Expression and stability of foreign tags inserted into nsp2 of porcine reproductive and respiratory syndrome virus (PRRSV)

Dal Young Kim, Jay G. Calvert, Kyeong Ok Chang, Kyle Horlen, Maureen Kerrigan, Raymond R.R. Rowland

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)106-114
Number of pages9
JournalVirus Research
Volume128
Issue number1-2
DOIs
StatePublished - Sep 1 2007
Externally publishedYes

Keywords

  • EGFP
  • nsp2
  • PRRSV

ASJC Scopus subject areas

  • Cancer Research
  • Virology
  • Infectious Diseases

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