Live-cell imaging of viral RNA genomes using a Pumilio-based reporter

Jens Tilsner, Olga Linnik, Nynne M. Christensen, Karen Bell, Ian M. Roberts, Christophe Lacomme, Karl J. Oparka

Research output: Contribution to journalArticlepeer-review

91 Citations (Scopus)

Abstract

We describe a method for localizing plant viral RNAs in vivo using Pumilio, an RNA-binding protein, coupled to bimolecular fluorescence complementation (BiFC). Two Pumilio homology domain (PUMHD) polypeptides, fused to either the N- or C-terminal halves of split mCitrine, were engineered to recognize two closely adjacent eight-nucleotide sequences in the genomic RNA of tobacco mosaic virus (TMV). Binding of the PUMHDs to their target sites brought the split mCitrine halves into close proximity, allowing BiFC to occur and revealing the localization of viral RNA within infected cells. The bulk of the RNA was sequestered in characteristic inclusion bodies known as viral replication complexes (VRCs), with a second population of RNA localized in discrete particles distributed throughout the peripheral cytoplasm. Transfer of the TMV Pumilio recognition sequences into the genome of potato virus X (PVX) allowed the PVX RNA to be localized. Unlike TMV, the PVX RNA was concentrated in distinctive 'whorls' within the VRC. Optical sectioning of the PVX VRCs revealed that one of the viral movement proteins was localized to the centres of the RNA whorls, demonstrating significant partitioning of viral RNA and proteins within the VRC. The utility of Pumilio as a fluorescence-based reporter for viral RNA is discussed.

Original languageEnglish
Pages (from-to)758-770
Number of pages13
JournalThe Plant Journal
Volume57
Issue number4
DOIs
Publication statusPublished - Feb 2009

Keywords

  • ribonucleic acid
  • live-cell imaging
  • virus
  • replication
  • Pumilio
  • bimolecular fluorescence complementation
  • TOBACCO-MOSAIC-VIRUS
  • BIMOLECULAR FLUORESCENCE COMPLEMENTATION
  • ENDOPLASMIC-RETICULUM
  • MOVEMENT PROTEIN
  • BINDING PROTEINS
  • GENE-EXPRESSION
  • MESSENGER-RNAS
  • TRANSLATIONAL CONTROL
  • REPLICATION COMPLEX
  • COAT PROTEIN

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