Richard, Charles-Adrien, Rincheval, Vincent, Lassoued, Safa, fix, Jenna, Cardone, Christophe, Esneau, Camille, Nekhai, Sergei, Galloux, Marie, Rameix-Welti, Marie-Anne, Sizun, Christina, Eléouët, Jean Francois, Sizun, Christina, Eléouët, Jean Francois, Unité de recherche Virologie et Immunologie Moléculaires (VIM (UR 0892)), Institut National de la Recherche Agronomique (INRA), Université Paris-Saclay, Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Center for Sickle Cell Disease and Department of Medicine, Howard University, Laboratoire de Microbiologie, Centre Hospitalier Intercommunal Castres-Mazamet, ANR Blanc 2011 « Bronchioliteaser » [ANR-11-BSV8-0024], ANR Blanc 2013 « Respisyncycell » [ANR-13-ISV3-0007], NIH Research Grant U19AI109664, Region Ile-de-France DIM Malinf, CNRS IR-RMN-THC FR3050, Matthias Johannes Schnell, Unité de recherche Virologie et Immunologie Moléculaires (VIM), Infection et inflammation chronique (2I), Centre National de la Recherche Scientifique (CNRS), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)
Respiratory syncytial virus (RSV) RNA synthesis occurs in cytoplasmic inclusion bodies (IBs) in which all the components of the viral RNA polymerase are concentrated. In this work, we show that RSV P protein recruits the essential RSV transcription factor M2-1 to IBs independently of the phosphorylation state of M2-1. We also show that M2-1 dephosphorylation is achieved by a complex formed between P and the cellular phosphatase PP1. We identified the PP1 binding site of P, which is an RVxF-like motif located nearby and upstream of the M2-1 binding region. NMR confirmed both P-M2-1 and P-PP1 interaction regions in P. When the P–PP1 interaction was disrupted, M2-1 remained phosphorylated and viral transcription was impaired, showing that M2-1 dephosphorylation is required, in a cyclic manner, for efficient viral transcription. IBs contain substructures called inclusion bodies associated granules (IBAGs), where M2-1 and neo-synthesized viral mRNAs concentrate. Disruption of the P–PP1 interaction was correlated with M2-1 exclusion from IBAGs, indicating that only dephosphorylated M2-1 is competent for viral mRNA binding and hence for a previously proposed post-transcriptional function., Author summary Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine and no potent antivirals are available against RSV, it is essential to better understand the mechanisms of viral replication to develop new antiviral strategies. Here we have investigated the mechanisms by which two essential components of the viral RNA polymerase machinery, the phosphoprotein P and the M2-1 transcription factor, interact and function. We identified the amino acid residues of P critical for this interaction and showed that they are required for P recruiting M2-1 to cytoplasmic inclusions, where viral polymerase complex proteins concentrate and viral RNA synthesis occurs. We also showed that M2-1 dephosphorylation, required for viral transcription, is achieved by a complex formed between P and the cellular phosphatase PP1. The region of P binding to PP1 is located nearby and upstream of the M2-1 binding domain. This is the first report showing that the phosphoprotein of a negative strand RNA virus can hijack a cellular phosphatase to modulate the phosphorylation state of its partners. These two P regions interacting with M2-1 and PP1 are also potential targets for future antiviral therapy.