SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells

Sasaki, Michihito; Uemura, Kentaro; Sato, Akihiko; Toba, Shinsuke; Sanaki, Takao; Maenaka, Katsumi; Hall, William W.; Orba, Yasuko; Sawa, Hirofumi

doi:10.1371/journal.ppat.1009233

Cited by 183 publications

(215 citation statements)

References 45 publications

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“…Deletion of the furin cleavage site has been shown to reduce SARS-CoV-2 replication in human respiratory cells, hamster and hACE2 mice ( 18 ). Previous work has demonstrated rapid emergence of spike variants at the furin-cleavage motif, including R685H, following five passages in type II transmembrane serine protease (TMPRSS2)-deficient cells ( 19 ). This explains the emergence of this variant in our study following propagation in Vero E6 cells, which do not express high levels of TMPRSS2 ( 20 ).…”

Section: Discussionmentioning

confidence: 99%

SARS-CoV-2 evolution in animals suggests mechanisms for rapid variant selection

Bashor

Gagne

Bosco‐Lauth

et al. 2021

Preprint

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SARS-CoV-2 spillback from humans into domestic and wild animals has been well-documented. We compared variants of cell culture-expanded SARS-CoV-2 inoculum and virus recovered from four species following experimental exposure. Five nonsynonymous changes in nsp12, S, N and M genes were near fixation in the inoculum, but reverted to wild-type sequences in RNA recovered from dogs, cats and hamsters within 1-3 days post-exposure. Fourteen emergent variants were detected in viruses recovered from animals, including substitutions at spike positions H69, N501, and D614, which also vary in human lineages of concern. The rapidity of in vitro and in vivo SARS-CoV-2 selection reveals residues with functional significance during host-switching, illustrating the potential for spillback reservoir hosts to accelerate evolution, and demonstrating plasticity of viral adaptation in animal models.

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Section: Discussionmentioning

confidence: 99%

SARS-CoV-2 evolution in animals suggests mechanisms for rapid variant selection

Bashor

Gagne

Bosco‐Lauth

et al. 2021

Preprint

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“…To discriminate the two S protein activation pathways, we included the . This explains why SARS-CoV-2 passaging in Vero E6 cells regularly leads to emergence of viruses bearing substitutions or deletions in the S1/S2 loop [45][46][47][48][49][50][51][52].…”

Section: Loop Deletion Mutants Of Sars-2-s Show Enhanced Cathepsin-dependent Entry Explaining Their Emergence In Vero Cellsmentioning

confidence: 99%

“…When unprimed, SARS-2-S pseudoviruses are strongly boosted towards the cathepsin B/L route. This likely explains the replication advantage of loop-deletion SARS-CoV-2 mutants in cathepsin L-rich Vero E6 cells [45][46][47][48][49][50][51][52]. The non-covalently linked S1/S2 form is less stable and a plausible disadvantage for endosomal entry.…”

Section: Plos Pathogensmentioning

confidence: 99%

“…For SARS-2-S, the determinants governing S1/S2 processing are still far from clear. Virus passaging in Vero cells commonly leads to substitutions or deletions in the S1/S2 cleavage loop [45][46][47][48][49][50][51][52]. In animal models, these viruses exhibit reduced transmission [53] or virulence [54], providing an explanation why severe mutations in the S1/S2 loop are only rarely detected in humans [48,53,55] (S1 and S2 Tables).…”

Section: Introductionmentioning

confidence: 99%

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The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

et al. 2021

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The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards the human respiratory tract. First, the S proteins exhibit an intrinsic temperature preference, corresponding with the temperature of the upper or lower airways. Pseudoviruses bearing the SARS-CoV-2 spike (SARS-2-S) were more infectious when produced at 33°C instead of 37°C, a property shared with the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV and MERS-CoV favored 37°C, in accordance with virus preference for the lower airways. Next, SARS-2-S-driven entry was efficiently activated by not only TMPRSS2, but also the TMPRSS13 protease, thus broadening the cell tropism of SARS-CoV-2. Both proteases proved relevant in the context of authentic virus replication. TMPRSS13 appeared an effective spike activator for the virulent coronaviruses but not the low pathogenic HCoV-229E virus. Activation of SARS-2-S by these surface proteases requires processing of the S1/S2 cleavage loop, in which both the furin recognition motif and extended loop length proved critical. Conversely, entry of loop deletion mutants is significantly increased in cathepsin-rich cells. Finally, we demonstrate that the D614G mutation increases SARS-CoV-2 stability, particularly at 37°C, and, enhances its use of the cathepsin L pathway. This indicates a link between S protein stability and usage of this alternative route for virus entry. Since these spike properties may promote virus spread, they potentially explain why the spike-G614 variant has replaced the early D614 variant to become globally predominant. Collectively, our findings reveal adaptive mechanisms whereby the coronavirus spike protein is adjusted to match the temperature and protease conditions of the airways, to enhance virus transmission and pathology.

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“…The S1/S2 furin cleavage site is unique to SARS-CoV-2 compared to other members of the betacoronavirus family and has been demonstrated to promote fusion between SARS-CoV-2 and host cell membranes [47]. Mutations in this region may promote viral entry into epithelial cells [48], increase transmissibility [49] or modulate the host immune response [46], [50].…”

Section: Resultsmentioning

confidence: 99%

Metagenomic sequencing of municipal wastewater provides a near-complete SARS-CoV-2 genome sequence identified as the B.1.1.7 variant of concern from a Canadian municipality concurrent with an outbreak

Landgraff

Wang

Buchanan

et al. 2021

Preprint

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Laboratory-based wastewater surveillance for SARS-CoV-2, the causative agent of the ongoing COVD-19 pandemic, can be conducted using RT-qPCR-based screening of municipal wastewater samples. Although it provides rapid viral detection and can inform SARS-CoV-2 abundance in wastewater, this approach lacks the resolution required for viral genotyping and does not support tracking of viral genome evolution. The recent emergence of several variants of concern, a result of mutations across the genome including the accrual of important mutations within the viral spike glycoprotein, has highlighted the need for a method capable of detecting the cohort of mutations associated with these and newly emerging genotypes. Here we provide an innovative methodology for the recovery of a near-complete SARS-CoV-2 sequence from a wastewater sample collected from across Canadian municipalities including one that experienced a significant outbreak attributable to the SARS-CoV-2 B.1.1.7 variant of concern. Our results demonstrate that a combined interrogation of genome consensus-level sequences and alternative alleles enables the identification of a SARS-CoV-2 variant of concern and the detection of a new allele within a viral accessory gene that may be representative of a recently evolved B.1.1.7 sublineage.

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SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells

Cited by 183 publications

References 45 publications

SARS-CoV-2 evolution in animals suggests mechanisms for rapid variant selection

SARS-CoV-2 evolution in animals suggests mechanisms for rapid variant selection

The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

Metagenomic sequencing of municipal wastewater provides a near-complete SARS-CoV-2 genome sequence identified as the B.1.1.7 variant of concern from a Canadian municipality concurrent with an outbreak

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