Computational Analysis of Short Linear Motifs in the Spike Protein of SARS-CoV-2 Variants Provides Possible Clues into the Immune Hijack and Evasion Mechanisms of Omicron Variant

Abstract

Short linear motifs (SLiMs) are short linear sequences that can mediate protein-protein interaction. Mimicking eukaryotic SLiMs to compete with extra- or intracellular binding partners, or to sequester host proteins is the crucial strategy of viruses to pervert the host system. Evolved proteins in viruses facilitate minimal protein-protein interactions that significantly affect intracellular signaling networks. Unfortunately, very little information about SARS-CoV-2 SLiMs is known, especially across SARS-CoV-2 variants. Through the ELM database-based sequence analysis of spike proteins from all the major SARS-CoV-2 variants, we identified four overriding SLiMs in the SARS-CoV-2 Omicron variant, namely, LIG_TRFH_1, LIG_REV1ctd_RIR_1, LIG_CaM_NSCaTE_8, and MOD_LATS_1. These SLiMs are highly likely to interfere with various immune functions, interact with host intracellular proteins, regulate cellular pathways, and lubricate viral infection and transmission. These cellular interactions possibly serve as potential therapeutic targets for these variants, and this approach can be further exploited to combat emerging SARS-CoV-2 variants.

Keywords: COVID-19; Omicron; SARS-CoV-2; SLiMs; coronaviruses; motifs; spike protein; variant.

Figure 1

Domain organization and SLiMs in SARS-CoV-2 variants. (A) Protein domain organization of spike protein. The S-protein contains the N-terminal domain (NTD), receptor binding domain (RBD), subdomain 1 (SD1), subdomain 2 (SD2), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), heptad repeat 2 (HR2), and cytoplasmic tail (CT). (B) Matrix of mutations on spike protein across SARS-CoV-2 variants. (C) Heatmap summarizing the identified SLiMs across SARS-CoV-2 variants. (D) Venn diagram showing the overlap of SLiMs of the spike protein from Wuhan, Delta, and Omicron variants. (E) A circular dendrogram showing the similarity of the spike protein from SARS-CoV-2 variants.

Figure 2

Structure and interaction networks of shelterin-complex-associated proteins. (A) Snapshot of the multiple sequence alignment of spike proteins from Wuhan, Delta, and Omicron, along with the specific mutations in the Omicron that led to the emergence of the novel LIG_TRFH_1 motif. (B) Snapshot of the multiple sequence alignment of spike protein from Omicron variant along with human proteins that contain this specific motif. (C) Domain organization of human TRF1, TRF2, and TIN2. (D) Interaction of the human-telomere-associated proteins. TIN2 bridges TRF1 and TRF2 that bind to the ds telomeric DNA. (E) Omicron SLiM LIG_TRFH_1 interacts with cellular TRF2 and TIN2. (F) LIG_TRFH_1 interaction with shelterin proteins protects the viral terminal repeats. Other proteins involved in the protective complex must be identified. TR-terminal repeats.

Figure 3

Representation of DNA damage tolerance pathway. (A,B) Snapshot of the multiple sequence alignment of spike proteins from Wuhan, Delta, and Omicron along with the specific mutations in the Omicron that lead to the emergence of SLiMs. (C) Schematic illustration of the domain structure of human Rev1. (D) Interactions of human Y-family polymerases in TLS. (E) Omicron SLiMs LIG_REV1ctd_RIR_1 and LIG_PCNA_TLS_4 interact with human REV1. (F) LIG_REV1ctd_RIR_1 and LIG_PCNA_TLS_4 motif involvement in the viral TLS.

Figure 4

The interaction of NSCaTE and IQ motifs in a Ca²⁺/CaM-mediated manner. Snapshot of the multiple sequence alignment of spike proteins from (A) SARS-CoV-2 (Wuhan), SARS1, and MERS1 viruses; and (B) Wuhan, Delta, and Omicron along with the specific mutations in the Omicron that lead to the emergence of SLiMs. (C) Ca²⁺ influx facilitates motif interactions. In resting state, motifs remain unbound, NSCaTE in the N-terminus, and the IQ motif in the C-terminus. Upon membrane depolarization and Ca²⁺ influx, a Ca²⁺/CaM-complex-mediated interaction of both motifs occurs in the Cav1 channel. (D) A cartoon representation of the involvement of increased LIG_CaM_NSCaTE_8 SLiM-mediated transmissibility in the SARS-CoV-2 Omicron variant. The spike glycoprotein on SARS-CoV-2 interacts with ACE2 to enter the host cells. Viral entry results in an intracellular hike on the Ca²⁺ level and hence the Ca²⁺/CaM complex in the cells. Ca²⁺/CaM complex-mediated ACE2 catalytic ectodomain shedding by ADAM-17 generates the soluble form of ACE2. SARS-CoV-2 can bind to the soluble ACE2, as it contains the viral binding site, but viral neutralization occurs without an intracellular environment and cannot duplicate. When the Omicron variant enters the cells, Ca²⁺/CaM-mediated binding transpires between the unique Omicron SLiM LIG_CaM_NSCaTE_8 and the LIG_CaM_IQ_9. This process hinders ACE2 ectodomain shedding due to the lack of Ca²⁺/CaM complex availability for the CaM binding site in the ACE2 cytoplasmic receptor. As a result, more active full-length ACE2 is expressed on the surface for viral binding.

Figure 5

Schematic representation of the Hippo signaling pathway and Omicron MOD_LATS_1 intervention. (A) Snapshot of the multiple sequence alignment of spike proteins from Wuhan, Delta, and Omicron along with the specific mutations in the Omicron that lead to the emergence of the SLiMs. (B) Modulation in the Hippo signaling pathway during Omicron infection. When the Hippo signaling pathway is active/on, YAP/TAZ proteins become phosphorylated by LATS1/2 kinases and remain in inactive form. However, during Omicron infection, cellular LATS1/2 kinases phosphorylate viral MOD_LATS_1 leaving active cytoplasmic YAP/TAZ, which can negatively regulate immune response and facilitate Omicron survival.

Figure 6

Schematic representation of Omicron spike protein organization and amino acid mutations. Omicron mutations are shown in a primary structure of SARS-CoV-2 S-protein. Amino acid mutations in SARS-CoV-2 Omicron spike proteins are A67V, Del69-70, T95I, Del142-144, Y145D, Del211, L212I, R214Insertion, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F. Selected SLiMs introduced due to the mutation in Omicron variants are marked at the bottom of the domain map.