Biology subjects

Teixeira-Silva, A.

Publications and source records attributed to Teixeira-Silva, A..

4 recordsLinked to original sources

bioRxiv2021

LRRC15 mediates an accessory interaction with the SARS-CoV-2 spike protein

The interactions between severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and human host factors enable the virus to propagate infections that lead to COVID-19. The spike protein is the largest structural component of the virus and mediates interactions essential for infection, including with the primary ACE2 receptor. We performed two independent cell-based systematic screens to determine whether there are additional proteins by which the spike protein of SARS-CoV-2 can interact with human cells. We discovered that in addition to ACE2, expression of LRRC15 also causes spike protein binding. This interaction is distinct from other known spike attachment mechanisms such as heparan sulfates or lectin receptors. Measurements of orthologous coronavirus spike proteins implied the interaction was restricted to SARS-CoV-2, suggesting LRRC15 represents a novel class of spike binding interaction. We localized the interaction to the C-terminus of the S1 domain, and showed that LRRC15 shares recognition of the ACE2 receptor binding domain. From analyzing proteomics and single-cell transcriptomics, we identify LRRC15 expression as being common in human lung vasculature cells and fibroblasts. Although infection assays demonstrated that LRRC15 alone is not sufficient to permit viral entry, we present evidence it can modulate infection of human cells. This unexpected interaction merits further investigation to determine how SARS-CoV-2 exploits host LRRC15 and whether it could account for any of the distinctive features of COVID-19. In briefWe present evidence from genome-wide screening that the spike protein of SARS-CoV-2 interacts with human cells expressing LRRC15. The interaction is distinct from previously known classes of spike attachment factors, and appears to have emerged recently within the coronavirus family. Although not sufficient for cell invasion, this interaction can modulate viral infection. Our data point to an unappreciated host factor for SARS-CoV-2, with potential relevance to COVID-19. Highlights- Two systematic cell-based screens for SARS-CoV-2 spike protein binding identify LRRC15 as a human host factor - Interaction with LRRC15 is reproducible in different human cell lines and independent of known glycan or ACE2 binding pathways - The C-terminal S1 domain of SARS-CoV-2 spike binds LRRC15 with sub-micromolar affinity, while related coronavirus spikes do not - LRRC15 is expressed in tissues with high ACE2 levels and may modulate infection

biochemistry↗

bioRxiv2021

Protease-activatable biosensors of SARS-CoV-2 infection for cell-based drug, neutralisation and virological assays

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.

microbiology↗

bioRxiv2018

Histone deposition plays an active role in recombination-dependent replication to balance genome2 stability upon replication stress

Replication stress poses a serious threat to genome and epigenome stability. Recombination-Dependent-Replication (RDR) ensures DNA synthesis resumption from arrested forks. Despite the identification of chromatin restoration pathways during DNA repair processes, crosstalk coupling RDR and chromatin assembly is largely unexplored. Here, we addressed the contribution of chromatin assembly to replication stress in fission yeast. We expressed a mutated histone (H3-H113D) to genetically impair replication-dependent chromatin assembly by destabilizing (H3-H4)2 tetramer. We established that DNA synthesis-dependent histone deposition is required for the completion of RDR. Histone deposition prevents joint-molecules from Rqh1-dependent disassembly, a crosstalk contributing to cell survival upon replication stress but channeling RDR towards deleterious events. Asf1 and CAF-1 act in RDR and CAF-1 recruitment to DNA synthesis associated to RDR requires the HR factor Rad52. Our data establish that CAF-1 counteracts Rqh1 activity at sites of replication stress by promoting repair synthesis-coupled histone deposition. These results demonstrate that histone deposition plays an active role in fine-tuning RDR, a benefit counterbalanced by stabilizing at-risk joint molecules for genome stability.

genetics↗

bioRxiv2017

The non-homologous end joining factor Ku orchestrates replication fork resection and fine-tunes Rad51-mediated fork restart

Replication requires Homologous Recombination (HR) to stabilize and restart terminally-arrested forks. HR-mediated fork processing requires single stranded DNA (ssDNA) gaps and not necessarily Double Strand Breaks. We used genetic and molecular assays to investigate fork-resection and restart at dysfunctional, unbroken forks in Schizosaccharomyces pombe. We found that fork-resection is a two-step process coordinated by the non-homologous end joining factor Ku. An initial resection mediated by MRN/Ctp1 removes Ku from terminally-arrested forks, generating ~ 110 bp sized gaps obligatory for subsequent Exo1-mediated long-range resection and replication restart. The lack of Ku results in slower fork restart, excessive resection, and impaired RPA recruitment. We propose that terminally-arrested forks undergo fork reversal, providing a single DNA end for Ku binding which primes RPA-coated ssDNA. We uncover an unprecedented role for Ku in orchestrating resection of unbroken forks and in fine-tuning HR-mediated replication restart.\n\nO_LIKu orchestrates a two-steps DNA end-resection of terminally-arrested and unbroken forks\nC_LIO_LIMRN/Ctp1 removes Ku from terminally-arrested forks to initiate fork-resection\nC_LIO_LIa ~110 bp sized ssDNA gap is sufficient and necessary to promote fork restart.\nC_LIO_LIThe lack of Ku decreases ssDNA RPA-coating, and slows down replication fork restart.\nC_LI

genetics↗