key: cord-0876477-yfj393rb
authors: Abbas, Qussai; Kusakin, Alexey; Sharrouf, Kinda; Jyakhwo, Susan; Komissarov, Aleksey S
title: Follow-up investigation and detailed mutational characterization of the SARS-CoV-2 Omicron variant lineages (BA.1, BA.2, BA.3 and BA.1.1)
date: 2022-02-26
journal: bioRxiv
DOI: 10.1101/2022.02.25.481941
sha: 5cc9d051a67fd7372931d7fa017d8e64a46ca3dc
doc_id: 876477
cord_uid: yfj393rb

Aided by extensive protein mutations, the SARS-CoV-2 Omicron (B.1.1.529) variant overtook the previously dominant Delta variant and rapidly spread around the world. It was shown to exhibit significant resistance to current vaccines and evasion from neutralizing antibodies. It is therefore critical to investigate the Omicron mutations’ trajectories. In this study, a literature search of published articles and SARS-CoV-2 databases was conducted, We explored the full list of mutations in Omicron BA.1, BA.1.1, BA.2, and BA.3 lineages. We described in detail the prevalence and occurrence of the mutations across variants, and how Omicron differs from them. We used GISAID as our primary data source, which provides open-access to genomics data of the SARS-CoV-2 virus, in addition to epidemiological and geographical data. We examined how these mutations interact with each other, their co-occurrence and clustering. Our study offers for the first time a comprehensive description of all mutations with a focus on non-spike mutations and demonstrated that mutations in regions other than the Spike (S) genes are worth investigating further. Our research established that the Omicron variant has retained some mutations reported in other SARS-CoV-2 variants, yet many of its mutations are extremely rare in other variants and unique to Omicron. Some of these mutations have been linked to the transmissibility and immune escape of the virus, and indicate a significant shift in SARS-CoV-2 evolution. The most likely theories for the evolution of the Omicron variant were also discussed.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, which emerged in December 2019 and it continued to evolve, creating different variants of progressively increased transmissibility between humans [1] . SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus with a genome size of about 30,000 base pairs of genus Betacoronavirus [2] . Genetic lineages of SARS-CoV-2 domain (RBD), possibly increasing human angiotensin-converting enzyme 2 (hACE2) binding [31] . It allows immune escape from monoclonal antibodies and polyclonal sera. ∆H69/∆V70 is also involved in the evasion of neutralising antibodies [30] . In other similar studies conducted by Borges V. et al. [28] and Karim F. et al. [27] it has been observed a concurrent accumulation of mutations de novo in the SARS-CoV-2 genome of immunocompromised patients (non-Hodgkin lymphoma patient under immunosuppressive therapy and advanced HIV patient with antiretroviral treatment failure, respectively) with a persistent SARS-CoV-2 infection over at least 6 months. The emerged mutations are potentially associated with immune evasion and/or enhanced transmission, primarily targeting the SARS-CoV-2 key host-interacting protein and antigen. Recent research has also demonstrated that immunocompromised paediatric and young adult patients are susceptible to prolonged viral infections with prolonged infectious virus shedding and mutation accumulation. The reported results support a potential correlation between host immune response and the emergence of viral variants, which may have the potential to escape antibody neutralisation [26] . These findings provide support to the hypothesis of intra-host evolution as one mechanism for the emergence of SARS-CoV-2 variants with immune evasion properties. Alternatively, the Omicron variant might have evolved in non-human reservoirs, such as animal source, and as of late spread from it to humans (Fig 1B) . Researchers have recently found evidence to support omicron's potential origin in mice. The result of the team of professor Jianguo Xu suggests that omicron variant may evolve in mouse host. They discover 5 mouse-adapted mutation sites (K417, E484, Q493, Q498, and N501) in viral S protein suggesting mouse as intermediate host [32] . During evolution in mice, omicron adapted in host body by acquiring amino acid mutation in spike protein that enhanced binding with mouse ACE2 receptor [33, 34] . A recent study suggests that the Omicron variant has been around for much longer than predicted, given its close relationship to the Alpha variant [35] . Instead, the virus may have began circulating and changing in underserved places with a tiny population, where it had the opportunity to mutate rapidly in comparison to variations outside of that bubble (Fig 1C) . It could then have been released into the larger population, where it could have spread to different groups. But that begs the issue of where Omicron's predecessors were for all of that period, and is there someplace isolated enough for this sort of virus to transmit for that long without resurfacing in other places. A recent study proposed that the Omicron "ins214EPE" insertion could have evolved in a co-infected individual. The study suggested that the nucleotide sequence encoding for ins214EPE could have been acquired by template switching involving the genomes of other viruses that infect the same host cells as SARS-CoV-2 or the human transcriptome of host cells infected with SARS-CoV-2 [7] .

A vast and unprecedented number of mutations has accumulated in the Omicron lineages (BA.1, BA.2, and BA.3). These mutations spread over the genome, including Spike, and non-Spike regions. Some of these mutations are not unique to the Omicron variant; rather, some can be found in other VOCs or VOIs. Out of the 82 mutations, 24 mutations were found to be present in at least one additional variant. Consequently, 58 mutations have not been identified in any VOCs or VOIs, which make them "Omicron specific mutations". In fact, the vast majority of mutations outside the Spike protein have not been identified in the previous VOCs or VOIs. After the investigation on the first occurrence place/time of each mutation (Table 1) , it was revealed that several mutations appeared for the first time in North America (24 mutations). Others appeared for the first time in Asia, Europe, Africa, South America, or Oceania (19, 18, 12, 5 , and 3 mutations, respectively). Interestingly, the bulk of these mutations first appeared in the first four months of 2020 (60 mutations in total). Analysis of the Omicron's deletions sites for non deletion mutations in other variants deletion mutation. In the Spike protein, the P26S substitution mutation has been identified in the Gamma variant, which occurs in the deletion site (25- Evaluation of the free energy perturbation (FEP) has shown that the effect of this mutation on antibody binding may not be significant, and it only affects class 2 mAbs. However, in the presence of other mutations (e.g. K417N), the neutralising activity of the antibodies may be reduced more significantly [45] . R346K is also found in the Mu variant of interest that emerged in early 2021.

Previous mutation variants found in this position include S371P, S371F, S371T and S371A [46] . The S371 residue is located in one of the cryptic binding epitopes that are aligned with conserved hinge positions. In addition, the S371-F541 region shows a lack of glycosylated sites. Identification of such regions is necessary to search for potential epitopes for neutralising antibodies [47] . On the other hand, changes in the amino acid residue at the S371 position result in reduced RBD recognition by antibodies [48] . For example, a single S371L mutation (which occurs in BA.1) at this position results in avoidance of some neutralising antibodies (by changing the conformation). Possibly, the S371F mutation leads to a similar effect [49, 50] .

This mutation decreases stability of the RBD and may reduce the ability of antibodies to recognise the Spike protein epitope [49, 51] .

This mutation may help to escape neutralising antibodies [52] .

This mutation probably first occurred in the Omicron. As it has effects on replacement of serin for phenylalanine, it may slightly change RBD conformation.

The T376 residue is located in the 2-strand, which may be an epitope for neutralising antibodies [53] . This mutation may be involved in evading recognition by neutralising antibodies, similar to other mutations in RBD [49] .

The D504N/Y mutation results in the escape of one of the monoclonal antibody groups that bind to the RBD base [54] . An important feature of this mutation is that it is located close to RBM, specifically the G504 residue. Hence can affect the RBM conformation [55] . In addition, residue D405 is involved in the Spike protein transition to the open form [56] .

The R408K mutation in this position has a similar effect to the D504N mutation in reducing epitope recognition in RBD by monoclonal antibodies [54] . R408 as well as D405 are involved in the RBD-opening transition [56] . Thus, the study of these residues is important to elucidate their role for viral entry into the cell and how mutations affect this ability. This mutation helps the virus to escape monoclonal antibodies [57] . This mutation alone showed a slightly lower or similar affinity for ACE 2 as wild type [58] . While reducing RBD stability it is increasing ACE2 binding affinity [59] . Might help to escape neutralising antibodies [60] . This substitution reduces protein's susceptibility to some monoclonal antibodies in neutralisation assay [64] . G496 is one of the key residues that interact with ACE2. For substitutions G496W and G496Y showed the largest binding energy change among all RBD mutations [65] . G496S substitution has the same effect of enhancing binding capacity [66] . Chen et al. showed that Y505 residue is a potential hotspot and has a most likely substitution Y505H [61] . Nevertheless this mutation may lead to reducing ACE binding affinity [59] .

Variants of mutations in this position for the first half year of the pandemics were T19P, T19I, T19S [67] . But the Delta variant already contained a T19R substitution, hence it is currently the most common [68] . The T19 site is located in the NTD in a region with a very high mutation density. As the NTD is located close to the RBD, it can therefore be an epitope for antibodies [69, 70] . Thus, it can be assumed that this high variability of this site is a mechanism of antibody recognition avoidance. This mutation might allosterically change S1 conformation and be supportive for mutations in RBD [71, 72] . These deletions are associated with increased virus replication and also involved in the evasion of neutralizing antibodies [30, 73] . One of the most prevalent mutations in the Delta variant [74] . Protein modeling has shown that by altering the NTD 3D structure may enhance G142 mutation and hence increase viral load [75] . Similar deletion (del141-144) was found in isolated culture on 70th day obtained from immunocompromised patient with cancer [76] . Because of the location of this deletion in the recurrent deletion region 2 (RDR2), it can affect the ability to escape neutralising antibodies [77] .

Spike protein with this mutation has shown ability to escape some neutralising antibodies [78] . This mutation locates in the recurrent deletion region 3 (RDR3) and may affect ability to escape some neutralising antibodies [77] . Cases of this mutation were already registered [52] , but it was first fixed in the Omicron. Both these mutations are located in the region of S1/S2 junction. Changing from neutral charged amino acids to the positive charged residues results in enhancing of coupling furin cleavage site with proteolytic enzyme hence that leads to increasing cleavability of spike S1/S2 site [83] . But at the same time, for a single N679K substitution, it is shown that it alone is not enough to increase transmissibility [84] . Appeared a few times independently [30, 85] , including a case of intra-host evolution in a immunocompromised (HIV) patient [27] . One of the most frequent mutations in M protein [90] . Locates in the exposed NTD region, mutation in which may affect the interactions with host cells [91] . There is no data on effects of these mutations yet. R203K and G204R mutations in nucleocapsid have been shown to be linked to increased subgenomic RNA expression [94] and increased viral loads [95] . In addition, these mutations are found in patients with inferior outcomes [96] .

N protein is highly phosphorylated and enriched with basic residues. Basic residues properties help to form a sustainable complex with viral RNA, while the role of phosphorylation is not defined. However, for phosphorylation null mutants viral particles have been shown to be assembled with comparable or slightly lower efficiency than the wild type [97] . Therefore, the loss of phosphorylation at some sites might negatively affect the process of packaging RNA into the nucleocapsid envelope. Residue S413 is one of the phosphorylation sites. From one side mutation S413R changes polar uncharged Serine for basic charged Arginine, on another side it removes phosphorylation from Serine. Hence it is unclear whether the S413R mutation has a positive effect on viral particle assembly (change to the major residue) or a negative effect (it eliminates phosphorylation).

Hydrogen-bond interaction analysis showed that S135 residue in NSP1 forms hydrogen bonds with C16 and C20 nucleotides of SARS-CoV-2 5'-UTR stem loop [98] . Substitution of Serine for Arginine may increase RNA compatibility and as result enhance recognition. This mutation was detected in some samples early in the pandemic [102, 103] , but has not become widespread since then.

L438F mutation was detected several times before, but has not become widespread [104] . One of the most frequently co-occurring with S:T478K non-spike mutation [62] . This deletion results in the replacement of amino acids 'SLSG' by 'S' (Serine). This Serine is directly followed by Phenylalanine, for which it has been shown to increase the affinity of NSP6 to the endoplasmic reticulum membrane. It is suggested that this binding helps to avoid the delivery of viral particles to the lysosomes. Hence, this deletion may be part of the mechanism of innate immunity evasion [106, 107] .

Is more likely to occur in P.1 (18.2%) [108] , but globally the deletion (NSP6:S106/G107/F108del) is more common in this position. According to GISAID (as on 1 September 2021), this deletion is found in 97.21% of the sequences [109] . Sun et al. suggested that this mutation may affect viral shedding and hence transmissibility, thereby altering viral load [110] . Although this assumption requires experimental verification. At least in Turkey this mutation has been found in a large number of samples [111] and presumably in combination with D614G mutation in the spike protein may have a correlation with severity of the disease [112] . [113] . In addition, this mutation results in the substitution of a positively charged amino acid for a polar uncharged residue. Thus, this mutation may lead to a slight change in protein folding. The [114] study also suggests the ability of this mutation to alter the secondary structure of the protein.

In humans, the R392C mutation is less frequent (<0.5% of human isolates) and is more common in the mink population (10.5% of mink isolates) [115] . The I42V mutation is not expected to have a significant effect on the protein structure as it consists of the substitution of one hydrophobic amino acid (Isoleucine) for another hydrophobic amino acid (Valine). The D61L mutation results in the substitution of a negatively charged residue (Aspartic acid) with a hydrophobic residue (Leucine). This substitution can lead to the formation of a stronger ORF6:IRF3 complex. This in turn leads to an increase in the antagonistic properties of ORF6 against IFN [88] .

2 Spike mutations aren't the only ones that matter Mutations in NSP1 are important NSP1 (or leader protein) interacts with 40S subunit of the ribosome, inhibits host gene expression, and also evades the immune system. Furthermore, NSP1 degrades host mRNA and facilitates viral gene expression [117, 118] . Leader protein contains a globular region that recognize the stem loop of the 5'-UTR region of SARS-CoV-2 RNAs hence viral genes continue to translate [98] . This protein has been proposed as a target for vaccine development and also for drug design. The majority of mutations occuring in NSP1 exhibited a destabilizing effect and increased flexibility [119] .

NSP3 has been proposed to work with nsp4 and nsp6 to induce double-membrane vesicles (DMVs), which serve as an important component of the replication/transcription complex (RTCs) [120] . Moreover NSP3 interacts with the N-terminal domain of the nucleocapsid phosphoprotein (N), which leads to binding of the nucleocapsid and RTCs [121] . NSP3 also antagonists type I interferon mediated immune response, and blocks NF-kappasignal transduction [122, 123] . Mutations in NSP3 had been linked with positive selection of viruses leading to evolution in beta-coronaviruses [124] .

NSP4 participates in assembly of cytoplasmic DMVs and helps in viral replication [120, 125] .

NSP5 or main protease (Mpro), also known as 3C-like protease (3CLpro) is essential for viral life cycle as it cleaves polyproteins pp1ab and pp1a into distinct functional proteins [126] . Therefore, this protein is a putative target for anticoronavirus therapy. NSP5 may be involved in evading the innate immune response by inhibiting mitochondrial antiviral signalling (MAVS) protein and IFN induction [127] . Mutations in this protein can affect its proteolytic activity.

Along with NSP3 and NSP4 is involved in the assembly of DMVs, which contain replication/transcription complexes. Furthermore, NSP6 has also been shown to have affinity to endoplasmic reticulum, which is provided by phenylalanine residues in the sequence of this protein [106] . Deletion mutation in NSP6 from 105-107 could aid in innate immune evasion, possibly by compromising cells' ability to degrade viral components [106] .

Mutations in NSP12 (RNA-dependent RNA polymerase) are important

Variants of RNA-dependent RNA polymerase (RdRp) emerged early during the COVID-19 outbreak in China, North America, Europe, and Asian countries and hence RdRp was considered as a mutation hotspot [128, 129] . The RdRp:P323L mutation was found to be associated with increasing point mutations in viral isolates in Europe during the early phase of COVID-19 outbreak [130] . Thus, it is possible that mutations in RdRp might alter the interaction of RdRp with the cofactors which could yield less effective proofreading activity leading to the emergence of multiple SARS-CoV-2 variants [128] .

In silico analysis predicted the docking site of antiviral drugs within a hydrophobic cleft located near the RdRp:P323L mutation site. This mutation was predicted to diminish the affinity of RdRp for existing antiviral drugs [128] .

NSP13 has several essential functions, including: (1) helicase activity in the 5'->3' direction, (2) the ability to unwind RNA/DNA duplex, (3) 5'm-RNA capping activity. Mutations in the active sites of this protein can affect the metabolism of the virus [126, 131] .

NSP14 acts as a 3'-5' exoribonuclease (N-terminal domain) for RNA replication proofreading. In addition, this protein has a second function acting as an N7-methyltransferase (C-terminal domain). Consequently, mutations in this protein may affect the proofreading of newly synthesised viral mRNAs or their stability [132] . Potentially can inhibit interferon signalling [133] .

NSP15 is an endoribonuclease that cleaves the 3'-end of uridylates [134] . This functionality allows coronaviruses to avoid an innate immune response by cleaving the 5'-polyuridines of the viral RNA, and hence preventing the activation of dsRNA sensors [126, 135] . NSP15 is an essential protein for the viral life cycle, as it has been shown that mutations in this protein can lead to a rapid antiviral response in macrophages, thereby suppressing infection in a short period of time [135] .

Forms the ion channel. Initiates the lysis process of the host cells to allow new viral particles shedding. Also involved in the autophagy inhibition and disruption of lysosomes. Therefore, it is an essential protein for the viral cycle [116, 131] . Because ORF3a is located on the surface of the membrane, it is able to induce a cellular and humoral immune response in infected individuals [136] .

Most conserved protein and most abundant structural protein. Have ability to interact with N, S proteins and viral RNA [90] . Mutations occurring in the M protein could influence the host cell interaction [137] .

The mutations found in the E protein may change the structural conformation of this protein and subsequently alter the associated functions such as viral assembly, replication, propagation, and pathogenesis as also previously observed in SARS-CoV [138, 139] .

It has been shown that ORF6 can act as a suppressor of interferon signalling by inhibiting primary interferon production. ORF6 can therefore interfere in the induction of innate immune response. Molecular docking and dynamics simulation analysis showed that the C-terminal region of the ORF6 protein is able to interact with the transcription factor IRF3 via hydrophobic bonds. Thus, ORF6 acts as an IFN antagonist [88] .

Nucleocapsid phosphoprotein interacts with M protein during viral assembling. Has N-terminal and C-terminal domains that can bind RNA. All SARS-CoV-2 variants of interest or concern defined by the WHO contain at least one mutation with >50% penetrance within seven amino acids On the RBD, among the 12 common mutations, 5 showed a higher binding affinity with hACE2, which was also affirmed by previous research conducted by Rehman et al. [143] . We created a 3D model of the spike RBD region using PyMOL software [144] . We labeled only unique mutations for each lineage that are not shared between lineages (Fig 5) . 

Virological characteristics of newly emerging SARS-CoV-2 variants, such as pathogenicity, transmissibility, resistance to antiviral drugs, and vaccine-induced immunity, are a critical global health concern. Omicron was first reported from South Africa at the end of November 2021. Then the Omicron lineages have rapidly spread worldwide and outcompeted other variants such as Delta. In this study, the mutational spectrum of Omicron lineages (BA.1, BA.1.1, BA.2, and BA.3) was explored in detail. We demonstrated where and when each of these mutations has been previously detected, which mutations were observed in the earlier variants, and how Omicron differs from them. Our study established that the Omicron variant has maintained several mutations that are found in other variants of concern and are thought to make the virus more infectious. We have also found that many of Omicron's mutations, are extremely rare in other SARS-CoV-2 variants, and represent a considerable jump in SARS-CoV-2 evolution. Accordingly, the mutation rate of the Omicron variant is exceeding the other variants. As well, it has enhanced transmissibility and immune evasion. This progression demonstrates the attempt of the Omicron variant to drive the SARS-CoV-2 for heightened viral fitness. Based on the Omicron mutation profile in the non-spike regions, we have clarified that it might have collectively enhanced infectivity relative to previous SARS-CoV-2 variants. These mutations have shown to be directly/indirectly associated with the transmission advantages and immune escape. Accordingly, these mutations are worth more studies and investigation. Certainly, more-fit variations can be anticipated to develop over time, and then the occurrence of which has to be monitored thoroughly, as these pose a possible public health threat. The good news is that nothing is infinite, and, eventually, new variants will provide no further advantage in infectivity. 

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Coronavirus biology and replication: implications for SARS-CoV-2

Structural insights into SARS-CoV-2 proteins

SARS-CoV-2 Nsp5 Demonstrates Two Distinct Mechanisms Targeting RIG-I and MAVS to Evade the Innate Immune Response. mBio

Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant

Decoding SARS-CoV-2 Transmission and Evolution and Ramifications for COVID-19 Diagnosis, Vaccine, and Medicine

SARS-CoV-2 mutations: the biological trackway towards viral fitness

Structure and Function of Major SARS-CoV-2 and SARS-CoV Proteins

Structural basis and functional analysis of the SARS coronavirus nsp14-nsp10 complex

SARS-CoV-2 nsp13, nsp14, nsp15 and orf6 function as potent interferon antagonists. Emerging Microbes and Infections

Crystal structure and mechanistic determinants of SARS coronavirus nonstructural protein 15 define an endoribonuclease family

Coronavirus nonstructural protein 15 mediates evasion of dsRNA sensors and limits apoptosis in macrophages

Humoral and cellular immune responses induced by 3a DNA vaccines against severe acute respiratory syndrome (SARS) or SARS-like coronavirus in mice. Clinical and Vaccine Immunology

Mutational analysis of structural proteins of SARS-CoV-2

The E Protein Is a Multifunctional Membrane Protein of SARS-CoV

Coronavirus virulence genes with main focus on SARS-CoV envelope gene

Rapid assessment of SARS-CoV-2-evolved variants using virus-like particles

SWISS-MODEL: homology modelling of protein structures and complexes

Insights from a computational analysis of the SARS-CoV-2 Omicron variant: Hostpathogen interaction, pathogenicity and possible therapeutics

A Computational Dissection of Spike protein of SARS-CoV-2 Omicron Variant

PyMOL -www.pymol.org

We gratefully acknowledge the authors, originating and submitting laboratories where the clinical specimens and/or virus isolates were obtained and published through GISAID on which this research was based. We also thank the BioRender's authors for their library icons which helps with figure design.

Aleksey Komissarov was financially supported by the ITMO Fellowship and Professorship Program.