Live Imaging of Influenza Viral Ribonucleoproteins Using Light-Sheet Microscopy

Methods Mol Biol. 2018;1836:303-327. doi: 10.1007/978-1-4939-8678-1_15.

Abstract

Influenza viruses exhibit a complex life cycle that is still poorly understood. It involves independent replication of each of the eight segments that make up its genome and subsequent coordinated assembly as they egress from the host cell. Fast, time-resolved volumetric live cell imaging offers a powerful tool for understanding the various host mechanisms hijacked by the virus. Here, we describe the methods necessary for generating influenza viruses that carry a fluorescently tagged polymerase complex, infection of biologically relevant cells with these viruses, and finally protocols for live cell imaging and analysis.

Keywords: Fluorescence imaging; Fluorescence microscopy; Fluorescent influenza virus; Fluorescent polymerase; Influenza genome; Light-sheet microscopy; Live cell imaging; Particle tracking.

MeSH terms

  • Animals
  • Cell Line
  • Cloning, Molecular
  • Gene Expression
  • Genes, Reporter
  • Humans
  • Image Processing, Computer-Assisted
  • Influenza A virus / physiology*
  • Microscopy, Fluorescence* / methods
  • Molecular Imaging* / instrumentation
  • Molecular Imaging* / methods
  • Plasmids / genetics
  • Recombinant Fusion Proteins
  • Ribonucleoproteins* / genetics
  • Ribonucleoproteins* / metabolism
  • Viral Plaque Assay
  • Viral Proteins* / genetics
  • Viral Proteins* / metabolism

Substances

  • Recombinant Fusion Proteins
  • Ribonucleoproteins
  • Viral Proteins