Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation

Zhang, Liping; Mann, Matthew; Syed, Zulfeqhar A.; Reynolds, Hayley M; Tian, E; Samara, N.L.; Zeldin, Darryl C.; Tabak, Lawrence A.; Hagen, Karl S.

doi:10.1101/2021.02.05.429982

Cited by 41 publications

(60 citation statements)

References 14 publications

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“…The structural and functional role of glycosylation in the SARS-CoV-2 spike protein has been widely investigated 52,[60][61][62][63][64][65][66] . Of note is the fact that 35% of the SARS-CoV-2 spike glycoprotein contains carbohydrate moieties, which have profound influence on the viral infectivity, susceptibility to antibody neutralization 67,68 . The N-linked glycosylation sites of spike proteins have been related to alterations in its open or closed state thus interfering in its capacity to bind to the receptor 69 .…”

Section: Discussionmentioning

confidence: 99%

SARS-CoV-2 activates ER stress and Unfolded protein response

Rosa-Fernandes

Macedo-da-Silvia

et al. 2021

Preprint

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Coronavirus disease-2019 (COVID-19) pandemic caused by the SARS-CoV-2 coronavirus infection is a major global public health concern affecting millions of people worldwide. The scientific community has joint efforts to provide effective and rapid solutions to this disease. Knowing the molecular, transmission and clinical features of this disease is of paramount importance to develop effective therapeutic and diagnostic tools. Here, we provide evidence that SARS-CoV-2 hijacks the glycosylation biosynthetic, ER-stress and UPR machineries for viral replication using a time-resolved (0-48 hours post infection, hpi) total, membrane as well as glycoproteome mapping and orthogonal validation. We found that SARS-CoV-2 induces ER stress and UPR is observed in Vero and Calu-3 cell lines with activation of the PERK-eIF2α-ATF4-CHOP signaling pathway. ER-associated protein upregulation was detected in lung biopsies of COVID-19 patients and associated with survival. At later time points, cell death mechanisms are triggered. The data show that ER stress and UPR pathways are required for SARS-CoV-2 infection, therefore representing a potential target to develop/implement anti-CoVID-19 drugs.

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Section: Discussionmentioning

confidence: 99%

SARS-CoV-2 activates ER stress and Unfolded protein response

Rosa-Fernandes

Macedo-da-Silvia

et al. 2021

Preprint

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“…Interestingly, WT residues S673, T676, T678 and S680 close to the furin site are all candidates for O-glycosylation which is dependent on proline P681 27 . It was shown that O-glycosylation of these residues negatively affects furin cleavage, contributing to S stability and infectivity 27 . P681 and S680 are deleted in BriSΔ, and P681 is mutated in other lineages, including the B.1.1.7 variant that emerged in Kent, UK 28 and rapidly spread globally.…”

Section: Resultsmentioning

confidence: 99%

“…We analyzed N-glycosylation of BriS and identified no significant changes compared to previous S structures (Supplementary information, Table S2). Interestingly, WT residues S673, T676, T678 and S680 close to the furin site are all candidates for O-glycosylation which is dependent on proline P681 27 . It was shown that O-glycosylation of these residues negatively affects furin cleavage, contributing to S stability and infectivity 27 .…”

Section: Cryo-em Structure Of Bris Glycoproteinmentioning

confidence: 99%

Structural basis for cell-type specific evolution of viral fitness by SARS-CoV-2

Gupta

Toelzer

Williamson

et al. 2021

Preprint

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As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants which is of particular concern due to their potential for increased transmissibility and pathology. In addition to this entrenched variant diversity in circulation, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriSΔ variant, originally identified as a viral subpopulation by passaging SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike glycoprotein an eight amino-acid deletion encompassing the furin recognition motif and S1/S2 cleavage site. Here, we analyzed the structure, function and molecular dynamics of this variant spike, providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity, allowing the virus to probe diverse trajectories in distinct cell types to evolve viral fitness.

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“…The spike protein is heavily glycosylated at both N- and O-linked sites, and impaired glycosylation would clearly affect the proper maturation of spike protein and concomitant virus/pseudovirus formation [ 43 ]. Conversely, glycosylation of the three new O-linked glycosylation sites adjacent to the polybasic furin site [ 2 ] decreases furin cleavage of S protein [ 44 ]. These findings underscore the critical importance of S protein glycosylation but do not detract from our overall conclusion that the novel ribosomal pausing site encoding the furin site acts as a brake on translation to allow proper modulation of spike protein cotranslational folding that is absent in the overexpressed optimized spike protein sequence.…”

Section: Discussionmentioning

confidence: 99%

The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

Postnikova

Uppal

Huang

et al. 2021

IJMS

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The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

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Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation

Cited by 41 publications

References 14 publications

SARS-CoV-2 activates ER stress and Unfolded protein response

SARS-CoV-2 activates ER stress and Unfolded protein response

Structural basis for cell-type specific evolution of viral fitness by SARS-CoV-2

The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

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