Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease.

SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp14/nsp10 exoribonuclease.

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is an urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp14 RNA cap methyltransferase.

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.

Unwinding of a eukaryotic origin of replication visualized by cryo-EM.

To prevent detrimental chromosome re-replication, DNA loading of a double hexamer of the minichromosome maintenance (MCM) replicative helicase is temporally separated from DNA unwinding. Upon S-phase transition in yeast, DNA unwinding is achieved in two steps: limited opening of the double helix and topological separation of the two DNA strands. First, Cdc45, GINS and Polε engage MCM to assemble a double CMGE with two partially separated hexamers that nucleate DNA melting. In the second step, triggered by Mcm10, two CMGEs separate completely, eject the lagging-strand template and cross paths. To understand Mcm10 during helicase activation, we used biochemical reconstitution with cryogenic electron microscopy. We found that Mcm10 splits the double CMGE by engaging the N-terminal homo-dimerization face of MCM. To eject the lagging strand, DNA unwinding is started from the N-terminal side of MCM while the hexamer channel becomes too narrow to harbor duplex DNA.

A DNA replication fork-centric view of the budding yeast DNA damage response.

The DNA damage response (DDR) checkpoint is activated when DNA is damaged or when DNA replication forks stall. The DDR checkpoint plays a critical role in preserving the integrity of stalled replication forks; this is essential for subsequent fork resumption, faithful and complete genome replication, and cell survival. The mechanisms by which the DDR checkpoint preserves stalled replication forks are still incompletely understood. Many substrates of the DDR checkpoint kinases have been identified over the years, but in many cases the functional consequences of phosphorylation are still unclear. Emerging as a complementary approach, recent advances in biochemical reconstitution of DNA replication have made it possible to characterise specific mechanisms of DNA replication regulation by the DDR checkpoint. In this review, we discuss the role of DNA replication in the activation of the DDR checkpoint and how this checkpoint regulates different aspects of DNA replication. We then distinguish between checkpoint action locally at the site of replication stalling and more globally, and we discuss how these functions contribute to coordinating complete replication of the genome in the face of replication stress.

Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation.

The Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

Mating in wild yeast: delayed interest in sex after spore germination.

Studies of laboratory strains of Saccharomyces cerevisiae have uncovered signaling pathways involved in mating, including information-processing strategies to optimize decisions to mate or to bud. However, lab strains are heterothallic (unable to self-mate), while wild yeast are homothallic. And while mating of lab strains is studied using cycling haploid cells, mating of wild yeast is thought to involve germinating spores. Thus, it was unclear whether lab strategies would be appropriate in the wild. Here, we have investigated the behavior of several yeast strains derived from wild isolates. Following germination, these strains displayed large differences in their propensity to mate or to enter the cell cycle. The variable interest in sex following germination was correlated with differences in pheromone production, which were due to both cis- and trans-acting factors. Our findings suggest that yeast spores germinating in the wild may often enter the cell cycle and form microcolonies before engaging in mating.

Imaging Polarization in Budding Yeast.

We describe methods for live-cell imaging of yeast cells that we have exploited to image yeast polarity establishment. As a rare event occurring on a fast time-scale, imaging polarization involves a trade-off between spatiotemporal resolution and long-term imaging without excessive phototoxicity. By synchronizing cells in a way that increases resistance to photodamage, we discovered unexpected aspects of polarization including transient intermediates with more than one polarity cluster, oscillatory clustering of polarity factors, and mobile "wandering" polarity sites.

To avoid a mating mishap, yeast focus and communicate.

During mating, yeast cells must perforate their rigid cell walls at the right place to allow cell-cell fusion. In this issue, Dudin et al. (2015; J. Cell Biol. http://dx.doi.org/jcb.201411124) image mating fission yeast cells with unprecedented spatiotemporal resolution. The authors find that when mating cells come into contact, they form aster-like actin structures that direct cell wall remodeling precisely to the point of contact.

Role of Polarized G Protein Signaling in Tracking Pheromone Gradients.

Yeast cells track gradients of pheromones to locate mating partners. Intuition suggests that uniform distribution of pheromone receptors over the cell surface would yield optimal gradient sensing. However, yeast cells display polarized receptors. The benefit of such polarization was unknown. During gradient tracking, cell growth is directed by a patch of polarity regulators that wanders around the cortex. Patch movement is sensitive to pheromone dose, with wandering reduced on the up-gradient side of the cell, resulting in net growth in that direction. Mathematical modeling suggests that active receptors and associated G proteins lag behind the polarity patch and act as an effective drag on patch movement. In vivo, the polarity patch is trailed by a G protein-rich domain, and this polarized distribution of G proteins is required to constrain patch wandering. Our findings explain why G protein polarization is beneficial and illuminate a novel mechanism for gradient tracking.

Cell polarity: netrin calms an excitable system.

In a tractable model for cell invasion, the Caenorhabditis elegans anchor cell migrates through basement membranes towards a polarity cue provided by netrin. A new study reveals that the anchor cell polarity network can break symmetry and oscillate in the absence of netrin, suggesting the presence of interlinked positive and negative feedback loops, which are common in polarity pathways.