The localization of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence

Abstract

The nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) possesses two regions in the N-terminal half of the protein that are enriched in basic amino acids. Presumably, these basic regions are important for packaging the RNA genome within the nucleocapsid of the virus. The PSORT computer program identified the same regions as nuclear localization signal (NLS) sequence motifs. N protein localization to the nucleus of infected MARC-145 and porcine pulmonary macrophages was observed following staining with SDOW-17 and SR-30 anti-N monoclonal antibodies. Furthermore, the co-localization of SR-30 antibody with human ANA-N autoimmune serum identified the nucleolus as the primary site for N protein localization within the nucleus. The localization of the N protein in the absence of infection was studied by following fluorescence in MARC-145 cells transfected with a plasmid, which expressed the nucleocapsid protein fused to an enhanced green fluorescent protein (N-EGFP). Similar to infected cells, N-EGFP localized to the cytoplasm and the nucleolus. Results following the transfection of cells with pEGFP fused to truncated portions of the N gene identified a region containing the second basic stretch of amino acids as the nucleolar localization signal (NoLS) sequence. Another outcome following transfection was the rapid disappearance of cells that expressed high levels of N-EGFP. However, cell death did not correlate with localization of N-EGFP to the nucleolus.