The Nucleolar Localization of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein and its Relationship to Apoptosis

Porcine reproductive and respiratory syndrome virus (PRRSV) represents the greatest threat to the health of swine and the livelihood of swine producers. As an RNA virus, PRRSV was thought to replicate and express its proteins exclusively in the cytoplasm. This was challenged by the observation of nucleocapsid protein (N) in the nucleoli of cells infected with PRRSV 23983, a North American isolate. Nucleolar localization was confirmed by the co-localization of anti-nucleocapsid and anti-nucleolar antibodies in infected cells. To determine if N alone was able to localize to nucleoli, the N-gene lacking the stop codon, was ligated into pEGFP, a eukaryotic vector expressing enhanced green fluorescent protein (EGFP). EGFP-N fluorescence was observed in the nucleoli and cytoplasm of transfected cells, similar to the distribution in infected cells. The EGFP-N cells showed similar effects to cells undergoing apoptotsis and the cytopathic effect associated with PRRSV infection. It was hypothesized that nucleolar localization of N occurred through an identifiable nucleolar localization signal (NoLS) sequence. and that the nucleolar localization of N induced apoptosis. The PSORT computer program identified two potential nuclear localization signal (NLS) sequences in N, regions of basic amino acids starting at 10 and 43, \u27pat 4\u27 and \u27 pat 7\u27 NLS sequences, respectively. These NLS sequences could also serve as part of an NoLS motif Transfection of cells with truncated segments of the N gene inserted into pEGFP, demonstrated a putative NoLS m the \u27pat 7\u27 NLS region. Apoptosis of23983 infected cells was demonstrated by all methods used. However, apoptosis of cells expressing EGFP-N fusion protein was not conclusively determined. Inconclusive results may have been due to low rates of transfection, and/or the rapid death of cells expressing high levels of EGFP-N. Nucleolar localization of N did not result in cell death (apoptosis). These studies showed that a region of amino acids downstream from the putative NoLS induced cell death (apoptosis). Future studies need to establish if N induces apoptosis, and the minimum sequence of its putative NoLS and death domain. Finally, the role of nucleolar localization of N in viral pathogenesis must be determined