. Analysis of more than 200,000 sequences of globally circulating SARS-CoV-2 variants show no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we also observed changes in the Spike (i.e., H69 E484, N501, H655) corresponding to mutations identified in emerging SARS-CoV-2 variants indicating that they can arise in vitro in the absence of immune selection. This study illustrates SARS-CoV-2 genome plasticity and offers new perspectives on surveillance of viral variants.One Sentence SummarySARS-CoV-2 drug resistance & genome plasticity

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science., To help meet the ensuing demand for COVID-19 reagents, this article presents an openly available ‘coronavirus toolkit’ (https://mrcppu-covid.bio) and describes the generation and validation of these tools, including a simple SARS-CoV-2 reverse genetics system and a near-comprehensive panel of SARS-CoV-2 antibodies.

COVID‐19 is caused by a novel coronavirus called severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2). Virus cell entry is mediated through a protein‐protein interaction (PPI) between the SARS‐CoV‐2 spike protein and angiotensin‐converting enzyme 2 (ACE2). A series of stapled peptide ACE2 peptidomimetics based on the ACE2 interaction motif were designed to bind the coronavirus S‐protein RBD and inhibit binding to the human ACE2 receptor. The peptidomimetics were assessed for antiviral activity in an array of assays including a neutralization pseudovirus assay, immunofluorescence (IF) assay and in‐vitro fluorescence polarization (FP) assay. However, none of the peptidomimetics showed activity in these assays, suggesting that an enhanced binding interface is required to outcompete ACE2 for S‐protein RBD binding and prevent virus internalization.