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

Sergiy Avilov,Stephen Cusack,Julie Magnus,Nadia Naffakh,Toru Takimoto

doi:10.1371/journal.pone.0149986

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.

Highlights

Influenza viruses are a global health concern because of the permanent threat of the emergence of novel strains potentially capable of causing pandemics [1, 2]
NP-mCherry appeared in the nuclei, and as puncta in the cytoplasm, with a high degree of co-localization with the NP immunostaining (Fig 2, bottom panels), as confirmed by co-localization analysis: the Pearson coefficient was 0.68±0.20 for the nuclei and 0.56±0.15 for the cytoplasm at 6–7 hours post-infection. This suggests that at least a sub-population of NP-mCherry is incorporated into Viral ribonucleoproteins (vRNPs), as neither free NP nor cRNPs accumulate in cytoplasmic puncta-like entities thought to be associated to recycling endosomes [27,28,29]
To independently visualize the PB2 influenza polymerase subunit and vRNPs in the infected cells, here we combined labeling of PB2 with split-GFP by using the recombinant WSN-PB2GFP11 virus [33] and labeling of virus-encoded RNPs by spontaneous incorporation of transiently expressed NP-mCherry fusion protein. The latter enabled us to bypass the problem of attenuation of recombinant influenza virus when fluorescent protein tags are fused to the NP

Summary

Introduction

Influenza viruses are a global health concern because of the permanent threat of the emergence of novel strains potentially capable of causing pandemics [1, 2]. There is a need for a deeper understanding of the molecular mechanisms

Methods

Results

Conclusion