key: cord-0996513-qn0snu74
authors: Pokhrel, Suman; Kraemer, Benjamin R.; Burkholz, Scott; Mochly-Rosen, Daria
title: Natural variants in SARS-CoV-2 S protein pinpoint structural and functional hotspots: implications for prophylaxis and therapeutic strategies
date: 2021-03-30
journal: bioRxiv
DOI: 10.1101/2021.01.04.425340
sha: e6aadacf74100fb81787db5ff27891071b513552
doc_id: 996513
cord_uid: qn0snu74

In December 2019, a novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of pneumonia with severe respiratory distress and outbreaks in Wuhan, China. The rapid and global spread of SARS-CoV-2 resulted in the coronavirus 2019 (COVID-19) pandemic. Earlier during the pandemic, there were limited genetic viral variations. As millions of people became infected, multiple single amino acid substitutions emerged. Many of these substitutions have no consequences. However, some of the new variants show a greater infection rate, more severe disease, and reduced sensitivity to current prophylaxes and treatments. Of particular importance in SARS-CoV-2 transmission are mutations that occur in the Spike (S) protein, the protein on the viral outer envelope that binds to the human angiotensin-converting enzyme receptor (hACE2). Here, we conducted a comprehensive analysis of 441,168 individual virus sequences isolated from humans throughout the world. From the individual sequences, we identified 3,540 unique amino acid substitutions in the S protein. Analysis of these different variants in the S protein pinpointed important functional and structural sites in the protein. This information may guide the development of effective vaccines and therapeutics to help arrest the spread of the COVID-19 pandemic.

To curb the COVID-19 pandemic, most efforts have focused on preventing entry of the virus by inhibiting the interaction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with its human receptor, angiotensin-converting enzyme 2 (hACE2) 1 . Interaction of SARS-CoV-2 with hACE2 occurs via the S protein on the viral envelope. Proteases cleave the S protein into S1 and S2 subunits 2, 3, 4 to enable viral binding to hACE2 5 and viral entry by membrane fusion 6 . The S protein is a homotrimer and the S1 subunit of each of the monomers of the S protein contains the receptor-binding domain (RBD; Fig. 1a , b) in either the 'open' (active) or 'closed' (inactive) conformations 7, 8, 9 (Supplemental Fig. 1a ).

Four main types of prophylaxis or therapeutic strategies, focusing on the S protein, have been employed: 1). Preventing proteolysis of the S protein 10 ; 2). Competing with S1 binding to hACE2, using S1 or hACE2 protein fragments or peptides 1, 11, 12 ; 3). Generating monoclonal or polyclonal antibodies against SARS-CoV-2 S protein or RBD, to be used as passive vaccines 13 ; and 4) Active vaccines that generate an immune response, usually to the S1 subunit 14, 15, 16, 17 .

Besides the RBD, the S protein of the coronaviruses, including SARS CoV-2, has several other regions that are predicted to be relatively conserved due to their critical role for S protein functions. These regions include the trimer interface of S 7,9 , furin proteolysis sites 5, 6 , glycosylation sites 18, 19 , neuropilin-binding sites 20, 21, 22 and linoleic acid (LA)-binding site 9, 23 .

These regions may be important for maintaining structural integrity, entry, and transmission of the virus and therefore are likely to serve as potential targets for development of prophylaxes and therapeutics.

Although SARS-CoV-2 undergoes mutations at a lower frequency than other viruses like influenza and HIV 24 , the emergence of several common variants of SARS-CoV-2 in human populations may generate resistance to current prophylaxis and therapeutics. Some of these mutations result in gain of fitness for the virus due to mutations in the S protein 25, 26, 27, 28 . Early in the pandemic, in February 2020, a single missense mutation resulting in a change from aspartate to glycine in position 614 (D614G) emerged in Europe and became the dominant variant of the virus. The D614G variant has spread throughout the world and increased the transmissibility of SARS-CoV-2 by conferring higher viral loads in young hosts without an apparent increase in the severity of the disease 29 . With the emergence of new variants, such as B.1.1.7 (also known as the UK variant) and B.1.351 (also known as the South African variant) that have greater transmissibility and escape antibody detection 25, 26, 27, 28 (Table 1) , it is imperative to map other substitutions in the S protein sequence. Such substitutions may contribute to future variants that lead to increased transmissibility or to variants that evade prophylaxis or therapeutics.

Particularly, amino acid substitutions in the RBD, including those that interact directly with hACE2 25, 26, 27, 28 (Fig. 1c) may have an impact. Here, we aimed to identify regions on the S protein that are relatively invariant to guide prophylaxis and therapeutic development more efficiently.

The SARS-CoV-2 S protein is 1273 amino acids long; it contains a signal peptide (amino acids 1-13), the S1 subunit (14-685 residues) that mediates receptor binding, and the S2 subunit (686-1273 residues) that mediates membrane fusion 30 . To identify areas in the S protein that are the least divergent as the virus evolves in humans, we obtained viral sequences from GISAID (Supplemental Table 1 As there are 3,540 variants, on average, each position in the 1273 amino acid protein sequence has approximately three variants (Fig. 1d) . However, some regions harbor 9 variants in a single amino acid position whereas others have no variants ( Fig. 1d ; Supplemental Table 3 ). Regions in S protein with 2 or fewer variants/position (marked in light blue, Fig. 1d , e) are more prevalent in the structurally critical trimer interface (46% of the amino acids; Fig. 1d , Supplementary Fig. 1b Table 3 ).

Much of the prophylaxis and therapeutic efforts are focused on the RBD (amino acids 331-524). Among the 3,540 variant sequences, we found only 22 invariant amino acids in the RBD (Fig. 1e , marked by dots under the position; Supplemental Table 3 ). Of those amino acid substitutions in the RBD, only 3% are predicted by PROVEAN software 32 to be structurally or functionally damaging (Supplemental Table 2 ). Using PROVEAN, we also examined the 

We next examined other regions in the S protein for which functions have been assigned.

Furin proteolysis at the S1-S2 boundary (681-685) and in S2 (811-815) exposes the RBD to enable hACE2 binding, and the S2 domain to initiate membrane fusion 5 . Recent studies show that these cleavage sites are not necessarily specific for furin-mediated proteolysis and that S may be processed by multiple proteases to open the RBD into the active conformation 2,3,4,33 .

Consistent with these observations, both the furin proteolysis consensus sites and the arginine that are critical for proteolysis are not conserved in the S protein (Fig. 2a) , in agreement with a prior analysis of furin site 1 34 .

The S protein also has 66 glycosylation sites in each trimer, which facilitate protein folding and may lead to host immune system evasion 19 , as 40% of the S protein's surface is shielded by glycans 18 . Surprisingly, with one exception, none of these glycosylation sites were invariable, suggesting that not all the glycosylation sites are essential for the S protein's functions (Fig. 2b,c) . The only asparagine serving as an invariable glycosylation site is N343 in the RBD domain, located more than 25Å away from hACE2-binding site, and therefore unlikely to mediate receptor binding.

Neuropilin-1 (NRP-1) is a transmembrane receptor that regulates angiogenesis 35 and immune response 36 and is expressed in many cell types 36 such as the endothelium 37 , immune cells 38 , and neurons 39 . Interaction between NRP-1 and S protein was proposed to regulate SARS-CoV-2 transmission 20, 21, 22 . Proteolysis of S1 index variant by furin was found to expose a C terminal motif, RXXR (where R is arginine and X is any amino acid), known to be the binding motif in NRP-1 20, 22 . For example, a monoclonal antibody against the RXXR-binding site on NRP-1 SARS-CoV-2 reduced infectivity in culture 22 . Nevertheless, we found that the NRP-1 interaction-site in S1 is not conserved (Fig. 2d) . Although the variants are predicted to have a neutral effect on the S1 protein structure (using PROVEAN analysis, Supplemental Table 2 

A fatty acid-binding pocket has been identified in the inactive conformation of S protein 9 ( Fig. 3a, b) . The amino acids that make this pocket are conserved in other coronaviruses 9 and are unchanged (less than 2 variants) in 75% of the position (Fig. 3a, b) . Furthermore, among the 20 amino acids that line this pocket, 71% of the identified variants are predicted to have a neutral effect using PROVEAN (Supplemental Table 2 ). Analysis of the LA-bonding site identifies a potential pharmacophore that may fit small molecules (Fig. 3c) , perhaps by mimicking ω-3 fatty acids 23 .

We also identified another less variable region between residues 541-612 (Fig. 4) ; 62% of the amino acid positions in this region have 2 or fewer variants and 12 positions are entirely invariable ('Hot Region'; Fig. 1a and Fig. 4a, b) . This less variable region is relatively hydrophobic, yet a substantial number of residues remain exposed in the open and closed conformations (Fig. 4c) . Six residues, V551, T553, C590, V595, V608, Y612, in this relatively invariable region form a part of the largest hydrophobic patch in the protein measuring 370 Å 2 ( Fig. 4d, e) . Five of these residues (excluding T553) along with other residues that make this hydrophobic patch tolerate very few mutations and almost all the mutations that are tolerated change to hydrophobic amino acids (Fig. 4d) . We examined this region using Site Finder in MOE 40 and found that there is a binding site with a positive score for the propensity of ligand binding 41 , which encompasses several residues from this region (i.e. Cys590, Ser591, Phe592, Gly593) (Supplemental Fig. 1e ). This hydrophobic region is also 81% identical between SARS-CoV and SARS-CoV-2, but less than 15% identical when comparing the SARS-CoV-2 sequence with that of MERS-CoV (Fig. 4f ).

While SARS-CoV-2 has a lower mutation rate than other viruses due to proof-reading mechanisms 24 , aspects such as a relatively high R 0 of 1.9 to 2.6 42 , comparatively long asymptomatic incubation and infection periods, and zoonotic origins, leads to high variability in mutations in specific regions compared to the original reference sequence. This has been illustrated with the divergence of 6 major lineages in the past few months (Table 1 ). Our analysis of the frequency of variants throughout the S protein of SARS-CoV-2 identified regions of high and low divergence, which may aid in developing effective prophylactic and therapeutic treatments. In this analysis of mutations in the S protein, we did not consider the frequency of a particular mutation or in how many countries the mutation was found. Such analysis, as was done for D614G 43 , may further aid in determining the potential improved viral fitness acquired by a particular mutation.

Protein glycosylation is essential for viral infection. 44 In SARS-CoV-2 S protein, there are 22 known N-glycosylation sites per monomer (Fig. 2b, c) , but only one, asparagine 343, appears to be conserved. Furthermore, we found 156 positions in S that mutate to an asparagine residue in the existing 3,540 variants that we analyzed, and many of them are exposed on the S protein (Supplemental Fig. 1d ). We propose that some of these new asparagine residues may create new glycosylation sites on the S protein that can contribute to immune evasion. Such an impact on the immune evasion by changes in the positions of glycosylation sites of viral envelope proteins have been described for influenza viruses; e.g., H3N2 has numerous new Nlinked glycans on the viral hemagglutinin that enabled the virus to escape antibody neutralization and evade the host's immune system 45 . The formation of new glycosylation positions may also affect viral susceptibility to existing antibodies and to the immune response of infected individuals. A cryo-electron microscopy study has already suggested that coronaviruses mask important immunogenic sites on their surface by glycosylation 46 . Furthermore, recent work suggests that changes in glycosylation sites may affect its recognition by other potential human proteins and receptors, inducing the toll-like receptors, calcitonin-like receptors, and heat shock protein GRP78, thus leading to a more severe inflammation that characterizes a more severe form of COVID-19 47 .

Additional sites on the S protein have been suggested to be critical for viral infectivity, including the trimer interface, the furin proteolysis sites and the NRP-1 binding site. However, our analysis suggests that not all these sites will be effective targets for prophylaxis and therapeutics. Specifically, the trimer interface is less accessible and therefore unlikely to be druggable. Another issue relates to the furin sites. As the viral S protein activation appears to require furin proteolysis 2,3,4 , protease-specific inhibitors are tested as a means to protect from infection 48 . However, our analysis suggests that this may not be an effective strategy, given the high variability of furin cleavage sites. This suggestion is consistent with previous data showing that other proteinases expressed throughout the body may work synergistically to activate the S protein 2,33 . Therefore, drugs that focus on inhibiting any single protease may not be effective preventative treatment against all SARS-CoV-2 variants. Similarly, the NRP1-binding site that is generated by proteolysis and the exposure of a C-terminal RXXR motif 20,22 may not be a good target for treatment against all SARS-CoV-2 variants, unless such a motif is created by other proteases.

Are there additional sites on the S1 protein that can be explored to identify new treatments of COVID-19 or prevention of infections by SARS-CoV-2? There might be a benefit in focusing on the LA-binding site that help stabilize the S in the inactive closed conformer.

Small molecules that mimic LA and bind into the LA pocket may stabilize the S protein in the closed/inactive conformation, thus reducing infectivity (Fig. 3) . Therefore, exploring the LA pharmacophore ( Fig. 3c) with small molecules that can hold the S-protein in closed conformation, thus preventing the presentation of RBD to hACE2, could be of great interest as this may reduce viral infectivity. Our data also suggest that it may be beneficial to develop passive and active vaccines that target the RBD, instead of the entire glycosylated S protein; the RBD is less variable relative to the whole S1 protein (compare Fig. 1e to 1d) . However, similar to some of the common viral isolates, such as the South African, B.1.351, new amino acid substitutions in the RBD may evade such therapeutics; e.g., loss of immunoreactivity to monoclonal antibodies 25 .

Finally, our study suggests that drugs and antibodies targeting region 541-612, a relatively conserved and exposed region on the protein's surface that we identified (Fig. 4) , warrant further study. Determining how druggable the pocket encompassing this region is (residues Cys590, Ser591, Phe592, Gly593), provided its solvent exposure, and whether modulating S protein by engaging this site will have a biological consequence is a challenge (Supplemental Fig. 1e ). Very recently, Q564 within this region (star in Fig. 4a) has been proposed to act as a 'latch', stabilizing the closed/inactive conformation of the S protein 49 

A FASTA formatted file containing 633,137 spike protein sequences was retrieved on 03/01 from the GISAID database. This file had previously been preprocessed by the database with the individual alignment of genomes to the WIV04 (MN996528.1 31 ) reference sequence, using mafft ) , were chosen by the strict quality thresholds to remove low quality and potentially error prone sequences based on those that were incomplete, contain uncommon deletions, insertions, and have an unusually high number of mutations.

The raw data for variants in the S protein (see Extended Material Table) was read into R studio 

Molecular Operating Environment (MOE) software 40 

The Spike protein sequences from SARS-CoV-2, SARS-CoV, and MERS-CoV were uploaded to Jalview 50 . The Mafft alignment was then performed to align each amino acid sequence.

PDB ID: 6ZB5 6 was opened and prepared using the QuickPrep functionality at the default settings in MOE as mentioned previously. Dummy atoms were created at the LA-binding site formed by chains 6ZB5.A and 6ZB5.C. AutoPH4 tool 51, 52 was used to generate the pharmacophore at the dummy atom site in the Apo generation mode. 

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Scientific Vector Language (SVL)

The authors declare no competing interests.