Zinc and Copper Ions Differentially Regulate Prion-Like Phase Separation Dynamics of Pan-Virus Nucleocapsid Biomolecular Condensates.

Anne Monette,Andrew J Mouland

doi:10.3390/v12101179

Abstract

Liquid-liquid phase separation (LLPS) is a rapidly growing research focus due to numerous demonstrations that many cellular proteins phase-separate to form biomolecular condensates (BMCs) that nucleate membraneless organelles (MLOs). A growing repertoire of mechanisms supporting BMC formation, composition, dynamics, and functions are becoming elucidated. BMCs are now appreciated as required for several steps of gene regulation, while their deregulation promotes pathological aggregates, such as stress granules (SGs) and insoluble irreversible plaques that are hallmarks of neurodegenerative diseases. Treatment of BMC-related diseases will greatly benefit from identification of therapeutics preventing pathological aggregates while sparing BMCs required for cellular functions. Numerous viruses that block SG assembly also utilize or engineer BMCs for their replication. While BMC formation first depends on prion-like disordered protein domains (PrLDs), metal ion-controlled RNA-binding domains (RBDs) also orchestrate their formation. Virus replication and viral genomic RNA (vRNA) packaging dynamics involving nucleocapsid (NC) proteins and their orthologs rely on Zinc (Zn) availability, while virus morphology and infectivity are negatively influenced by excess Copper (Cu). While virus infections modify physiological metal homeostasis towards an increased copper to zinc ratio (Cu/Zn), how and why they do this remains elusive. Following our recent finding that pan-retroviruses employ Zn for NC-mediated LLPS for virus assembly, we present a pan-virus bioinformatics and literature meta-analysis study identifying metal-based mechanisms linking virus-induced BMCs to neurodegenerative disease processes. We discover that conserved degree and placement of PrLDs juxtaposing metal-regulated RBDs are associated with disease-causing prion-like proteins and are common features of viral proteins responsible for virus capsid assembly and structure. Virus infections both modulate gene expression of metalloproteins and interfere with metal homeostasis, representing an additional virus strategy impeding physiological and cellular antiviral responses. Our analyses reveal that metal-coordinated virus NC protein PrLDs initiate LLPS that nucleate pan-virus assembly and contribute to their persistence as cell-free infectious aerosol droplets. Virus aerosol droplets and insoluble neurological disease aggregates should be eliminated by physiological or environmental metals that outcompete PrLD-bound metals. While environmental metals can control virus spreading via aerosol droplets, therapeutic interference with metals or metalloproteins represent additional attractive avenues against pan-virus infection and virus-exacerbated neurological diseases.

Highlights

The recent re-classification of the eukaryotic cellular phenomena of phase separation of protein condensates as the underlying mechanism creating membraneless organelles (MLOs) for cellular compartmentalization is initiated by a liquid demixing program
We hypothesized that pan-virus assembly and budding and maintenance of infectious particles within aerosol droplets is nucleated by phase separation events initiated by viral protein PrLDs gaining structure to bind viral genomic RNA (vRNA) via metal loading of their juxtaposing RNA-recognition motifs (RRMs)
In some cases where identification of functional orthologs of Gag was complicated by diverging viruses having vastly different replication intermediate steps, we broadened our inclusion criteria to accept viral proteins that bind viral RNAs, or viral proteins possessing characterized zinc fingers (ZnFs)

Summary

Introduction

The recent re-classification of the eukaryotic cellular phenomena of phase separation of protein condensates as the underlying mechanism creating membraneless organelles (MLOs) for cellular compartmentalization is initiated by a liquid demixing program. When exacerbated by a chronic stimulus including cellular stress, LLPS gives rise to the assembly and persistence of stress granules (SGs) associated with pathological disease onset [6]. LLPS gives rise to aggregates found in cells derived from patients with neurodegenerative diseases [7]. Self-aggregating mutated proteins that are hallmarks of neurological diseases, including SOD1, G3BP1, TIAR, TIA-1, DDX6, TDP-43, FUS/TLS, Tau, Amyloid β (Aβ), and hnRNP proteins [9], are characterized as RNA-binding proteins, as helicases or chaperones, and as components of SGs or processing bodies (PBs) [10,11]

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