QTQTN motif upstream of the furin-cleavage site plays a key role in SARS-CoV-2 infection and pathogenesis
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Abstract
The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site—the FCS, loop length, and glycosylation—are required for efficient SARS-CoV-2 replication and pathogenesis.
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SciScore for 10.1101/2021.12.15.472450: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: All studies were conducted under a protocol approved by the UTMB Institutional Animal Care and Use Committee and complied with USDA guidelines in a laboratory accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Sex as a biological variable Hamster infection studies: Male golden Syrian hamsters (3-4 weeks old) were purchased from Envigo. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Membranes were probed with SARS-CoV S-specific antibodies (Novus Biologicals #NB100-56578) and followed with horseradish peroxidase (HRP)-conjugated anti-rabbit … SciScore for 10.1101/2021.12.15.472450: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: All studies were conducted under a protocol approved by the UTMB Institutional Animal Care and Use Committee and complied with USDA guidelines in a laboratory accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Sex as a biological variable Hamster infection studies: Male golden Syrian hamsters (3-4 weeks old) were purchased from Envigo. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Membranes were probed with SARS-CoV S-specific antibodies (Novus Biologicals #NB100-56578) and followed with horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (Cell Signaling Technology #7074S). anti-rabbitsuggested: (Cell Signaling Technology Cat# 7074, RRID:AB_2099233)Experimental Models: Cell Lines Sentences Resources Calu-3 2B4 cells were grown in DMEM supplemented with 10% defined FBS (HyClone #SH30070.03), 1% antibiotic-antimycotic, and 1 mg/ml sodium pyruvate. Calu-3 2B4suggested: RRID:CVCL_YZ47)Vero E6 cells were infected with a total MOI of 0.01 (WT alone, 1:1 WT:ΔQTQTN, or ΔQTQTN alone) as described in ‘In vitro infection’. Vero E6suggested: RRID:CVCL_XD71)Software and Algorithms Sentences Resources Data quality was determined to be sufficient by generating and loading bedgraph files into the UCSC Genome Browser 22. UCSC Genome Browsersuggested: (UCSC Genome Browser, RRID:SCR_005780)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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