RNA promotes liquid-liquid phase separation (LLPS) to build membrane-less compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is unknown. Here we report that secondary structure allows mRNAs to self-associate and determines if an mRNA is recruited to or excluded from liquid compartments. The polyQ-protein Whi3 induces conformational changes in RNA structure and generates distinct molecular fluctuations depending on the RNA sequence. These data support a model in which structure-based, RNA-RNA interactions promote assembly of distinct droplets and protein-driven, conformational dynamics of the RNA maintain this identity. Thus, the shape of RNA can promote the formation and coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.

We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with viral RNA. N-protein condenses with specific RNA genomic elements under physiological buffer conditions and condensation is enhanced at human body temperatures (33 C and 37 C) and reduced at room temperature (22 C). RNA sequence and structure in specific genomic regions regulate N-protein condensation while other genomic regions promote condensate dissolution, potentially preventing aggregation of the large genome. At low concentrations, N-protein preferentially crosslinks to specific regions characterized by single-stranded RNA flanked by structured elements and these features specify the location, number, and strength of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is RNA sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules, and therefore presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2.

A mechanistic understanding of the SARS-CoV-2 viral replication cycle is essential to develop new therapies for the COVID-19 global health crisis. In this study, we show that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with the viral genome, and propose a model of viral packaging through LLPS. N-protein condenses with specific RNA sequences in the first 1000 nts (5'-End) under physiological conditions and is enhanced at human upper airway temperatures. N-protein condensates exclude non-packaged RNA sequences. We comprehensively map sites bound by N-protein in the 5'-End and find preferences for single-stranded RNA flanked by stable structured elements. Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules thus presenting screenable processes for identifying antiviral compounds effective against SARS-CoV-2. IntroductionThe outbreak of COVID-19, caused by the severe acute respiratory syndrome-related coronavirus SARS-CoV-2, is a global public health crisis. Coronaviruses, including SARS-CoV-2, are RNA viruses with ~30 kb genomes that are replicated and packaged in host cells. Packaging is thought to be highly specific for the complete viral genome (gRNA), and excludes host RNA and abundant virus-produced subgenomic RNAs (1). Viral replication and gRNA packaging depends on the nucleocapsid protein (N-protein) (2, 3). The N-protein has two RNA-binding domains, forms multimers (4) and is predicted to contain intrinsically disordered regions ( Figure 1A). N-protein thus has hallmarks of proteins that undergo liquid-liquid phase separation (LLPS), a process which may provide selectivity and efficiency to viral replication and packaging. N-protein phase separates with viral RNA in a length, sequence and concentration dependent mannerWe reconstituted purified N-protein under physiological buffer conditions with viral RNA segments and observed that N-protein produced in mammalian cells (post-translationally modified) or bacteria (unmodified) phase separated with viral RNA segments. However, unmodified protein yielded larger and more abundant droplets ( Figure S1A). Since N-protein in SARS-CoV1 virions is hypophoshorylated (5) and packaging (initiated by binding of N-protein to gRNA) first occurs in the cytoplasm of coronaviruses (6,7), where N-protein is thought to be in its unphosphorylated state (8), we used unmodified protein for subsequent experiments.Pure N-protein demixed into droplets on its own and phase separation was enhanced by fulllength genomic SARS-CoV-2 RNA ( Figure 1B). To determine if certain segments of SARS-CoV-2 genome had preferential ability to drive phase separation, we identified regions of the gRNA under synonymous codon constraints. We hypothesized that LLPS occurs specifically with gRNA carrying a viral packaging signal(s), whose ex...

Bartonella bacilliformis, which causes the human diseases Oroya fever and verruga peruana, binds to human erythrocytes in vitro and produces substantial and long-lasting deformations in erythrocyte membranes, including cone-shaped depressions, trenches, and deep invaginations. The deforming force is probably provided by the polar flagella of these highly motile bacteria. Deep invaginations containing bacteria are commonly seen, and membrane fusion at the necks of the invaginations leads to the formation of intracellular vacuoles containing bacteria. Fluorescent compounds present externally render the vacuoles fluorescent and, occasionally, lightly fluorescent cells are seen, suggesting that the vacuoles sometimes rupture to admit the bacteria to the cytoplasm. Vacuoles present in fluorescent erythrocytes prepared by preloading the erythrocytes with fluorescent compounds are seen as dark areas from which the fluorescent marker is excluded. Entry of the bacteria appears to be the result of a process of forced endocytosis.

Nucleocapsid protein (N-protein) is required for multiple steps in betacoronaviruses replication. SARS-CoV-2-N-protein condenses with specific viral RNAs at particular temperatures making it a powerful model for deciphering RNA sequence specificity in condensates. We identify two separate and distinct double-stranded, RNA motifs (dsRNA stickers) that promote N-protein condensation. These dsRNA stickers are separately recognized by N-protein's two RNA binding domains (RBDs). RBD1 prefers structured RNA with sequences like the transcription-regulatory sequence (TRS). RBD2 prefers long stretches of dsRNA, independent of sequence. Thus, the two N-protein RBDs interact with distinct dsRNA stickers, and these interactions impart specific droplet physical properties that could support varied viral functions. Specifically, we find that addition of dsRNA lowers the condensation temperature dependent on RBD2 interactions and tunes translational repression. In contrast RBD1 sites are sequences critical for sub-genomic (sg) RNA generation and promote gRNA compression. The density of RBD1 binding motifs in proximity to TRS-L/B sequences is associated with levels of sub-genomic RNA generation. The switch to packaging is likely mediated by RBD1 interactions which generate particles that recapitulate the packaging unit of the virion. Thus, SARS-CoV-2 can achieve biochemical complexity, performing multiple functions in the same cytoplasm, with minimal protein components based on utilizing multiple distinct RNA motifs that control N-protein interactions.