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The mutation P681H in the B.1.1.7 variant of SARS-CoV-2 probably enhances viral entry and replication

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Halim Maaroufi at Université Laval
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Abstract

The B.1.1.7 variant of SARS-CoV-2 detected in United Kingdom (UK), and today, observed in many countries, indicating further spread. The mutation P681H is in 680-SPRRAR↓SV-687 insertion, forming a cleavage site for protease furin-like enzymes. This cleavage site is important for efficient viral entry into host cells. It has been reported that phosphorylated S680 ([ST]P-x-[RK]) inhibits, in vitro, the furin cleavage of the site S1/S2. Therefore, the P681H mutation abolishes this phospho-inhibition, suggesting an enhancement of virus entry into cells. It also introduces a potential LATS1 phosphorylation site in S686 (H-x-[KR]-x-x-[ST]). It has been demonstrated that host LATS kinases play an important role in optimal RNA replication of the hepatitis C virus through the phosphorylation of its NS5A protein. Hence, it is probable that the phosphorylation of S protein by LATS1 improves the replication of the B.1.1.7 variant. One Sentence Summary: Probably P681H improves viral entry and replication.
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1
REPORT
The mutation P681H in the B.1.1.7 variant of SARS-CoV-2 probably enhances
viral entry and replication
Author: Halim Maaroufi*
Affiliation: Institut de biologie intégrative et des systèmes (IBIS). Université Laval. Quebec
City, Quebec, Canada.
*Correspondence to: Halim.maaroufi@ibis.ulaval.ca
Abstract: The B.1.1.7 variant of SARS-CoV-2 detected in United Kingdom (UK), and today,
observed in many countries, indicating further spread. The mutation P681H is in
680SPRRAR↓SV687 insertion, forming a cleavage site for protease furin-like enzymes. This
cleavage site is important for efficient viral entry into host cells. It has been reported that
phosphorylated S680 ([ST]P-x-[RK]) inhibits, in vitro, the furin cleavage of the site S1/S2.
Therefore, the P681H mutation abolishes this phospho-inhibition, suggesting an enhancement of
virus entry into cells. It also introduces a potential LATS1 phosphorylation site in S686 (H-x-[KR]-
x-x-[ST]). It has been demonstrated that host LATS kinases play an important role in optimal RNA
replication of the hepatitis C virus through the phosphorylation of its NS5A protein. Hence, it is
probable that the phosphorylation of S protein by LATS1 improves the replication of the B.1.1.7
variant.
One Sentence Summary: Probably P681H improves viral entry and replication.
2
Keywords: B.1.1.7 variant; P681H; S1/S2 cleavage site; phospho-regulation
Main Text: The B.1.1.7 variant of SARS-CoV-2 detected in Kent, United Kingdom (UK) on
December, 2020, and today, observed in many countries in the world, indicating further spread due
to higher infection rates. This variant has an unusually great number of mutations, especially in the
spike (S) protein that contains three deletions (H69, V70 and Y144) and six mutations (N501Y,
A570D, P681H, T716I, S982A and D1118H) (1). The mutation P681H was previously observed
independently. Indeed, GISAID reported the first P681H mutation on March 12, 2020
(EPI_ISL_430887) (2). And at December 31, 2020 a total of 5,955 strains with P681H mutation
have been reported, revealing that this mutation is starting to take a significant exponential increase
in worldwide (3). In addition, recently a new variant in Nigeria (B.1.207)(EPI_ISL_729975) has
been defined by the P681H mutation (4).
The mutation P681H is in an intrinsically disordered, solvent-exposed loop, 680SPRRAR↓SV687,
forming a cleavage site RxxR for protease furin-like enzymes at the boundary of S1/S2 subunits
(5). Of note, the region with S1/S2 site is also found in S protein of Infectious Bronchitis Virus
(IBV) (Gammacoronaviruses) with conserved phosphosites corresponding to S/T680 and S686
(Fig. 1). IBVs are known to be highly contagious and intringly present similarities with SARS-
CoV-2 (6).
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Fig. 1. Multiple amino acid sequence alignment of spike proteins (region at the boundary of S1/S2
subunits cleavage site) of Betacoronaviruses SARS-CoV-2 (ID: YP_009724390.1), MERS-CoV
(ID: ALK80251.1), SARS-CoV (ID: P59594.1) and Gammacoronaviruses Infectious Bronchitis
Virus (IBV, ID: APZ73816.1 and P11223) using Clustal omega
(https://www.ebi.ac.uk/Tools/msa/clustalo/). P681 is indicated by black arrow and S680, S686 are
indicated by red stars. S1/S2 cleavage site is represented by green arrow (of note, SARS-CoV has
not a cleavage site). The figure was prepared with ESPript (http://espript.ibcp.fr).
In SARS-CoV-2, this cleavage site is important for efficient viral entry into host cells (7).
Additionally, this insertion contains two phosphorylation sites S680 ([ST]P-x-[RK]) and S686 (R-
x-x-S) that are phosphorylatable, in vitro, by cyclin B-Cdk1 complex (CDK) and basophilic protein
kinase PKA (cAMP-dependent protein kinase), respectively (8). CDKs (Cyclin-dependent kinases)
are proline-directed kinases that phosphorylate a Ser/Thr residue preceding a Pro residue. Of
particular interest is phosphorylated S680 and S686 inhibit furin cleavage, in vitro, of the site S1/S2
(8). Thus, the P681H mutation abolishes the CDK phosphorylation site, preventing the phospho-
inhibition of the cleavage at S1/S2 site known to be important for efficient viral entry into human
cells. Importantly, the P681H mutation does not only abolish the CDK phosphorylation site in S680
but also introduces a potential LATS1 (Large Tumor Suppressor 1) kinase phosphorylation site
S686 in 681HRRARSV687 motif. Of note, the position P681 is occupied by R533 (ID: P11223) or
H536 (ID: APZ73816.1) in IBV (Fig. 1). H536 introduces, as in SARS-CoV-2, a potential LATS1
phosphosite. This novel potential phosphosite overlaps with this of PKA. Interestingly, it has been
reported that the kinase activity of LATS1 is increased upon PKA activation (9). LATS1 is a
member of basophilic AGC kinases (10), but it is distinguishable of them because it requires a
histidine at -5 position (H-x-[KR]-x-x-[ST]). LATS kinases are core components of the mammalian
Hippo pathway that has a role in virus infection and pathogenesis (11). It has been reported that
4
host LATS kinases play an important role in optimal genome replication of the hepatitis C virus
(HCV) through the phosphorylation of non-structural protein 5A (NS5A) (12-14). Of note, HCV
such as SARS-CoV-2 is an enveloped, positive-sense single-stranded RNA virus. During the
SARS-CoV-2 replication cycle, S protein (except the short C-terminal tail) is supposed to be facing
the endoplasmic reticulum or the Golgi lumen (15). Thus, it is probable, as shown in HCV, that the
phosphorylation of S protein by LATS1 improves the replication of the B.1.1.7 variant. The
mutation P681H by enhancing the viral entry (by impairment of S680 phosphorylation that inhibits
furin cleavage in the boundary of S1/S2 subunits) and the viral genome replication (creation of
potential LATS1 phosphorylation site S686) could explain, at least in part, why the B.1.1.7 variant
is more infectious than other strains of SARS-CoV-2. Therefore, LATS1 inhibitors could be tested
against SARS-CoV-2 variants with the mutation P681H (16).
References
1. A. Rambaut, N. Loman, O. Pybus et al., Preliminary genomic characterisation of an
emergent SARS-CoV-2 lineage in the UK defined by a novel set of spike mutations
(2020).
https://virological.org/t/preliminary-genomic-characterisation-of-an-emergent-sars-cov-
2-lineage-in-the-uk-defined-by-a-novel-set-of-spike-mutations/563
2. P. Roychoudhury, X. Hong, K. Jerome, A. Greninger, Virus Name: HCoV-19/USA/WA-
UW-555/2020 / Accession ID: EPI_ISL_430887. UW Virology Lab; (2020).
https://www.epicov.org/epi3/frontend#3ba4e8
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3. D. P. Maison, L. L. Ching, C. M. Shikuma, V. R. Nerurkar, Genetic Characteristics and
Phylogeny of 969-bp S Gene Sequence of SARS-CoV-2 from Hawaii Reveals the
Worldwide Emerging P681H Mutation. bioRxiv, (2021).
4. P. E. Oluniyi, Virus Name: HCoV-19/Nigeria/OS-CV296/2020 / Accession ID:
EPI_ISL_729975. Nigeria Centre for Disease Control (NCDC); (2020).
https://www.epicov.org/epi3/frontend#376f5
5. B. Coutard et al., The spike glycoprotein of the new coronavirus 2019-nCoV contains a
furin-like cleavage site absent in CoV of the same clade. Antiviral Res 176, 104742
(2020).
6. W. H. Al-Jameel, S. S. Al-Mahmood, Similarities and differences of COVID-19 and
avian infectious bronchitis from molecular pathologist and poultry specialist view point.
Iraqi J. Vet. Sci. 34, 223-231 (2020).
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furin and phospho-regulation in SARS-CoV2 but not in SARS-CoV1 or MERS-CoV. Sci
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9. F. X. Yu et al., Protein kinase A activates the Hippo pathway to modulate cell
proliferation and differentiation. Genes Dev 27, 1223-1232 (2013).
10. J. Avruch et al., Protein kinases of the Hippo pathway: regulation and substrates. Semin
Cell Dev Biol 23, 770-784 (2012).
11. Z. Wang et al., The Hippo Pathway and Viral Infections. Front Microbiol 10, 3033
(2019).
12. M. J. Evans, C. M. Rice, S. P. Goff, Phosphorylation of hepatitis C virus nonstructural
protein 5A modulates its protein interactions and viral RNA replication. Proc Natl Acad
Sci U S A 101, 13038-13043 (2004).
13. Y. Huang, K. Staschke, R. De Francesco, S. L. Tan, Phosphorylation of hepatitis C virus
NS5A nonstructural protein: a new paradigm for phosphorylation-dependent viral RNA
replication? Virology 364, 1-9 (2007).
14. H. Meistermann et al., A novel immuno-competitive capture mass spectrometry strategy
for protein-protein interaction profiling reveals that LATS kinases regulate HCV
replication through NS5A phosphorylation. Mol Cell Proteomics 13, 3040-3048 (2014).
15. E. Lontok, E. Corse, C. E. Machamer, Intracellular targeting signals contribute to
localization of coronavirus spike proteins near the virus assembly site. J Virol 78, 5913-
5922 (2004).
7
16. N. Kastan et al., Small-molecule inhibition of Lats kinases promotes Yap-dependent
proliferation in postmitotic mammalian tissues. bioRxiv, (2020).
Acknowledgments: I would like to thank the IBIS (Université Laval) bioinformatics group for
their help; Funding: no funding and Competing interests: no conflicts of interest.
ResearchGate has not been able to resolve any citations for this publication.
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