In order to understand the transmission and virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to understand the functions of each of the gene products encoded in the viral genome. One feature of the SARS-CoV-2 genome that is not present in related, common coronaviruses is ORF10, a putative 38-amino acid protein-coding gene. Proteomic studies found that ORF10 binds to an E3 ubiquitin ligase containing Cullin-2, Rbx1, Elongin B, Elongin C, and ZYG11B (CRL2ZYG11B). Since CRL2ZYG11B mediates protein degradation, one possible role for ORF10 is to “hijack” CRL2ZYG11B in order to target cellular, antiviral proteins for ubiquitylation and subsequent proteasomal degradation. Here, we investigated whether ORF10 hijacks CRL2ZYG11B or functions in other ways, for example, as an inhibitor or substrate of CRL2ZYG11B. While we confirm the ORF10−ZYG11B interaction and show that the N terminus of ORF10 is critical for it, we find no evidence that ORF10 is functioning to inhibit or hijack CRL2ZYG11B. Furthermore, ZYG11B and its paralog ZER1 are dispensable for SARS-CoV-2 infection in cultured cells. We conclude that the interaction between ORF10 and CRL2ZYG11B is not relevant for SARS-CoV-2 infection in vitro.

The adenosine analogue remdesivir has emerged as a frontline antiviral treatment for SARS-CoV-2, with preliminary evidence that it reduces the duration and severity of illness1. Prior clinical studies have identified adverse events1,2, and remdesivir has been shown to inhibit mitochondrial RNA polymerase in biochemical experiments7, yet little is known about the specific genetic pathways involved in cellular remdesivir metabolism and cytotoxicity. Through genome-wide CRISPR-Cas9 screening and RNA sequencing, we show that remdesivir treatment leads to a repression of mitochondrial respiratory activity, and we identify five genes whose loss significantly reduces remdesivir cytotoxicity. In particular, we show that loss of the mitochondrial nucleoside transporter SLC29A3 mitigates remdesivir toxicity without a commensurate decrease in SARS-CoV-2 antiviral potency and that the mitochondrial adenylate kinase AK2 is a remdesivir kinase required for remdesivir efficacy and toxicity. This work elucidates the cellular mechanisms of remdesivir metabolism and provides a candidate gene target to reduce remdesivir cytotoxicity.

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The SARS-CoV-2 pandemic has caused widespread illness, loss of life, and socioeconomic disruption that is unlikely to resolve until vaccines are widely adopted, and effective therapeutic treatments become established. Here, a well curated and annotated library of 6710 clinical and preclinical molecules, covering diverse chemical scaffolds and known host targets was evaluated for inhibition of SARS-CoV-2 infection in multiple infection models. Multi-concentration, high-content immunocytofluorescence-based screening identified 172 strongly active small molecules, including 52 with submicromolar potencies. The active molecules were extensively triaged by in vitro mechanistic assays, including human primary cell models of infection and the most promising, obatoclax, was tested for in vivo efficacy. Structural and mechanistic classification of compounds revealed known and novel chemotypes and potential host targets involved in each step of the virus replication cycle including BET proteins, microtubule function, mTOR, ER kinases, protein synthesis and ion channel function. In the mouse disease model obatoclax effectively reduced lung virus load by 10-fold. Overall, this work provides an important, publicly accessible, foundation for development of novel treatments for COVID-19, establishes human primary cell-based pharmacological models for evaluation of therapeutics and identifies new insights into SARS-CoV-2 infection mechanisms.