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

ARÁN MARTÍN; CONCI, JULIETA; CAPOREALE G; DE OLIVEIRA, GA; SALGUEIRO M; PELLIZZA LEONARDO; PRAT-GAY, GONZALO DE

San Luis

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

Mononegavirales order includes viruses of medical relevance, such as ebola, rabies, andrespiratory syncytial virus (RSV). Throughout their infective cycle, these viruses formgranules where the polymerase complex and viral RNA concentrate. The complexconsists of a nucleoprotein (N), a phosphoprotein cofactor (P), the RNA polymerase and atranscription factor. Within the granules viral replication and transcription occurs and, forthis reason, they have been called viral factories. Coexpression of N and P is theminimum required system for the formation of viral factories in cells. Like several otherbiomolecular condensates described in recent years, viral factories form by liquid-liquidphase 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 secondarystructure 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 thatRSV P drives the formation of viral factories. Here we show that RSV P condenses at highconcentrations in the presence of a crowding agent, forming spherical, dynamic andreversible droplets. LLPS is favored at low temperatures and occurs at physiological orhigher concentrations of NaCl. Stabilization of preformed α-helices, induced by TFE,favors LLPS; this suggests that contacts between metastable helices are necessary forcondensation. In addition, we observe a strong correlation between a low temperaturerange conformational transition and the LLPS; likewise, the presence of N dramaticallydecreases the critical concentration of P for LLPS. These results provide thepysicochemical grounds for understanding how viruses exploit the LLPS phenomenon forcompartmentalizing the components and reactions involved in replication andtranscription.