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[Preprint]. 2020 Aug 26:2020.08.26.268854.
doi: 10.1101/2020.08.26.268854.

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

Bryan A Johnson  1 , Xuping Xie  2 , Birte Kalveram  3 , Kumari G Lokugamage  1 , Antonio Muruato  1 , Jing Zou  2 , Xianwen Zhang  2 , Terry Juelich  3 , Jennifer K Smith  3 , Lihong Zhang  3 , Nathen Bopp  3 , Craig Schindewolf  1 , Michelle Vu  1 , Abigail Vanderheiden  4   5   6 , Daniele Swetnam  2 , Jessica A Plante  1 , Patricia Aguilar  3 , Kenneth S Plante  1 , Benhur Lee  7 , Scott C Weaver  1   8 , Mehul S Suthar  4   5   6 , Andrew L Routh  2 , Ping Ren  3 , Zhiqiang Ku  9 , Zhiqiang An  9 , Kari Debbink  10 , Pei Yong Shi  2   8 , Alexander N Freiberg  3   8 , Vineet D Menachery  1   8
Affiliations

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

Bryan A Johnson et al. bioRxiv. .

Update in

  • Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis.
    Johnson BA, Xie X, Bailey AL, Kalveram B, Lokugamage KG, Muruato A, Zou J, Zhang X, Juelich T, Smith JK, Zhang L, Bopp N, Schindewolf C, Vu M, Vanderheiden A, Winkler ES, Swetnam D, Plante JA, Aguilar P, Plante KS, Popov V, Lee B, Weaver SC, Suthar MS, Routh AL, Ren P, Ku Z, An Z, Debbink K, Diamond MS, Shi PY, Freiberg AN, Menachery VD. Johnson BA, et al. Nature. 2021 Mar;591(7849):293-299. doi: 10.1038/s41586-021-03237-4. Epub 2021 Jan 25. Nature. 2021. PMID: 33494095 Free PMC article.

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 .

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Conflict of interest statement

Competing interests

X.X., V.D.M., and P.-Y.S. have filed a patent on the reverse genetic system and reporter SARS-CoV-2. Other authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Distinct replication, spike cleavage, and competition for ΔPRRA.
A) Generation of a SARS-CoV-2 mutant deleting the furin cleavage site insertion from the spike protein. B) Structure of the SARS-CoV-2 spike trimer with a focus on the furin cleavage site (inset). Modeled using the SARS-CoV-1 trimer structure (PDB 6ACD) (14), the WT SARS-CoV-2 trimer (grey) with SARS-CoV-2 PRRA deletion mutant monomer overlay (red). The loop (inset), which is unresolved on SARS-CoV-2 structures (AA 691–702), is shown in cyan on SARS-CoV-2 with the PRRA sequence in blue. The loop region in the PRRA deletion mutant is shown in pink. C) Viral titer from Vero E6 cells infected with WT SARS-CoV-2 (black) or ΔPRRA (blue) at MOI 0.01 (N=3). D) Purified SARS-CoV, SARS-CoV-2 WT, and ΔPRRA virions were probed with anti-spike or anti-nucleocapsid antibody. Full length (FL), S1/S2 cleavage form, and S2’ annotated. E) Competition assay between SARS-CoV-2 WT (black) and ΔPRRA (blue) showing RNA percentage based on quantitative RT-PCR at 50:50, 90:10, 10:90, 99:1, and 1:99 WT/ΔPRRA ratio (N=3 per group). F) Viral titer from Calu3 2B4 cells infected with WT SARS-CoV-2 (black) or ΔPRRA (blue) at MOI 0.01 (N=3). G) Purified SARS-CoV, SARS-CoV-2 WT, and ΔPRRA virions were probed with anti-spike or anti-nucleocapsid antibody. Full length (FL), S1/S2 cleavage form, and S2’ annotated. P-values based on Student T-test and are marked as indicated: *<0.05 ***<0.001.
Figure 2.
Figure 2.. In vivo attenuation of ΔPRRA mutant.
A) Primary SARS-CoV-2 challenge schematic. Two groups of male hamsters (N=4) were challenged with 105 plaque forming units of either SARS-CoV-2 WT or ΔPRRA mutant and evaluated over a 28 day time course for B) weight loss, C) disease score, D) viral titer from nasal wash, and E) viral RNA from oral swabs. F) Schematic for rechallenge of previously infected hamsters. Twenty eight DPI, hamsters from SARS-CoV-2 WT and ΔPRRA were rechallenged with 105 PFU of SARS-CoV-2 WT and evaluated for G) weight loss, H) disease score, I) viral titer from nasal wash, and E) viral RNA from oral swabs. P-values based on Student T-test and are marked as indicated: *<0.05 **<0.01 ***<0.001.
Figure 3.
Figure 3.. Antibody neutralization of ΔPRRA mutant.
A) Schematic for SARS-CoV-2 ΔPRRA reporter virus expressing mNeonGreen (mNG) gene in place of ORF7 equivalent to previously described WT SARS-CoV-2 mNG virus. B) Plaque reduction neutralization (PRNT50) values as measured by changes to mNG expression. PRNT50 values plotted as Log (1/serum dilution) with ΔPRRA on Y axis and WT-SARS-CoV-2. C-E) Representative curves from C) low, D) intermediate, and E) high neutralizing COVID-19 patient sera. F-H) Neutralization curves from mAB-1 (F), mAB-2 (G), and mAB-3 (H), N=3.
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