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.