Functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association

PRRSV (porcine reproductive and respiratory syndrome virus) nucleocapsid (N) protein is the most abundant structural protein of the virus. During infection, the N protein is specifically localized to the nucleus and nucleolus in addition to its normal cytoplasmic distribution. Previously, a nuclear localization signal (NLS, 41-PGKK(N/S)KKKN)-null mutant virus (41-PGGGNKKKN) showed reduced viremia and increased production of neutralizing antibodies in infected pigs. However, the mutagenized NLS underwent strong selection pressure in the pig that resulted in partial or complete reversion and reacquisition of NLS function, and thus the biological effect of the NLS-null mutation needed further investigation. In the present study, a total of 9 "reversion resistant" mutants were generated by amino acid deletions and substitutions using an infectious cDNA clone. Two mutant clones (PG--SKKKS and PG--S-KKS) that produced progeny viruses were genetically stable for at least 20 passages in cell culture. Infection of pigs with those mutants induced neutralizing antibodies to higher titers than with wild-type virus. Both mutant viruses induced viremia of lower titer and of shorter duration than wild-type virus. RT-PCR from tonsils showed that both mutants persisted at a reduced level. Virus transmission to contact pigs was also lower in the mutant virus infected groups. No reversion to functional NLS was detected in either mutant from any pig. These data demonstrate that N protein nuclear localization is indeed associated with viral pathogenesis and host response to PRRS.