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Diagnostic testing continues to be an integral component of the strategy to contain the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) global pandemic, the causative agent of Coronavirus Disease 2019 (COVID-19). The SARS-CoV-2 genome encodes the 3C-like protease (3CLpro) which is essential for coronavirus replication. This study adapts an in vitro colorimetric gold nanoparticle (AuNP) based protease assay to specifically detect the activity of SARS-CoV-2 3CLpro as a purified recombinant protein and as a cellular protein exogenously expressed in HEK293T human cells. We also demonstrate that the specific sensitivity of the assay for SARS-CoV-2 3CLpro can be improved by use of an optimised peptide substrate and through hybrid dimerisation with inactive 3CLpro mutant monomers. These findings highlight the potential for further development of the AuNP protease assay to detect SARS-CoV-2 3CLpro activity as a novel, accessible and cost-effective diagnostic test for SARS-CoV-2 infection at the point-of-care. Importantly, this versatile assay could also be easily adapted to detect specific protease activity associated with other viruses or diseases conditions.

Funder: NIHR Cambridge BRC, Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.Author summaryTechniques for measuring infection with SARS-CoV-2 in the laboratory are laborious and time-consuming, and different laboratories use different approaches. There is therefore no generally agreed way to quantitate neutralising antibodies against SARS-CoV-2, which block infection with the virus and protect people from COVID-19. In this study, we describe a new way to measure SARS-CoV-2 infection, which is much simpler and faster than existing methods. It relies on the production of a specific protease enzyme by the virus, which is able to cleave and activate an engineered protein biosensor in infected cells. This biosensor emits light in the presence of viral infection, and the amount of light released is used as a readout for the amount of infectious SARS-CoV-2 present. The signal is very sensitive, so the number of infected cells required is very small, and the method can be scaled-up to test many samples at once. In particular, we demonstrate how it can be used to detect different variants of SARS-CoV-2, and quantitate neutralising antibodies against these viruses.

During the translation surveillance mechanism known as ribosome-associated quality control, the ASC-1 complex (ASCC) disassembles ribosomes stalled on the mRNA. Here, we show that there are two distinct classes of stalled ribosome. Ribosomes stalled by translation elongation inhibitors or methylated mRNA are short lived in human cells because they are split by the ASCC. In contrast, although ultraviolet light and 4-nitroquinoline 1-oxide induce ribosome stalling by damaging mRNA, and the ASCC is recruited to these stalled ribosomes, we found that they are refractory to the ASCC. Consequently, unresolved UV- and 4NQO-stalled ribosomes persist in human cells. We show that ribosome stalling activates cell-cycle arrest, partly through ZAK-p38

The translation machinery is composed of a myriad of proteins and RNAs whose levels must be coordinated to produce efficiently proteins without wasting energy or substrate. However, protein synthesis is clearly not always perfectly tuned to its environment as disruption of translation machinery components can lengthen lifespan and stress survival. While much has been learned from bacteria and yeast about translational regulation, much less is known in metazoans. In a screen for mutations protecting C. elegans from hypoxic stress, we isolated multiple genes impacting protein synthesis: a ribosomal RNA helicase gene, tRNA biosynthesis genes, and a gene controlling amino acid availability. To define better the mechanisms by which these genes impact protein synthesis, we performed a second screen for suppressors of the conditional developmental arrest phenotype of the RNA helicase mutant and identified genes involved in ribosome biogenesis. Surprisingly, these suppressor mutations restored normal hypoxic sensitivity and protein synthesis to the tRNA biogenesis mutants, but not to the mutant reducing amino acid uptake. Proteomic analysis demonstrated that reduced tRNA biosynthetic activity produces a selective homeostatic reduction in ribosomal subunits, thereby offering a mechanism for the suppression results. Our study uncovers an unrecognized higher order translation regulatory mechanism in a metazoan whereby ribosome biogenesis genes communicate with genes controlling tRNA abundance matching the global rate of protein synthesis with available resources.

Following cell stress, a wide range of molecular pathways are initiated to orchestrate the stress response and enable adaptation to an environmental or intracellular perturbation. The post-transcriptional regulation strategies adopted during the stress response result in a substantial reorganization of gene expression, designed to prepare the cell for either acclimatization or programmed death, depending on the nature and intensity of the stress. Fundamental to the stress response is a rapid repression of global protein synthesis, commonly mediated by phosphorylation of translation initiation factor eIF2α. Recent structural and biochemical information have added unprecedented detail to our understanding of the molecular mechanisms underlying this regulation. During protein synthesis inhibition, the translation of stress-specific mRNAs is nonetheless enhanced, often through the interaction between RNA-binding proteins and specific RNA regulatory elements. Recent studies investigating the unfolded protein response (UPR) provide some important insights into how posttranscriptional events are spatially and temporally fine-tuned in order to elicit the most appropriate response and to coordinate the transition from an early, acute stage into the chronic state of adaptation. Importantly, cancer cells are known to hi-jack adaptive stress response pathways, particularly the UPR, to survive and proliferate in the unfavorable tumor environment. In this review, we consider the implications of recent research into stress-dependent post-transcriptional regulation and make the case for the exploration of the stress response as a strategy to identify novel targets in the development of cancer therapies. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution Translation > Translation Mechanisms > Translation Regulation.