Altered Sub-Genomic RNA Expression in SARS-CoV-2 B.1.1.7 Infections (preprint)

SARS-CoV-2 lineage B.1.1.7 viruses are more transmissible, may lead to greater clinical severity, and result in modest reductions in antibody neutralization. subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome and is a crucial step in the SARS-CoV-2 life cycle. Applying our tool (periscope) to ARTIC Network Oxford Nanopore genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA expression profiles are significantly increased in B.1.1.7 infections (n=879). This increase is seen over the previous dominant circulating lineage in the UK, B.1.177 (n=943), which is independent of genomic reads, E gene cycle threshold and day of illness when sampling occurred. A noncanonical subgenomic RNA which could represent ORF9b is significantly enriched in B.1.1.7 SARS-CoV-2 infections, potentially as a result of a triple nucleotide mutation leading to amino acid substitution D3L in nucleocapsid in this lineage which increases complementarity with the genomic leader sequence. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles in sequence data to evaluate emerging potential variants of concern.

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

We have developed periscope, a tool for the detection and quantification of sub-genomic RNA in ARTIC network protocol generated Nanopore SARS-CoV-2 sequence data. We applied periscope to 1155 SARS-CoV-2 sequences from Sheffield, UK. 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 sub-genomic RNA. We were able to detect all canonical sub-genomic RNAs at expected abundances, with the exception of ORF10, suggesting that this is not a functional ORF. A number of recurrent non-canonical sub-genomic RNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sub-genomic RNA analysis. Finally variants found in genomic RNA are transmitted to sub-genomic RNAs with high fidelity in most cases. This tool can be applied to tens of thousands of sequences worldwide to provide the most comprehensive analysis of SARS-CoV-2 sub-genomic RNA to date.

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

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.