title: Expression and stability of foreign tags inserted into nsp2 of porcine reproductive and respiratory syndrome virus (PRRSV).
abstract: The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the single largest protein produced during infection. The cDNA of the pCMV-129 infectious PRRSV clone was modified for accepting foreign tags by first creating unique Mlu I and SgrA I restrictions sites at nucleotide (nt) positions 3219 and 3614, respectively, within the C-terminal region of nsp2. cDNAs encoding oligo- and polypeptide tags, including FLAG, enhanced green fluorescent protein (EGFP) and luciferase were inserted into the newly created restriction sites. The results showed that only the EGFP-containing genomes were properly expressed and produced virus. EGFP fluorescence, but not EGFP immunoreactivity, was lost during passage of recombinant EGFP viruses in culture. Sequencing of a fluorescent-negative EGFP virus showed that the EGFP remained intact, except for the appearance of arginine to cysteine mutation at position 96, which may interfere with chromophore formation or function.
authors.affiliation: Department of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, KS 66506, USA.
authors.forename: Dal-Young
authors.initials: DY
authors.lastname: Kim
authors.forename: Jay G
authors.initials: JG
authors.lastname: Calvert
authors.forename: Kyeong-Ok
authors.initials: KO
authors.lastname: Chang
authors.forename: Kyle
authors.initials: K
authors.lastname: Horlen
authors.forename: Maureen
authors.initials: M
authors.lastname: Kerrigan
authors.forename: Raymond R R
authors.initials: RR
authors.lastname: Rowland
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine / Q000821:virology; D014779:Virus Replication
chemical_list: D001426:Bacterial Proteins; D009842:Oligopeptides; D010455:Peptides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C085706:FLAG peptide; D008156:Luciferases; C035041:endodeoxyribonuclease MluI; C064435:endodeoxyribonuclease SgrAI; D015252:Deoxyribonucleases, Type II Site-Specific; D003546:Cysteine Endopeptidases; C078306:nsP2 proteinase