QTQTN motif upstream of the furin-cleavage site plays a key role in SARS-CoV-2 infection and pathogenesis

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Significance

This study demonstrates that in addition to the furin cleavage site (FCS), both the length/composition of the exterior loop and glycosylation of the QTQTN motif are necessary for efficient SARS-CoV-2 infection and pathogenesis. Disruption of any of these three elements reduces SARS-CoV-2 replication, alters entry pathway utilization, and attenuates in vivo disease. Together, the work highlights the complexity of spike activation beyond just the presence of an FCS.

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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Most cited references 30

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Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Alexandra C Walls, Young-Jun Park, M. Alejandra Tortorici … (2020)

Summary The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We determined cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.

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The Human Genome Browser at UCSC

a. Haussler, W. J. Kent, C. Sugnet … (2002)

As vertebrate genome sequences near completion and research refocuses to their analysis, the issue of effective genome annotation display becomes critical. A mature web tool for rapid and reliable display of any requested portion of the genome at any scale, together with several dozen aligned annotation tracks, is provided at http://genome.ucsc.edu. This browser displays assembly contigs and gaps, mRNA and expressed sequence tag alignments, multiple gene predictions, cross-species homologies, single nucleotide polymorphisms, sequence-tagged sites, radiation hybrid data, transposon repeats, and more as a stack of coregistered tracks. Text and sequence-based searches provide quick and precise access to any region of specific interest. Secondary links from individual features lead to sequence details and supplementary off-site databases. One-half of the annotation tracks are computed at the University of California, Santa Cruz from publicly available sequence data; collaborators worldwide provide the rest. Users can stably add their own custom tracks to the browser for educational or research purposes. The conceptual and technical framework of the browser, its underlying MYSQL database, and overall use are described. The web site currently serves over 50,000 pages per day to over 3000 different users.

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The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade

B. Coutard, C. Valle, X de Lamballerie … (2020)

In 2019, a new coronavirus (2019-nCoV) infecting Humans has emerged in Wuhan, China. Its genome has been sequenced and the genomic information promptly released. Despite a high similarity with the genome sequence of SARS-CoV and SARS-like CoVs, we identified a peculiar furin-like cleavage site in the Spike protein of the 2019-nCoV, lacking in the other SARS-like CoVs. In this article, we discuss the possible functional consequences of this cleavage site in the viral cycle, pathogenicity and its potential implication in the development of antivirals.

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Author and article information

Journal

Journal ID (nlm-ta): Proc Natl Acad Sci U S A

Journal ID (iso-abbrev): Proc Natl Acad Sci U S A

Journal ID (hwp): pnas

Journal ID (publisher-id): PNAS

Title: Proceedings of the National Academy of Sciences of the United States of America

Publisher: National Academy of Sciences

ISSN (Print): 0027-8424

ISSN (Electronic): 1091-6490

Publication date (Electronic): 26 July 2022

Publication date (Print): 9 August 2022

Publication date PMC-release: 26 July 2022

Volume: 119

Issue: 32

Electronic Location Identifier: e2205690119

Affiliations

[1] ^aDepartment of Microbiology and Immunology, University of Texas Medical Branch , Galveston, TX 77555;

[2] ^bInstitute for Human Infection and Immunity, University of Texas Medical Branch , Galveston, TX 77555;

[3] ^cWorld Reference Center of Emerging Viruses and Arboviruses, University of Texas Medical Branch , Galveston, TX 77555;

[4] ^dDepartment of Biochemistry and Molecular Biology, University of Texas Medical Branch , Galveston, TX 77555;

[5] ^eInstitute for Translational Sciences, University of Texas Medical Branch , Galveston, TX;

[6] ^fDepartment of Pathology, University of Texas Medical Branch , Galveston, TX 77555;

[7] ^gDepartment of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University , Baltimore, MD 21211;

[8] ^hCenter for Biodefense and Emerging Infectious Disease, University of Texas Medical Branch , Galveston, TX 77555

Author notes

¹To whom correspondence may be addressed. Email: vimenach@ 123456utmb.edu .

Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, New York; received March 31, 2022; accepted May 26, 2022

Author contributions: M.N.V., K.G.L., B.A.J., S.C.W., D.H.W., W.K.R., A.L.R., and V.D.M. designed research; M.N.V., K.G.L., J.A.P., D.S., A.O.B., S.S., D.M.S., B.A.J., C.S., R.E.A., P.A.C.-V., K.D., D.H.W., W.K.R., A.L.R., K.S.P., and V.D.M. performed research; K.G.L., A.O.B., K.D., S.C.W., and V.D.M. contributed new reagents/analytic tools; M.N.V., J.A.P., A.O.B., S.S., D.M.S., P.A.C.-V., K.D., D.H.W., W.K.R., A.L.R., K.S.P., and V.D.M. analyzed data; and M.N.V. and V.D.M. wrote the paper.