title: Optimized P2A for reporter gene insertion into Nipah virus results in efficient ribosomal skipping and wild-type lethality.
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.
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California-Los Angeles, Los Angeles, CA, USA.|Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
authors.forename: Arnold
authors.initials: A
authors.lastname: Park
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Yun
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Terence E
authors.initials: TE
authors.lastname: Hill
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA.|Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA.|Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Ikegami
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Terry L
authors.initials: TL
authors.lastname: Juelich
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Jennifer K
authors.initials: JK
authors.lastname: Smith
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Lihong
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA.|Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA.|Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Freiberg
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.|Department of Microbiology, Immunology and Molecular Genetics, University of California-Los Angeles, Los Angeles, CA, USA.
authors.forename: Benhur
authors.initials: B
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005260:Female; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D045464:Henipavirus Infections / Q000235:genetics* / Q000401:mortality / Q000473:pathology / Q000821:virology; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008647:Mesocricetus; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D045405:Nipah Virus / Q000235:genetics* / Q000472:pathogenicity; D010590:Phalloidine / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D016019:Survival Analysis; D014158:Transcription, Genetic; D014779:Virus Replication / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D010590:Phalloidine; D012269:Ribosomal Proteins; D000097573:Red Fluorescent Protein