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

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.

Liquid–Liquid Phase Separation by Intrinsically Disordered Protein Regions of Viruses: Roles in Viral Life Cycle and Control of Virus–Host Interactions

“…Again, a role for structural disorder and protein multivalence has been hypothesized, since the NC ZnF overlaps with a prion-like domain (PLD), which is conserved in other retroviral Gag proteins capable of undergoing LLPS [199]. As already mentioned above, evidence of PLD-driven LLPS has long been recognized in proteins involved in neurodegeneration, with a possible mechanistic link between coacervate formation and amyloid fibrillation [121].…”

Liquid Biomolecular Condensates and Viral Lifecycles: Review and Perspectives

“…NC contains two highly conserved ZnF domains responsible for many functions essential for the replication of the virus including binding nucleic acids (single-stranded DNA and RNA), as well as condensing and annealing them [209]. It was recently shown that in in vitro assays, NC as well as the uncleaved Gag can undergo LLPS, forming liquid structures that are spherical, divide, fuse, and the process is governed by the ZnF domain of NC [210]. It is accepted that proteins that undergo LLPS typically contain IDR or LCD and can bind DNA and RNA scaffolds through ZnFs or RNA-recognition motifs [85].…”

Formation and Function of Liquid-Like Viral Factories in Negative-Sense Single-Stranded RNA Virus Infections

“…Distinctly, the IB scaffolds of NNS RNA viruses, nucleoproteins and/or phosphoproteins, contain a high content of IDRs [ 109 ]. Recent bioinformatics analyses of viral proteins further revealed that many of the viral IDRs are prion-like disordered domains (PrLD) [ 110 ]. PrLDs are intimately linked to many neurodegenerative diseases because they are prone to misfold and form insoluble solid-like protein aggregates via aberrant phase transition [ 111 ].…”