The SARS-CoV-2 variants associated with 1 infections in India, B.1.617, show 2 enhanced spike cleavage by furin 3

Abstract


Introduction
Unlike its closest known relatives, the SARS-CoV-2 spike (S) protein contains a furin cleavage site at the S1/S2 junction that enhances SARS-CoV-2 replication in airway cells and contributes to virus pathogenicity and transmissibility (1)(2)(3)(4)(5)(6).Pre-cleavage of the S protein in producer cells allows SARS-CoV-2 to enter target cells at the cell surface avoiding endosomal restriction factors (4,7).However, the cleavage site of the early SARS-CoV-2 isolates that emerged in late 2019 are suboptimal, leaving the potential for evolution of variants with increased transmission as a result of an optimised cleavage site (4).
Towards the end of 2020 the SARS-CoV-2 pandemic entered a new phase with repeated emergence of 'variants of concern' lineages with altered viral properties such as transmissibility, pathogenicity, and antigenicity (8).The most widespread and best characterised of these variants is the B.1.1.7 lineage, first found in the UK, which has increased transmissibility and pathogenicity compared to other circulating strains (9)(10)(11).We and others have previously described that the S1/S2 cleavage site of B.1.1.7 S contains a P681H mutation that enhances post-translational S1/S2 cleavage during virus budding (12,13).Other widely circulating variants that arose around the same time include the B.1.351and P.1 lineages, first found in South Africa and Brazil, respectively, that show antigenic escape but do not contain alterations at the furin cleavage site (14).As of May 2021, an increasing number of variant lineages have been described, one of which is the B.1.617lineage.The emergence of this lineage in India coincided with a period of record disease burden across the country, leading to partial collapse of its health infrastructure (15).Early evidence from the UK suggests one B.1.617sublineage (B.1.617.2) likely has enhanced transmissibility, comparable to, or greater than B.1.1.7 (16).B.1.617and its sublineages contain several S mutations, some shared with other variants and associated with antigenic escape (see Table 1).One S substitution shared by all B.1.617sublineages is P681R which we hypothesise further optimises the furin cleavage site (681PRRAR/S686 to 681RRRAR/S686, Figure 1a).In this report we characterise the impact of P681R on the S1/S2 cleavage site.

Results and discussion
To investigate whether the S protein of B.1.617undergoes a higher degree of posttranslational cleavage at S1/S2 than previously circulating strains, we isolated several B.1.617lineage viruses (1 x B.1.617.1 and 2 x B.1.617.2) and compared their S1/S2 cleavage to that of a previously circulating strain of lineage B.1.238,which contains only D614G.The B.1.617lineage S proteins were all more highly cleaved (≥50% cleaved), with a higher proportion of cleaved S2 and a lower proportion of full-length S detectable than the control virus (~33% cleaved) (Figure 1b, c).
To characterise which amino change in the B.1.617S is responsible for its enhanced cleavage, we generated pseudovirus containing the SARS-CoV-2 full B.1.617.1 S and compared it to pseudovirus with D614G spike (WT).As we had previously observed, SARS-CoV-2 spike expressed on pseudovirus contains a larger proportion of cleaved spike (4).While WT S displayed both full length (~20%) and cleaved (~80%) S, B.1.617.1 S showed significantly enhanced cleavage (~95%), with an almost complete lack of full-length protein (Figure 1d,e).P681R alone (on a D614G backbone) was sufficient to convey this phenotype (~96% cleaved), with cleavage enhanced to a similar level as for a previously described S protein carrying the fully optimised furin cleavage site from an H5N1 avian influenza virus haemagglutinin (~97% cleaved) (4).This suggests P681R alone is responsible for the enhanced S cleavage seen in the B.1.617lineages viruses.
We then performed assays to determine whether the optimised cleavage site found in the B.1.617S enables better cleavage directly by furin.We measured the ability of recombinant furin to cleave fluorescently labelled peptides corresponding to the S1/S2 cleavage site of SARS-CoV-2 testing peptides containing 681P (WT), 681R, or a monobasic mutant (monoCS) whereby two of the arginines are substituted to non-basic residues (see Figure 1a) (4).As expected, monoCS was poorly cleaved by recombinant furin compared to the WT peptide which was efficiently cleaved by furin as previously described (Figure 1f) (2).P681R significantly enhanced the ability of furin to cleave the peptide confirming that the arginine substitution is responsible for the enhanced cleavage of the B.1.617S protein.
To conclude, we speculate that enhanced S1/S2 cleavage seen in B.1.617and B.1.1.7 (which contains P681H (12)) may be contributing to the enhanced transmissibility of these SARS-CoV-2 variants.As well as B.1.1.7 and B.1.617,several other emerging lineages contain mutations in the furin cleavage site (8).We would advise that these lineages be kept under close monitoring for any early evidence of more rapid transmission or higher pathogenesis.
Upper respiratory tract swabs used to isolate viruses were collected for routine clinical diagnostic use and sequenced using the ARTIC network protocol (https://artic.network/ncov-2019)to confirm the presence of B.1.617lineage virus, under approval by the Public Health England Research Ethics and Governance Group for the COVID-19 Genomics UK consortium (R&D NR0195).Virus was isolated by inoculating 100 µL of neat swab material onto Vero cells, incubating at 37 o C for 1 h before replacing with growth media supplemented with 1x penicillin/streptomycin and 1x amphotericin B.
Cells were incubated for 5-7 days until cytopathic effect was observed.Isolates were passaged a further two times in Vero E6-ACE2-TMPRSS2 cells (17), the supernatant clarified by centrifugation and concentration for western blot analysis viruses by centrifuging in an Amicon® Ultra-15 Centrifugal Filter Unit followed by an Amicon® Ultra-0.5 Centrifugal Filter Unit with 50 kDa exclusion size.
All spike expression plasmids used in this study contain D614G and K1255*STOP (that results in deletion of the C-terminal cytoplasmic tail of spike containing the endoplasmic retention signal, aka the Δ19 spike truncation).