NP-RNA LLPS biochemistry
purified NP is tightly associated with RNA - forms condensates
(Carlson MolCell 2020 PMID: 33248025) - they denature + renature the NP for their experiments in order to get rid of the RNA
Biophysical properties of condensates in vitro and vivo
(Lu, Corbett NatCom 2021 PMID: 33479198) N + RNA condensates formed in vitro show much higher viscosity than in vivo.
In vitro analysis of N11SD suggested that the viscosity of N + RNA condensates is regulated by phosphorylation of the S/R-rich region, with phosphorylation strongly decreasing viscosity.
We found that N11SD behaves similarly to wild-type N in cells, forming condensates in a minority of cells (20% in wildtype N vs. 45% in N11SD; Fig. 5b) that show fast fluorescence recovery in FRAP analysis, indicative of low viscosity (Fig. 5c, d and Supplementary Fig. 5a, b). In contrast, N14SA and NΔSR formed large, dense condensates in nearly 100% of cells (Fig. 5b).
Dephosphorylated N is more prone to condensation in cells. Together, these data strongly support the idea that the N protein is highly phosphorylated in its S/R-rich region when expressed in cells, and that this phosphorylation regulates both its propensity to form condensates and the viscosity of those condensates. The different behavior of phosphorylated vs. unphosphorylated N may reflect an adaptation to its distinct roles, with low-viscosity phosphorylated condensates promoting viral replication and host immune evasion (see below), and high-viscosity unphosphorylated condensates mediating viral RNA packaging into virions55.
Concentrations and kinds of droplets
(Guseva Blackledge 2020 SciAdv PMID: 32270045)
N protein formed small puncta only at the highest protein concentration tested (72 μM) under physiological salt concentration (150 mM NaCl)
N formed spherical droplets at lower salt with half maximal inhibition constant (IC50) of 94 ± 1 µM NaCl
The addition of 50 ng/µL polyC homopolymer RNA substrate triggered phase separation of N even at physiological salt with a half saturation constant (ksat) of 11.0 ± 0.7 µM, ± s.e.).
These condensates were dissolved under higher salt concentrations (IC50 = 163 ± 8 mM NaCl), suggesting that they are liquid-like rather than solid structures (Figure 1C).
unstructured and flexible polyanions form complex coacervates with the positively charged N protein (20) and trigger formation of liquid condensates. The structure of the RNA affects N phase separation, with secondary structures of RNA triggering formation of gel-like droplets (13, 23, 24). Phase separation of N with RNA was also dependent on protein-RNA stoichiometry. Increasing the concentration of the N protein promoted phase separation, whereas high concentrations of RNA dissolved these N condensates (Figure 1E, Figure 1_figure supplement 2A,B), presumably by reducing the local concentration of the N protein on an RNA scaffold (25, 26).
N protein formed asymmetric gel-like condensates with viral RNA fragments in vitro(Figure 1F, Figure 1_figure supplement 1B,C). The shapes of these asymmetric structures were consistent with remodeling of vRNPs into “beads on a string”, as observed by cryoEM (6). These asymmetric structures formed across a wide range of protein and RNA concentrations, did not change shape over time, and were not strongly affected by raising the temperature from 20 °C to 37 °C
changes in protein-protein interaction during LLPS formation
(Guseva Blackledge 2020 SciAdv PMID: 32270045)
phosphorylation of NP in LLPS formation
see above - dynamics of condensates
(Guseva Blackledge 2020 SciAdv PMID: 32270045)
PMID: 33248025 /2020 :
Phosphorylation of the central disordered region promotes the protein's transcriptional function, but the underlying mechanism is not known.
Unmodified N protein forms partially ordered gel-like condensates and discrete 15-nm particles based on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces these interactions, generating a more liquid-like droplet. We propose that distinct oligomeric states support the two functions of the N protein: unmodified protein forms a structured oligomer that is suited for nucleocapsid assembly, and phosphorylated protein forms a liquid-like compartment for viral genome processing.