Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis.

• Type of Material: Article
• Authors: Bryan A. Johnson, University of Texas Medical Branch; Xuping Xie, University of Texas Medical Branch; Birte Kalveram, University of Texas Medical Branch; Kumari G. Lokugamage, University of Texas Medical Branch; Antonio Muruato, University of Texas Medical Branch; Jing Zou, University of Texas Medical Branch; Xianwen Zhang, University of Texas Medical Branch; Terry Juelich, University of Texas Medical Branch; Jennifer K. Smith, University of Texas Medical Branch; Lihong Zhang, University of Texas Medical Branch; Nathen Bopp, University of Texas Medical Branch; Craig Schindewolf, University of Texas Medical Branch; Michelle Vu, University of Texas Medical Branch; Abigail Vanderheiden, Emory University; Daniele Swetnam, University of Texas Medical Branch; Jessica A. Plante, University of Texas Medical Branch; Patricia Aguilar, University of Texas Medical Branch; Kenneth S. Plante, University of Texas Medical Branch; Benhur Lee, Icahn School of Medicine at Mount Sinai; Scott C. Weaver, University of Texas Medical Branch; Mehul Suthar, Emory University; Andrew L. Routh, University of Texas Medical Branch; Ping Ren, University of Texas Medical Branch; Zhiqiang Ku, University of Texas Health Science Center; Zhiqiang An, University of Texas Health Science Center; Kari Debbink, Bowie State University; Pei Yong Shi, University of Texas Medical Branch; Alexander N. Freiberg, University of Texas Medical Branch; Vineet D. Menachery, University of Texas Medical Branch
• Language: English
• Date: 2020-08-26
• Publisher: National Institutes of Health
• Publication Version: Preprint (Prior to Peer Review)
• Copyright Statement: The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1101/2020.08.26.268854
• Title of Journal or Parent Work: bioRxiv
• Volume: 1
• Grant/Funding Information: Research was also supported by STARs Award provided by the University of Texas System to VDM and trainee funding provided by the McLaughlin Fellowship Fund at UTMB.; R01AI123449 to AF and BL; R24AI120942 (WRCEVA) to SCW).; AI134907, AI145617, and UL1TR001439 to P-YS;; P-YS was also supported by CDC grant for the Western Gulf Center of Excellence for Vector-Borne Diseases, and awards from the Sealy & Smith Foundation, Kleberg Foundation, John S. Dunn Foundation, Amon G. Carter Foundation, Gilson Longenbaugh Foundation, and Summerfield Robert Foundation.; Research was supported by grants from NIA and NIAID of the NIH to (AI153602 and AG049042 to VDM AI142759,
• Keywords: Biochemistry & Molecular Biology; Pathology; SARS-CoV-2; Cleavage site; Immunology; Microbiology; Pathogenesis; Furin
• Research Categories: Health Sciences, Immunology; Chemistry, Biochemistry; Biology, Microbiology

Abstract

SARS-CoV-2 has resulted in a global pandemic and shutdown economies around the world. Sequence analysis indicates that the novel coronavirus (CoV) has an insertion of a furin cleavage site (PRRAR) in its spike protein. Absent in other group 2B CoVs, the insertion may be a key factor in the replication and virulence of SARS-CoV-2. To explore this question, we generated a SARS-CoV-2 mutant lacking the furin cleavage site (ΔPRRA) in the spike protein. This mutant virus replicated with faster kinetics and improved fitness in Vero E6 cells. The mutant virus also had reduced spike protein processing as compared to wild-type SARS-CoV-2. In contrast, the ΔPRRA had reduced replication in Calu3 cells, a human respiratory cell line, and had attenuated disease in a hamster pathogenesis model. Despite the reduced disease, the ΔPRRA mutant offered robust protection from SARS-CoV-2 rechallenge. Importantly, plaque reduction neutralization tests (PRNT 50 ) with COVID-19 patient sera and monoclonal antibodies against the receptor-binding domain found a shift, with the mutant virus resulting in consistently reduced PRNT 50 titers. Together, these results demonstrate a critical role for the furin cleavage site insertion in SARS-CoV-2 replication and pathogenesis. In addition, these findings illustrate the importance of this insertion in evaluating neutralization and other downstream SARS-CoV-2 assays. Importance: As COVID-19 has impacted the world, understanding how SARS-CoV-2 replicates and causes virulence offers potential pathways to disrupt its disease. By removing the furin cleavage site, we demonstrate the importance of this insertion to SARS-CoV-2 replication and pathogenesis. In addition, the findings with Vero cells indicate the likelihood of cell culture adaptations in virus stocks that can influence reagent generation and interpretation of a wide range of data including neutralization and drug efficacy. Overall, our work highlights the importance of this key motif in SARS-CoV-2 infection and pathogenesis. Article Summary: A deletion of the furin cleavage site in SARS-CoV-2 amplifies replication in Vero cells, but attenuates replication in respiratory cells and pathogenesis in vivo. Loss of the furin site also reduces susceptibility to neutralization in vitro .