Unique mutations in SARS-CoV-2 Omicron subvariants' non-spike proteins: Potential impacts on viral pathogenesis and host immune evasion

Abstract

SARS-CoV-2 is the causative agent behind the ongoing COVID-19 pandemic. This virus is a cumulative outcome of mutations, leading to frequent emergence of new variants and their subvariants. Some of them are a matter of high concern, while others are variants of interest for studying the mutational effect. The major five variants of concern (VOCs) are Alpha (B.1.1.7), Beta (B.1.315), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529.*/BA.*). Omicron itself has >100 subvariants at present, among which BA.1 (21K), BA.2 (21L), BA.4 (22A), BA.5 (22B), and BA.2.12.1 (22C) are the dominant ones. Undoubtedly, these variants and sometimes their progeny subvariants have significant differences in their spike region that impart them the unique properties they harbor. But alongside, the mutations in their non-spike regions could also be responsible elements behind their characteristics, such as replication time, virulence, survival, host immune evasion, and such. There exists a probability that these mutations of non-spike proteins may also impart epistatic effects that are yet to be brought to light. The focus of this review encompasses the non-spike mutations of Omicron, especially in its widely circulating subvariants (BA.1, BA.2, BA.4, BA.5, and BA.2.12.1). The mutations such as in NSP3, NSP6, NSP13, M protein, ORF7b, and ORF9b are mentioned few of all, which might have led to the varying properties, including growth advantages, higher transmission rate, lower infectivity, and most importantly better host immune evasion through natural killer cell inactivation, autophagosome-lysosome fusion prevention, host protein synthesis disruption, and so on. This aspect of Omicron subvariants has not yet been explored. Further study of alteration of expression or interaction profile of these non-spike mutations bearing proteins, if present, can add a great deal of knowledge to the current understanding of the viral properties and thus effective prevention strategies.

Keywords: Accessory proteins; Epistasis; Immune-evasion; Non-spike mutations; Non-structural protein; Omicron; Subvariants; Variants of concern.

Fig. 1

Unique amino acid substitution in the non-spike protein for SARS-CoV-2 B.1.1.529 (Omicron variant), the aa substitution shown here resides in the interacting domains, which might have an impact on the functionality for the Omicron variant; a) The ORF1ab polyprotein has 19 substitutions for the Omicron variant, the mutations for NSP1, NSP3, and NSP6 are shown; b) The non-spike structural protein, Membrane, Nucleocapsid, and Envelope protein have 3, 5, and 1 aa substitution; and c) The accessory proteins ORF3a, ORF6, and ORF9b have 1, 1 and 4 aa substitution, respectively. The red in the labelling circle represents BA.1 subvariant, the sky blue, green and violet is for BA.2/BA.2.12.1, BA.4 and BA.5 subvariants, respectively.

Fig. 2

Probable impacts of non-spike mutations in Omicron in the replication cycle (A) and immune evasion (B). Here, NSP, N protein, E protein, M protein, ORF denotes Non-Structural protein, nucleocapsid protein, Envelope protein, and Membrane protein. (A)Schematic representation of the role of NSPs, E protein, M protein, and N protein harboring significant mutations in the replicative pathway. Viral NSP1 inhibits host mRNA from translating, which can be interfered with by S135R mutation in Omicron (K141-,S142-, F143- mutations are present only in BA.4). NSP3 possesses some unique mutations (T24I, G489S, K38R) in the region involved in binding ss RNA and nucleocapsid. Viral polyproteins cleavage is conducted by NSP3 and NSP5(P132H). Also, NSP3(S1265-, L1266I, A1892T) along with NSP4(L264F, T327I) and NSP6 forming a subcomplex mediates Double Membrane Vesicle (DMV) formation, which is required for viral RNA replication. NSP6 (L105-, I189V) inhibits autophagosome formation. NSP13(R392C) is the RNA helicase has role in double stranded RNA unwinding. The proofreading mechanism is facilitated by NSP14 (I42V). Structural proteins such as E protein (T9I), N protein (P13L, E31-, R32-, S33-), M protein (D3G, D3N, Q19E, A63T) have a role in post-translational modification, nucleosome formation, and viral assembly respectively. (B)Schematic representation of possible effects of proteins of interest in distinctive immune evasion pathways. Cytokine storm is mediated by ORF3a (T223I), whereas ORF6(D61L), ORF7b (L11F), and ORF9b (P10S, E27-, N28-, A29-) are potent Interferon (IFN) antagonists. NSP1, NSP3, NSP6, N protein, and M protein also show some role in immune evasion through distinctive pathways. ‘*’ denotes mutations in these proteins may alter their corresponding functions. Abbreviation- RER: Rough Endoplasmic Reticulum, TLR3: Toll-Like Receptor 3, TRAF-3: Tumor Necrosis Factor Receptor-Associated Factor-3, TBK1: TANK Binding Kinase 1, IKKε: IkappaB kinase ε, MDA5: Melanoma Differentiation -Associated protein 5, RIG-1: Retinoic acid-inducible Gene-1, MAVs: Mitochondrial Antiviral signaling proteins, IRF: Interferon Regulatory factor, ISG: Interferon-Stimulated Gene, TOM70: Translocase of Outer Membrane 70, EIR: Early IFN Response, LIR: Late IFN Response.