title: Time-Resolved Visualisation of Nearly-Native Influenza A Virus Progeny Ribonucleoproteins and Their Individual Components in Live Infected Cells.
abstract: Influenza viruses are a global health concern because of the permanent threat of novel emerging strains potentially capable of causing pandemics. Viral ribonucleoproteins (vRNPs) containing genomic RNA segments, nucleoprotein oligomers, and the viral polymerase, play a central role in the viral replication cycle. Our knowledge about critical events such as vRNP assembly and interactions with other viral and cellular proteins is poor and could be substantially improved by time lapse imaging of the infected cells. However, such studies are limited by the difficulty to achieve live-cell compatible labeling of active vRNPs. Previously we designed the first unimpaired recombinant influenza WSN-PB2-GFP11 virus allowing fluorescent labeling of the PB2 subunit of the viral polymerase (Avilov et al., J.Virol. 2012). Here, we simultaneously labeled the viral PB2 protein using the above-mentioned strategy, and virus-encoded progeny RNPs through spontaneous incorporation of transiently expressed NP-mCherry fusion proteins during RNP assembly in live infected cells. This dual labeling enabled us to visualize progeny vRNPs throughout the infection cycle and to characterize independently the mobility, oligomerization status and interactions of vRNP components in the nuclei of live infected cells.
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France.|University Grenoble Alpes-CNRS-EMBL International Unit (UMI 3265) for Virus Host-Cell Interactions, UMI 3265, Grenoble, France.
authors.forename: Sergiy
authors.initials: S
authors.lastname: Avilov
authors.affiliation: Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, Département de Virologie, Paris, France.|CNRS, UMR 3569, Paris, France.|Université Paris Diderot, Sorbonne Paris Cité, Unité de Génétique Moléculaire des Virus à ARN, Paris, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Magnus
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France.|University Grenoble Alpes-CNRS-EMBL International Unit (UMI 3265) for Virus Host-Cell Interactions, UMI 3265, Grenoble, France.
authors.forename: Stephen
authors.initials: S
authors.lastname: Cusack
authors.affiliation: Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, Département de Virologie, Paris, France.|CNRS, UMR 3569, Paris, France.|Université Paris Diderot, Sorbonne Paris Cité, Unité de Génétique Moléculaire des Virus à ARN, Paris, France.
authors.forename: Nadia
authors.initials: N
authors.lastname: Naffakh
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D009980:Influenza A virus / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D012261:Ribonucleoproteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D012261:Ribonucleoproteins; D049452:Green Fluorescent Proteins