Expression and stability of foreign tags inserted into nsp2 of porcine reproductive and respiratory syndrome virus (PRRSV).
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.
Kim
Dal-Young
DY
Department of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, KS 66506, USA.
Calvert
Jay G
JG
Chang
Kyeong-Ok
KO
Horlen
Kyle
K
Kerrigan
Maureen
M
Rowland
Raymond R R
RR
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
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