Green, L. R. | Search bioRxiv

bioRxiv2020

periscope: sub-genomic RNA identification in SARS-CoV-2 ARTIC Network Nanopore Sequencing Data

We have developed periscope, a tool for the detection and quantification of sub-genomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed "sub-genomic RNAs". sgRNAs are produced through discontinuous transcription which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L which is located in the 5 UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5 end of all sgRNA. We applied periscope to 1,155 SARS-CoV-2 genomes from Sheffield, UK and validated our findings using orthogonal datasets and in vitro cell systems. Using a simple local alignment to detect reads which contain the leader sequence we were able to identify and quantify reads arising from canonical and non-canonical sgRNA. We were able to detect all canonical sgRNAs at expected abundances, with the exception of ORF10. A number of recurrent non-canonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/- cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing datasets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.

bioinformatics↗

bioRxiv2020

Unfractionated heparin potently inhibits the binding of SARS-CoV-2 spike protein to a human cell line

The SARS-CoV-2 spike protein is known to bind to the receptor, ACE2, on the surface of target cells. The spike protein is processed by membrane proteases, including TMPRSS2, and either internalises or fuses directly with the cell, leading to infection. We have identified a human cell line that expresses both ACE2 and TMPRSS2, the RT4 urinary bladder transitional carcinoma, and used it to develop a proxy assay for viral interactions with host cells. A tagged recombinant form of the spike protein, containing both the S1 and S2 domains, interacted strongly with RT4 cells as determined by flow cytometry, whereas the S1 domain and the receptor binding domain (RBD) interacted weakly. S1S2 interaction was temperature dependent and increased sharply at 37{degrees}C, suggesting that processing of the intact spike protein is likely to be important in the interaction. S1S2 protein could associate with cells with a low dependence on ACE2 expression, while RBD required the presence of ACE2 for interaction. As the spike protein has previously been shown to bind heparin, a soluble glycosaminoglycan, we used a flow cytometric assay to determine the effect of heparin on spike protein interaction with RT4 cells. Unfractionated heparin inhibited spike protein interaction with an IC50 value of <0.05U/ml whereas two low molecular weight heparins were much less effective. A mutant form of the spike protein, lacking the Arg-rich region proposed to be a furin cleavage site, interacted very weakly with cells and had a lower affinity for unfractionated and lower molecular weight heparin than the wild type spike protein. This indicates that the furin cleavage site might also be a heparin binding site and potentially important in interactions with host cells. Taken together, our data suggest that heparin, particularly unfractionated forms, could be considered to reduce clinical manifestations of COVID-19 by inhibiting continuing viral infection. Author SummarySince the emergence of SARS-CoV-2 in 2019, the world has faced a vast public health crisis. SARS-CoV-2 associates with human cells through interaction of the viral spike protein with the host receptor, ACE2. In the absence of a vaccine, new treatments are required to reduce the morbidity and mortality of SARS-CoV-2. Here, we use a novel technique to demonstrate spike protein interactions with human cells with low levels of ACE2 at the cell surface, suggesting a secondary receptor. We demonstrate the importance of a new heparin-binding site within the viral spike protein for these interactions. We also found that unfractionated heparin was able to bind to the viral spike protein and therefore, potently inhibit viral spike protein interactions with human cells. Our data demonstrate that ACE2 is not absolutely required for spike protein interactions with human cells and furthermore, that unfractionated heparin should be considered as a treatment to reduce SARS-CoV-2 viral infection.

microbiology↗