Related Experiment Video
Updated: Mar 27, 2026
CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites
Published on: November 14, 2018
Optimized P2A for reporter gene insertion into Nipah virus results in efficient ribosomal skipping and wild-type
Arnold Park1,2, Tatyana Yun3, Terence E Hill3
1Department of Microbiology, Immunology and Molecular Genetics, University of California-Los Angeles, Los Angeles, CA, USA.
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.
