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.

Recent advances in cell biology enable precise molecular perturbations. The spatiotemporal organization of cells and organisms, however, also depends on physical processes such as diffusion or cytoplasmic flows, and strategies to perturb physical transport inside cells are not yet available. Here, we demonstrate focused-light-induced cytoplasmic streaming (FLUCS). FLUCS is local, directional, dynamic, probe-free, physiological, and is even applicable through rigid egg shells or cell walls. We explain FLUCS via time-dependent modelling of thermoviscous flows. Using FLUCS, we demonstrate that cytoplasmic flows drive partitioning-defective protein (PAR) polarization in Caenorhabditis elegans zygotes, and that cortical flows are sufficient to transport PAR domains and invert PAR polarity. In addition, we find that asymmetric cell division is a binary decision based on gradually varying PAR polarization states. Furthermore, the use of FLUCS for active microrheology revealed a metabolically induced fluid-to-solid transition of the yeast cytoplasm. Our findings establish how a wide range of transport-dependent models of cellular organization become testable by FLUCS.

Highlights d Ded1p phase-separates in response to heat and pH to form gel condensates d Condensation inactivates Ded1p and represses housekeeping mRNAs d Ded1p condensation promotes stress protein production and limits cell growth d Ded1p condensation is adapted to the maximum growth temperature of a species

A wide range of systems containing proteins have been shown to undergo liquid-liquid phase separation (LLPS) forming membraneless compartments, such as processing bodies1, germ granules2, stress granules3 and Cajal bodies4. The condensates resulting from this phase transition control essential cell functions, including mRNA regulation, cytoplasm structuring, cell signalling and embryogenesis1-4. RNA-binding Fused in Sarcoma (FUS) protein is one of the most studied systems in this context, due to its important role in neurodegenerative diseases5-7. It has recently been discovered that FUS condensates can undergo an irreversible phase transition which results in fibrous aggregate formation6. Gelation of protein condensates is generally associated with pathology. One case where liquid-to-solid transition (LST) of liquid-liquid phase separated proteins is functional, however, is that of silk spinning8,9, which is largely driven by shear, but it is not known what factors control the pathological gelation of functional condensates. Here we show that four proteins and one peptide system not related to silk, and with no function associated with fibre formation, have a strong propensity to undergo LST when exposed to even low levels of mechanical shear comparable to those found inside a living cell, once present in their liquidliquid phase separated forms. Using microfluidics to control the application of mechanical shear, we generated fibres from single protein condensates and characterized their structures and .

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...

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.

Betacoronavirus SARS-CoV-2 infections caused the global Covid-19 pandemic. The nucleocapsid protein (N-protein) is required for multiple steps in the betacoronavirus replication cycle. SARS-CoV-2-N-protein is known to undergo liquid-liquid phase separation (LLPS) with specific RNAs at particular temperatures to form condensates. We show that N-protein recognizes at least two separate and distinct RNA motifs, both of which require double-stranded RNA (dsRNA) for LLPS. These motifs are separately recognized by N-protein's two RNA binding domains (RBDs). Addition of dsRNA accelerates and modifies N-protein LLPS in vitro and in cells and controls the temperature condensates form. The abundance of dsRNA tunes N-protein-mediated translational repression and may confer a switch from translation to genome packaging. Thus, N-protein's two RBDs interact with separate dsRNA motifs, and these interactions impart distinct droplet properties that can support multiple viral functions. These experiments demonstrate a paradigm of how RNA structure can control the properties of biomolecular condensates.

This protocol is associated to the publication "Non-invasive perturbations of intracellular flow reveal physical principles of cell organization" by Mittasch et al., published in Nature Cell Biology. In the following, we describe the procedure for maintenance and preparation of C. elegans zygotes for imaging and application of FLUCS. Preparation of S. cerevisiae cells for microscopy and intracellular flow perturbations. S. cerevisiae cells were grown into logarithmic stage. Subsequently the S. cerevisiae cells were either grown for 2 hours or alternatively energy depleted through addition of 2-Desoxy-D-Glucose (inhibition of glycolysis) and antimycin A (inhibition of mitochondrial ATP production) for 2 h prior to flow induction. This treatment causes a more than 95% reduction in cellular ATP 2. Potentially this protocol is suitable to energy deplete all S. cerevisiae strains and closely related yeast species. A specific marker such as µNS-GFP1is needed to observe subsequent flow perturbations by microscopy.