SARS-CoV-2 N Proteins Hijacks Human 14-3-3 Proteins With Implications Of Future Rise In Neurological And Psychiatric Issues

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A new international study by researchers from the Russian Academy of Sciences-Moscow, the University of York-United Kingdom and the Oregon State University-USA have found that the SARS-CoV-2 nucleocapsid protein (N) is able to hijack the functions of the human 14-3-3 Proteins.

As these proteins are predominantly found in the human host brain tissues and are involved in neural signaling, neuronal development and neuroprotection, a disruption of their functions will ultimately lead to a variety of neurological and neuropsychiatric issues arising in those that have been infected!
 
The study findings were published on a preprint server ad are currently being peer reviewed. 

https://www.biorxiv.org/content/10.1101/2021.12.23.474009v1

The current pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2), exposed a number of weaknesses in our approach to tackle viral outbreaks.

The lack of understanding host-virus interactions at the molecular level slowed the development of novel antiviral therapies and understanding the causes of virus pathogenicity, resulting in lack of preparedness and, consequently, in major health, economic and social impacts for the society.

One of the most abundant virus proteins, the 46-kDa nucleoprotein (N), is common for single- stranded RNA viruses, including the positive-sense coronaviruses. It is responsible for the replication, packaging and storage of viral genomic RNA 1,2.

N is one of the most abundant coronavirus proteins in the infected cells (reaching ~1 % of a total number of cell proteins) 3–5. In SARS-CoV-2, N has been proposed to be the major factor of the enhanced pathogenicity 6.

Sharing 89.1% sequence identity with SARS-CoV N, SARS-CoV-2 N consists of the structured N-terminal RNA-binding domain 7, the structured C-terminal dimerization domain 8, disordered terminal tails and the long unstructured interdomain linker (residues 176-247) (Fig. 1a) 9,10.

The latter linker domain contains a Ser/Arg-rich region (residues ~176-210) 11–13 that becomes heavily phosphorylated upon host infection 4,14–16 and is also a hotspot of viral mutations 17–19.

This interdomain linker contributes to higher-order oligomerization of N molecules 20, to the N-M interactions 21, and to liquid-liquid phase separation with RNA 11,17 via the tentative amyloid-like aggregation of specific peptide stretches 179-184, 217-222 and 243-248 22.

Phosphorylation of several Ser/Thr residues in the SR-rich region controls the flexibility, electrostatics, and functions of SARS-CoV-2 N 17,23,24.

Apart from affecting properties of N itself, phosphorylation can also regulate N interaction with other proteins such as 14-3-3 25,26 – the universal and abundant phosphopeptide-binding protein-protein interaction hub represented by seven specific isoforms in human (β, γ, ε, ζ, η, σ, τ) 27,28.

In line with this, SARS-CoV N undergoes phosphorylation-dependent association with human 14-3-3 in the cytoplasm of the transfected cells, which is proposed to control nucleocytoplasmic shuttling of N and hijack 14-3-3 functions via sequestration 25,26.

In previous work, we have shown that upon phosphorylation of the SR-rich region, SARS-CoV-2 N is specifically recognized by human 14-3-3 proteins, to form a 2:2 complex 26. The use of truncated N variants narrowed down the binding site to phospho-Ser197 26.

This site is conserved between SARS-CoV and SARS-CoV-2 N proteins (Fig. 1a), as well as several  animal coronaviruses, and represents a nearly canonical 14-3-3-binding motif SRNpS197TP 26. Nonetheless, other phosphosites within SARS-CoV-2 N promoting 14-3-3 binding were also proposed 26. Despite biochemical advances on SARS-CoV-2 N interaction with 14-3-3 proteins, the structural basis for such interaction remained elusive.

Production of phosphorylated N is challenged by the dense distribution of phosphorylatable residues and associated heterogeneity of poly-phosphorylated N forms (Fig. 1b) 26,29. Here, we first used genetic code expansion to verify phosphoserine exclusively at position Ser197 in SARS-CoV-2 N is sufficient to enable binding to 14-3-3.

Intriguingly, when poly-phosphorylated by a kinase, we found that the naturally occuring SARS-CoV-2 N variant S197L still displayed a strong phosphorylation-dependent interaction with 14-3-3.

We therefore identified an adjacent phosphosite at position Thr205, that also contributes to 14-3-3 binding and is absent in SARS- CoV N. Using fluorescence anisotropy, we determined micromolar dissociation constants for the interaction of both phospho-motifs with all seven human isoforms of 14-3-3, which revealed the characteristic affinity profiles reflecting the affinity hierarchy of the seven 14-3-3 isoforms 28 and a remarkable selectivity of 14-3-3 towards the SARS-CoV-2 N phosphopeptides.

Crystal structures of the corresponding 14-3-3 complexes helped rationalize the site-selective binding of the SARS-CoV-2 N phosphopeptides to 14-3-3. This also captured for the first time residues 193-210 of the functional SR-rich region, providing a structural framework for the analysis of the existing and emerging SARS-CoV-2 mutations.

Fig. 1. SARS-CoV-2 N site-specifically phosphorylated at Ser197 interacts with human 14-3-3γ. a. Schematic representation of the SARS-CoV-2 N domain structure. NTD and CTD, interdomain linker, Ser/Arg-rich region are labeled, Ser197 and Thr205 phosphorylation sites are marked by red circles, and blue rectangles indicate peptide fragments prone to amyloid aggregation 22. b. Local alignment of the SR-regions of SARS-CoV-2 and SARS-CoV N proteins is shown, with the phosphosites of interest marked by red font and potential 14-3-3-binding sites

by bold font. Note that SARS-CoV N has only the first potential 14-3-3-binding site. c. Phosphorylation of SARS-CoV-2 N variants analyzed by a Phos-tag gel electrophoresis. d. The same samples as in c were analyzed by SDS-PAGE. e,f. The interaction of human 14-3-3γ with singly phosphorylated SARS-CoV-2 N.S197TAG (e) or the unphosphorylated SARS-CoV-2 N counterpart (f) analyzed by SEC when 14-3-3 was added to N in excess at 220 mM NaCl in the samples and running buffer, with SDS-PAGE of all fractions along the elution profiles. M – molecular mass markers (indicated in kDa to the left). Positions of proteins are indicated by arrows on the right. Note that fractions 4-6 (dashed rectangles) contained the 14-3-3:N complex only in the case of S197TAG.

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