scholarly journals SARS-CoV-2 variants reveal features critical for replication in primary human cells

PLoS Biology ◽  
2021 ◽  
Vol 19 (3) ◽  
pp. e3001006
Author(s):  
Marie O. Pohl ◽  
Idoia Busnadiego ◽  
Verena Kufner ◽  
Irina Glas ◽  
Umut Karakus ◽  
...  
Keyword(s):  
Large Scale ◽  
Human Cells ◽  
Sequencing Data ◽  
Furin Cleavage ◽  
Bronchial Epithelial ◽  
Naturally Occurring ◽  
Furin Cleavage Site ◽  
Low Passage ◽  
Viral Sequencing ◽  
Primary Human Cells

Since entering the human population, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; the causative agent of Coronavirus Disease 2019 [COVID-19]) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication, or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from nonhuman Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific replication and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants of concern.

scholarly journals Distinct Phenotypes of SARS-CoV-2 Isolates Reveal Viral Traits Critical for Replication in Primary Human Respiratory Cells

2020 ◽  
Author(s):  
Marie O. Pohl ◽  
Idoia Busnadiego ◽  
Verena Kufner ◽  
Stefan Schmutz ◽  
Maryam Zaheri ◽  
...  
Keyword(s):  
Virus Replication ◽  
Large Scale ◽  
Sequencing Data ◽  
Furin Cleavage ◽  
New Host ◽  
Sequence Features ◽  
Furin Cleavage Site ◽  
Respiratory Cells ◽  
Low Passage ◽  
Viral Sequencing

ABSTRACTSince entering the human population, SARS-CoV-2 (the causative agent of COVID-19) has spread across the world, causing >40 million infections and >1 million deaths. While large-scale sequencing efforts have identified numerous genetic mutations in SARS-CoV-2 during its circulation, it remains largely unclear whether these changes impact adaptation, replication or transmission of the virus in its new host. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all the major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, viral stocks and passaging experiments, with kinetic virus replication data from non-human Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 sequence features that associate with distinct phenotypes. Notably, naturally-occurring substitutions in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G substitution generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E, and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin-cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human respiratory cells. Overall, our study uncovers natural sequence features in the SARS-CoV-2 genome that determine efficient virus replication and tropism for the human respiratory epithelium.

scholarly journals Large-scale discovery of recombinases for integrating DNA into the human genome

2021 ◽  
Author(s):  
Matthew G Durrant ◽  
Alison Fanton ◽  
Josh Tycko ◽  
Michaela Hinks ◽  
Sita Chandrasekaran ◽  
...  
Keyword(s):  
Human Genome ◽  
Large Scale ◽  
Genome Engineering ◽  
Human Cells ◽  
Sequencing Data ◽  
Dna Breaks ◽  
Serine Recombinases ◽  
Attachment Sites ◽  
Dna Attachment ◽  
Computational Discovery

Recent microbial genome sequencing efforts have revealed a vast reservoir of mobile genetic elements containing integrases that could be useful genome engineering tools. Large serine recombinases (LSRs), such as Bxb1 and PhiC31, are bacteriophage-encoded integrases that can facilitate the insertion of phage DNA into bacterial genomes. However, only a few LSRs have been previously characterized and they have limited efficiency in human cells. Here, we developed a systematic computational discovery workflow that searches across the bacterial tree of life to expand the diversity of known LSRs and their cognate DNA attachment sites by >100-fold. We validated this approach via experimental characterization of LSRs, leading to three classes of LSRs distinguished from one another by their efficiency and specificity. We identify landing pad LSRs that efficiently integrate into native attachment sites in a human cell context, human genome-targeting LSRs with computationally predictable pseudosites, and multi-targeting LSRs that can unidirectionally integrate cargos with similar efficiency and superior specificity to commonly used transposases. LSRs from each category were functionally characterized in human cells, overall achieving up to 7-fold higher plasmid recombination than Bxb1 and genome insertion efficiencies of 40-70% with cargo sizes over 7 kb. Overall, we establish a paradigm for the large-scale discovery of microbial recombinases directly from sequencing data and the reconstruction of their target sites. This strategy provided a rich resource of over 60 experimentally characterized LSRs that can function in human cells and thousands of additional candidates for large-payload genome editing without double-stranded DNA breaks.

scholarly journals ACE2-independent interaction of SARS-CoV-2 spike protein to human epithelial cells can be inhibited by unfractionated heparin

2020 ◽  
Author(s):  
Lynda J. Partridge ◽  
Lucy Urwin ◽  
Martin J.H. Nicklin ◽  
David C. James ◽  
Luke R. Green ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Protein Interactions ◽  
Human Cells ◽  
Host Cells ◽  
Spike Protein ◽  
Heparin Binding ◽  
Furin Cleavage ◽  
Heparin Binding Site ◽  
Furin Cleavage Site

AbstractThe 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°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.

scholarly journals SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected, hospitalized COVID-19 patients

2020 ◽  
Author(s):  
William B. Klimstra ◽  
Natasha L. Tilston-Lunel ◽  
Sham Nambulli ◽  
James Boslett ◽  
Cynthia M. McMillen ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Serum Samples ◽  
Furin Cleavage ◽  
Furin Cleavage Site ◽  
Similar Disease ◽  
Antibody Titers ◽  
Low Passage

AbstractSARS-CoV-2, the causative agent of COVID-19, emerged at the end of 2019 and by mid-June 2020, the virus has spread to at least 215 countries, caused more than 8,000,000 confirmed infections and over 450,000 deaths, and overwhelmed healthcare systems worldwide. Like SARS-CoV, which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low passage (P) strains of SARS-CoV-2 (Wash1: P4 and Munich: P1) were cultured twice in Vero-E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1: P6 and minor variants in the Munich: P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero-E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies we investigated the development of neutralizing antibody titers in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

scholarly journals Coronaviruses with a SARS-CoV-2-like receptor-binding domain allowing ACE2-mediated entry into human cells isolated from bats of Indochinese peninsula

2021 ◽  
Author(s):  
Sarah Temmam ◽  
Khamsing Vongphayloth ◽  
Eduard Baquero Salazar ◽  
Sandie Munier ◽  
Max Bonomi ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Human Cells ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Furin Cleavage ◽  
Animal Reservoir ◽  
Angiotensin Converting Enzyme 2 ◽  
Furin Cleavage Site ◽  
Spike Sequence ◽  
Early Human

Abstract The animal reservoir of SARS-CoV-2 is unknown despite reports of various SARS-CoV-2-related viruses in Asian Rhinolophus bats, including the closest virus from R. affinis, RaTG13. Several studies have suggested the involvement of pangolin coronaviruses in SARS-CoV-2 emergence. SARS-CoV-2 presents a mosaic genome, to which different progenitors contribute. The spike sequence determines the binding affinity and accessibility of its receptor-binding domain (RBD) to the cellular angiotensin-converting enzyme 2 (ACE2) receptor and is responsible for host range. SARS-CoV-2 progenitor bat viruses genetically close to SARS-CoV-2 and able to enter human cells through a human ACE2 pathway have not yet been identified, though they would be key in understanding the origin of the epidemics. Here we show that such viruses indeed circulate in cave bats living in the limestone karstic terrain in North Laos, within the Indochinese peninsula. We found that the RBDs of these viruses differ from that of SARS-CoV-2 by only one or two residues, bind as efficiently to the hACE2 protein as the SARS-CoV-2 Wuhan strain isolated in early human cases, and mediate hACE2-dependent entry into human cells, which is inhibited by antibodies neutralizing SARS-CoV-2. None of these bat viruses harbors a furin cleavage site in the spike. Our findings therefore indicate that bat-borne SARS-CoV-2-like viruses potentially infectious for humans circulate in Rhinolophus spp. in the Indochinese peninsula.

scholarly journals Delta variant with P681R critical mutation revealed by ultra-large atomic-scale ab initio simulation: Implications for the fundamentals of biomolecular interactions

2021 ◽  
Author(s):  
Puja Adhikari ◽  
Bahaa Jawad ◽  
Praveen Rao ◽  
Rudolf Podgornik ◽  
Wai-Yim Ching
Keyword(s):  
Amino Acid ◽  
Ab Initio ◽  
Infection Rate ◽  
Large Scale ◽  
Atomic Scale ◽  
Spike Protein ◽  
Furin Cleavage ◽  
Double Mutation ◽  
Furin Cleavage Site ◽  
High Infection

ABSTRACTSARS-CoV-2 Delta variant is emerging as a globally dominant strain. Its rapid spread and high infection rate are attributed to a mutation in the spike protein of SARS-CoV-2 allowing the virus to invade human cells much faster and with increased efficiency. Particularly, an especially dangerous mutation P681R close to the furin cleavage site has been identified as responsible for increasing the infection rate. Together with the earlier reported mutation D614G in the same domain, it offers an excellent instance to investigate the nature of mutations and how they affect the interatomic interactions in the spike protein. Here, using ultra large-scale ab initio computational modeling, we study the P681R and D614G mutations in the SD2-FP domain including the effect of double mutation and compare the results with the wild type. We have recently developed a method of calculating the amino acid-amino acid bond pairs (AABP) to quantitatively characterize the details of the interatomic interactions, enabling us to explain the nature of mutation at the atomic resolution. Our most significant find is that the mutations reduce the AABP value, implying a reduced bonding cohesion between interacting residues and increasing the flexibility of these amino acids to cause the damage. The possibility of using this unique mutation quantifiers in a machine learning protocol could lead to the prediction of emerging mutations.

scholarly journals SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients

2020 ◽  
Vol 101 (11) ◽  
pp. 1156-1169 ◽  
Author(s):  
William B. Klimstra ◽  
Natasha L. Tilston-Lunel ◽  
Sham Nambulli ◽  
James Boslett ◽  
Cynthia M. McMillen ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Serum Samples ◽  
Furin Cleavage ◽  
Furin Cleavage Site ◽  
Antibody Titres ◽  
Low Passage

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

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