Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

Lamers, Mart M.; Mykytyn, Anna Z; Breugem, Tim I; Wang, Yiquan; Wu, Douglas C.; Riesebosch, Samra; Doel, Petra B. van den; Schipper, Debby; Bestebroer, Theo M.; Wu, Nicholas C.; Haagmans, Bart L.

doi:10.1101/2021.01.22.427802

Cited by 25 publications

(36 citation statements)

References 33 publications

(25 reference statements)

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“…Finally, as also observed by others [32-34, 53], we found that in-frame deletions of the furin-cleavage site in S could be selected for and enriched in SARS-CoV-2 isolates during passage in Vero-CCL81 cells. This was apparent even for very low passage stocks as described here (passage 3 from original patient material), but importantly was not a universal feature of all SARS-CoV-2 isolates: deletions of the S furin-cleavage site were only detected in 2 out of 14 of our primary isolates.…”

Section: Discussionsupporting

confidence: 86%

“…Such findings, together with our additional observations relating to functional E substitutions during SARS-CoV-2 growth in Vero-CCL81 cells, underscore the need for care during stock preparation, particularly where viruses are to be compared with one another for phenotypes. Thus, it may be that other cell-lines, including human Calu-3 [53], are more suitable for SARS-CoV-2 propagation, and should be thoroughly characterized in the future as a possible substrate to limit cell culture adaptation in SARS-CoV-2 genes such as S and E. Nevertheless, we found that in-frame deletions in the S furin-cleavage site attenuated SARS-CoV-2, most notably in primary human bronchial epithelial cells. However, passaging of SARS-CoV-2 preparations containing the S furin-cleavage site deletions in primary human bronchial epithelial cells provided a very high selection pressure to enrich only for viruses that retained the full S furin-cleavage site.…”

Section: Discussionmentioning

confidence: 99%

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SARS-CoV-2 Variants Reveal Features Critical for Replication in Primary Human Cells

Pohl

Busnadiego

Kufner

et al. 2020

Preprint

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Since 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.

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Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

SARS-CoV-2 Variants Reveal Features Critical for Replication in Primary Human Cells

Pohl

Busnadiego

Kufner

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Finally, as also observed by others [32][33][34]53], we found that in-frame deletions of the furin cleavage site in S could be selected for and enriched in SARS-CoV-2 isolates during passage in Vero-CCL81 cells. This was apparent even for very low-passage stocks as described here (passage 3 from original patient material), but importantly was not a universal feature of all SARS--CoV-2 isolates: Deletions of the S furin cleavage site were only detected in 2 out of 14 of our primary isolates.…”

Section: Plos Biologysupporting

confidence: 87%

“…Such findings, together with our additional observations relating to functional E substitutions during SARS-CoV-2 growth in Vero-CCL81 cells, underscore the need for care during stock preparation, particularly where viruses are to be compared with one another for phenotypes. Thus, it may be that other cell lines, including human Calu-3 [53], are more suitable for SARS-CoV-2 propagation and should be thoroughly characterized in the future as a possible substrate to limit cell culture adaptation in SARS-CoV-2 genes such as S and E. Nevertheless, we found that in-frame deletions in the S furin cleavage site attenuated SARS-CoV-2, most notably in primary human BEpCs. However, passaging of SARS-CoV-2 preparations containing the S furin cleavage site deletions in primary human BEpCs provided a very high selection pressure to enrich only for viruses that retained the full S furin cleavage site.…”

Section: Plos Biologymentioning

confidence: 79%

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

et al. 2021

View full text Add to dashboard Cite

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.

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“…Epithelial organoids have gained traction as a physiological platform for personal medicine because these organoids retain patient-specific traits 19 . A handful of studies have used alveolospheres and organoids to study SARS-CoV-2 epithelial response 20,21,22,23,24,25 and tropism 7, 26,27,28,29,30 . A comprehensive analysis of SARS-CoV-2 infection in a large panel of lung organoids has not been reported to date.…”

Section: Introductionmentioning

confidence: 99%

SARS-CoV-2 infection studies in lung organoids identify TSPAN8 as novel mediator

Hysenaj

Little²,

Kulhanek³

et al. 2021

Preprint

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SARS coronavirus-2 (SARS-CoV-2) is causing a global pandemic with large variation in COVID-19 disease spectrum. SARS-CoV-2 infection requires host receptor ACE2 on lung epithelium, but epithelial underpinnings of variation are largely unknown. We capitalized on comprehensive organoid assays to report remarkable variation in SARS-CoV-2 infection rates of lung organoids from different subjects. Tropism is highest for TUBA- and MUC5AC-positive organoid cells, but levels of TUBA-, MUC5A-, or ACE2- positive cells do not predict infection rate. We identify surface molecule Tetraspanin 8 (TSPAN8) as novel mediator of SARS-CoV-2 infection, which is not downregulated by this specific virus. TSPAN8 levels, prior to infection, strongly correlate with infection rate and TSPAN8-blocking antibodies diminish SARS-CoV-2 infection. We propose TSPAN8 as novel functional biomarker and potential therapeutic target for COVID-19.

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Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

Cited by 25 publications

References 33 publications

SARS-CoV-2 Variants Reveal Features Critical for Replication in Primary Human Cells

SARS-CoV-2 Variants Reveal Features Critical for Replication in Primary Human Cells

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

SARS-CoV-2 infection studies in lung organoids identify TSPAN8 as novel mediator

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