Liquid-liquid phase separation of an RNA polymerase phosphoprotein cofactor drives the formation of viral factories

SALGUEIRO, M.; CAMPOREALE, G.; ARAN, M; PELLIZA, L; DE PRAT GAY, G

Congreso; Sociedad Argentina de Biofisica; 2019

Liquid-liquid phase separation of an RNA polymerase phosphoprotein cofactor drives the formation of viral factoriesSalgueiro, Ma Camporeale, Ga Arán, Ma Pelliza, La de Oliveira, GAb Conci, Ja Prat Gay, GaMononegavirales order includes viruses of medical relevance, such as ebola, rabies, and respiratory syncytial virus (RSV). Throughout their infective cycle, these viruses form granules where the polymerase complex and viral RNA concentrate. The complex consists of a nucleoprotein (N), a phosphoprotein cofactor (P), the RNA polymerase and a transcription factor. Within the granules viral replication and transcription occurs and, for this reason, they have been called viral factories. Coexpression of N and P is the minimum required system for the formation of viral factories in cells. Like several other biomolecular condensates described in recent years, viral factories form by liquid-liquid phase separation (LLPS). The proteins involved in LLPS have features such as modularity, multivalence, intrinsic disorder, weak interactions and often interact with nucleic acids. RSV P is modular, tetrameric and intrinsically disordered. It has transient secondary structure elements that are more stable at low temperatures and in a tetrameric context. In addition, RSV P establishes local and remote internal contacts. Our hypothesis is that RSV P drives the formation of viral factories. Here we show that RSV P condenses at high concentrations in the presence of a crowding agent, forming spherical, dynamic and reversible droplets. LLPS is favored at low temperatures and occurs at physiological or higher concentrations of NaCl. Stabilization of preformed α-helices, induced by TFE, favors LLPS; this suggests that contacts between metastable helices are necessary for condensation. In addition, we observe a strong correlation between a low temperature range conformational transition and the LLPS; likewise, the presence of N dramatically decreases the critical concentration of P for LLPS. These results provide the pysicochemical grounds for understanding how viruses exploit the LLPS phenomenon for compartmentalizing the components and reactions involved in replication and transcription.