Diversity of Short Linear Interaction Motifs in SARS-CoV-2 Nucleocapsid Protein

Abstract

SUMMARYMolecular mimicry of short linear interaction motifs has emerged as a key mechanism for viral proteins binding host domains and hijacking host cell processes. Here, we examine the role of RNA-virus sequence diversity in the dynamics of the virus-host interface, by analyzing the uniquely vast sequence record of viable SARS-CoV-2 species with focus on the multi-functional nucleocapsid protein. We observe the abundant presentation of motifs encoding several essential host protein interactions, alongside a majority of possibly non-functional and randomly occurring motif sequences absent in subsets of viable virus species. A large number of motifs emergeex nihilothrough transient mutations relative to the ancestral consensus sequence. The observed mutational landscape implies an accessible motif space that spans at least 25% of known eukaryotic motifs. This reveals motif mimicry as a highly dynamic process with the capacity to broadly explore host motifs, allowing the virus to rapidly evolve the virus-host interface.

Modulation of Biophysical Properties of Nucleocapsid Protein in the Mutant Spectrum of SARS-CoV-2

“…The N-protein amino acid sequences exhibit ≈43 million instances of mutations distributed across ≈92% of its residues. We have previously characterized this dataset with regard to the amino acid mutational landscape of N-protein, and found mutation frequencies that are strongly dependent on position and largely time-invariant, except for the defining mutations arising in variants of concern, the latter comprising ≈36% Delta-variant and ≈49% Omicron-variant sequences (Schuck and Zhao 2023). A histogram of the number of different amino acids mutations that are found at each residue is shown in Figure 1B .…”

“…Exploiting the N-protein mutational landscape and sequence data, previous work in our laboratory has focused on local amino acid sequence properties such as mutation effects on transient structural features in the linker IDR (Zhao et al 2023) and the creation of short linear motifs (Schuck and Zhao 2023). However, nonlocal biophysical properties may also be functionally critical and evolutionarily conserved despite amino acid sequence heterogeneity in IDRs (Zarin et al 2017; Zarin et al 2021).…”

“…The vast majority of mutations, however, seem inconsequential in that they usually do not lead to any fixed substitutions. Nonetheless, the mutant spectrum exhaustively describes a landscape of amino acids that may occupy any position in the viral proteins, as in a natural deep mutational scan (Zhao et al 2022; Bloom and Neher 2023; Schuck and Zhao 2023). Biophysical constraints implicit in the shape of such landscapes are key to understand the function and molecular evolution of viral proteins (Starr and Thornton 2016; Wang et al 2021).…”

“…Generally, intrinsic disorder and loose packing is a common characteristic of many RNA virus proteins (Tokuriki et al 2009), which is thought to promote functional promiscuity, permit greater diversity, and enhance evolvability to adopt new functions with few mutations (Tokuriki and Tawfik 2009; Gitlin et al 2014; Charon et al 2018). One possible mechanism is viral mimicry of host-protein short linear motifs (SLiMs) that allow binding to host protein domains and cause subversion of host cellular pathways (Davey et al 2011; Hagai et al 2014; Davey et al 2015; Kruse et al 2021; Shuler and Hagai 2022; Mihalič et al 2023; Schuck and Zhao 2023). It was also shown how nonlocal biophysical properties, such as the charge of intrinsically disordered regions (IDRs), can be relevant evolutionary traits (Zarin et al 2017; Zarin et al 2021).…”

Protein structure–function continuum model: Emerging nexuses between specificity, evolution, and structure

“…Recently, liquid–liquid phase separation of viral proteins has been shown to be involved in the viral life cycle within the host cells (Brocca et al, 2020; Saito et al, 2021). The intrinsic disorder is also exploited by SARS‐CoV‐2 to evade immune systems by mutating preferentially at disordered sites of spike and nuclear proteins (Quaglia et al, 2022), as well as to hijack cellular machinery utilizing a multitude of SLiMs in SARS‐CoV‐2 nucleocapsid protein (Schuck & Zhao, 2023).…”