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

The nucleocapsid (N) protein of porcine reproductive and respiratory syndro me virus (PRRSV) possesses two regions in the N-terminal half of the protei n that are enriched in basic amino acids. Presumably, these basic regions a re important for packaging the RNA genome within the nucleocapsid of the vi rus. The PSORT computer program identified the same regions as nuclear loca lization signal (NLS) sequence motifs. N protein localization to the nucleu s of infected MARC-145 and porcine pulmonary macrophages was observed follo wing staining with SDOW-17 and SR-30 anti-N monoclonal antibodies. Furtherm ore, the co-localization of SR-30 antibody with human ANA-N autoimmune seru m identified the nucleolus as the primary site for N protein localization w ithin the nucleus. The localization of the N protein ill the absence of inf ection was studied by following fluorescence in MARC-145 cells transfected with a plasmid, which expressed the nucleocapsid protein fused to an enhanc ed green fluorescent protein (N-EGFP). Similar to infected cells, N-EGFP lo calized to the cytoplasm and the nucleolus. Results following the transfect ion of cells with pEGFP fused to truncated portions of the N gene identifie d a region containing the second basic stretch of amino acids as the nucleo lar localization signal (NoLS) sequence. Another outcome following transfec tion was the rapid disappearance of cells that expressed high levels of N-E GFP. However, cell death did not correlate with localization of NEGFP to th e nucleolus. (C) 1999 Elsevier Science B.V. All rights reserved.