Biology subjects

James, L. C.

Publications and source records attributed to James, L. C..

5 recordsLinked to original sources

bioRxiv2020

SARS-CoV-2 infects brain choroid plexus and disrupts the blood-CSF-barrier

Coronavirus disease-19 (COVID-19), caused by the SARS-CoV-2 virus, leads primarily to respiratory symptoms that can be fatal, particularly in at risk individuals. However, neurological symptoms have also been observed in patients, including headache, seizures, stroke, and fatigue. The cause of these complications is not yet known, and whether they are due to a direct infection of neural cells, such as neurons and astrocytes, or through indirect effects on supportive brain cells, is unknown. Here, we use brain organoids to examine SARS-CoV-2 neurotropism. We examine expression of the key viral receptor ACE2 in single-cell RNA sequencing (scRNA-seq) revealing that only a subset of choroid plexus cells but not neurons or neural progenitors express this entry factor. We then challenge organoids with both SARS-CoV-2 spike protein pseudovirus and live virus to demonstrate high viral tropism for choroid plexus epithelial cells but not stromal cells, and little to no infection of neurons or glia. We find that infected cells of the choroid plexus are an apolipoprotein and ACE2 expressing subset of epithelial barrier cells. Finally, we show that infection with live SARS-CoV-2 leads to barrier breakdown of the choroid plexus. These findings suggest that neurological complications may result from effects on the choroid plexus, an important barrier that normally prevents entry of immune cells and cytokines into the cerebrospinal fluid (CSF) and brain.

neuroscience↗

bioRxiv2020

Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor Ace2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors will not prevent viral spread.

microbiology↗

bioRxiv2020

Substrate-induced clustering activates Trim-Away of pathogens and proteins

Trim-Away is a powerful new technology that exploits off-the-shelf antibodies and the E3 RING ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically-activated upon substrate engagement during either its normal immune function or when re-purposed for targeted protein degradation is unknown. Here we show that a mechanism of substrate-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce an antiviral response or drive Trim-Away. We harness this mechanism to expand the Trim-Away toolbox with highly-active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has important implications for targeted protein degradation technologies.

molecular biology↗

bioRxiv2020

Learning to count: determining the stoichiometry of bio-molecular complexes using fluorescence microscopy and statistical modelling

Cellular biology occurs through myriad interactions between diverse molecular components, many of which assemble in to specific complexes. Various techniques can provide a qualitative survey of which components are found in a given complex. However, quantitative analysis of the absolute number of molecules within a complex (known as stoichiometry) remains challenging. Here we provide a novel method that combines fluorescence microscopy and statistical modelling to derive accurate molecular counts. We have devised a system in which a given biomolecule is differentially labelled with spectrally distinct fluorescent dyes (label A or B), which are then mixed such that B-labelled molecules are vastly outnumbered by those with label A. Complexes, containing this component, are then simply scored as either being positive or negative for label B. The frequency of positive complexes is directly related to the stoichiometry of interaction and molecular counts can be inferred by statistical modelling. We demonstrate this method using complexes of Adenovirus particles and monoclonal antibodies, achieving counts that are in excellent agreement with previous estimates. Beyond virology, this approach is readily transferable to other experimental systems and, therefore, provides a powerful tool for quantitative molecular biology. The statistical models used in our analysis are available here: https://github.com/sophiamersmann/molecular-counting, the raw data used for molecular counting can be found here: 10.5281/zenodo.3955142.

bioinformatics↗

bioRxiv2020

A thermostable, closed, SARS-CoV-2 spike protein trimer.

The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. S exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor binding site, and subsequently from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S which allow production of thermostable, crosslinked, S protein trimers that are trapped in the closed, pre-fusion, state. We have determined the structures of crosslinked and non-crosslinked proteins, identifying two distinct closed conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.

biochemistry↗