Time-Resolved Visualisation of Nearly-Native Influenza A Virus Progeny Ribonucleoproteins and Their Individual Components in Live Infected Cells

PLoS One. 2016 Mar 15;11(3):e0149986. doi: 10.1371/journal.pone.0149986. eCollection 2016.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cell Membrane / metabolism
  • Cell Nucleus / metabolism
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Influenza A virus / metabolism*
  • Microscopy, Fluorescence
  • Protein Transport
  • Ribonucleoproteins / metabolism*
  • Spectrometry, Fluorescence

Substances

  • Ribonucleoproteins
  • Green Fluorescent Proteins

Grants and funding

S.A. was supported by a Marie Curie Intra-European Fellowship within the 7th European Community Framework Programme (Grant Agreement PIEF-GA-2009-236959), and by an EIPOD fellowship (EMBL). This work was supported in part by the EU FP7 funded FLUPHARM project (FP7-INFLUENZA-2010 grant 259751, http://flupharm.eu). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.