Optimized P2A for reporter gene insertion into Nipah virus results in efficient ribosomal skipping and wild-type lethality

J Gen Virol. 2016 Apr;97(4):839-843. doi: 10.1099/jgv.0.000405. Epub 2016 Jan 18.

Abstract

Incorporation of reporter genes within virus genomes is an indispensable tool for interrogation of virus biology and pathogenesis. In previous work, we incorporated a fluorophore into a viral ORF by attaching it to the viral gene via a P2A ribosomal skipping sequence. This recombinant Nipah virus, however, was attenuated in vitro relative to WT virus. In this work, we determined that inefficient ribosomal skipping was a major contributing factor to this attenuation. Inserting a GSG linker before the P2A sequence resulted in essentially complete skipping, significantly improved growth in vitro, and WT lethality in vivo. To the best of our knowledge, this represents the first time a recombinant virus of Mononegavirales with integration of a reporter into a viral ORF has been compared with the WT virus in vivo. Incorporating the GSG linker for improved skipping efficiency whenever functionally important is a critical consideration for recombinant virus design.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Female
  • Gene Expression Regulation
  • Genes, Reporter*
  • Genetic Engineering / methods*
  • Henipavirus Infections / genetics*
  • Henipavirus Infections / mortality
  • Henipavirus Infections / pathology
  • Henipavirus Infections / virology
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mesocricetus
  • Molecular Sequence Data
  • Mutagenesis, Insertional
  • Nipah Virus / genetics*
  • Nipah Virus / pathogenicity
  • Phalloidine / genetics
  • Phalloidine / metabolism
  • Red Fluorescent Protein
  • Ribosomal Proteins / genetics*
  • Ribosomal Proteins / metabolism
  • Ribosomes / genetics
  • Ribosomes / metabolism
  • Survival Analysis
  • Transcription, Genetic
  • Virus Replication / genetics

Substances

  • Luminescent Proteins
  • Ribosomal Proteins
  • Phalloidine