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.

Introduction

Porcine reproductive and respiratory virus (PRRSV) is a positive polarity single-stranded RNA virus belonging to the family, Arteriviridae. This group also includes lactate dehydrogenase-elevating virus (LDV) of mice, equine arteritis virus (EAV), and simian hemorrhagic fever virus (SHFV; Plagemann, 1996). Recently, the Arteriviridae, along with the Coronaviridae, have been placed in a single order, Nidovirales (Cavanagh, 1997). Two distinct strains of PRRSV are represented by VR-2332 of the United States (Benfield et al., 1992, Collins et al., 1992) and the Lelystad virus from the Netherlands (Wensvoort et al., 1991, Meulenberg et al., 1993). Even though the Lelystad virus and VR-2332 possess similar biochemical properties and cause similar disease symptoms, they share only 56% identity at the nucleotide level of the structural genes, ORFs 2–7 (Murtaugh et al., 1995). PRRSV replication in the infected pig is primarily restricted to a subpopulation of permissive macrophages (Pol et al., 1991, Wensvoort et al., 1991, Rossow et al., 1996, Lawson et al., 1997). Primary cultures of porcine alveolar macrophages and MARC-145 cells, a subclone of monkey kidney MA-104 cells, support PRRSV replication to high levels (Wensvoort et al., 1991, Kim et al., 1993).

The N gene, also known as ORF7, codes for a non-glycosylated nucleocapsid (N) protein, which is relatively conserved among North American field isolates (Kapur et al., 1996). The N protein does not contain a signal peptide; therefore, it is translated and maintained as a mature 123 amino acid, 14 kd protein (Mardassi et al., 1995, Mounir et al., 1995). Regions enriched in basic amino acids appear to be important for packaging the PRRSV RNA genome within the nucleocapsid of the mature virion (Mounir et al., 1995, Mardassi et al., 1996, Plagemann, 1996).

Stretches of basic amino acids are the principal components of nuclear localization signal (NLS) sequences (Silver, 1991), which are generally classified as one of three types (reviewed in Nakai and Kanehisa, 1992, Hicks and Raikhel, 1995). The ‘pat4’ motif consists of a continuous stretch of four basic amino acids (lysine or arginine) or three basic amino acids associated with histidine or proline. The ‘pat7’ sequence starts with proline and is followed within three residues by a segment containing three basic residues out of four. The third type of NLS, known as a ‘bipartite’ motif, consists of two basic amino acids, a ten amino acid spacer, and a five amino acid segment containing at least three basic residues. The actual participation of a particular NLS sequence in nuclear localization of a protein requires several additional considerations (reviewed in Silver, 1991). First, proteins without a NLS sequence can localize to the nucleus by co-transport with other nuclear proteins. Secondly, the presence of an NLS sequence does not guarantee that a protein is nuclear, especially if the NLS sequence is not exposed on the surface of the protein. And finally, small proteins less than 70 kd can passively diffuse into the nucleoplasm through the nuclear pore complex.

Nucleolar localization signal (NoLS) sequence motifs are less well understood; however, NoLS sequences usually incorporate an NLS sequence. For example, the NoLS sequence, GRKKRRQRRRAHQN, in the HIV Tat protein contains two stretches of basic amino acids separated by a single glycine. The minimal sequence, RKKR, is sufficient to accumulate Tat in the nucleus, but the protein is excluded from the nucleolus (Siomi et al., 1990). NoLS motifs of other viral proteins follow a similar pattern, except that proline may be substituted for glycine (Siomi et al., 1988).

NLS and NoLS sequences are frequently found in the proteins of DNA and RNA viruses that require the nucleus for replication. For RNA viruses that replicate in the cytoplasm the purpose of targeting viral proteins to the nucleus is less clear. One possibility is that viral proteins targeted to the nucleolus during replication may regulate host cell biosynthesis. For example, the nucleocapsid protein of Semliki Forest virus (SFV), a togavirus, contains two NoLS sequences, which are sufficient to localize a reporter protein to the nucleolus (Favre et al., 1994, Jakob, 1995). Nucleolar localization of the Semliki Forest virus nucleocapsid protein during infection appears to be the involved in the regulation of protein synthesis, perhaps by regulating the synthesis of ribosomal RNA and/or assembly of ribosomes. Low amounts of SFV nucleocapsid protein electroporated into cells enhance protein synthesis; whereas, high concentrations inhibit translation (Elgizoli et al., 1989, Michel et al., 1990).

In this report we describe the localization of the PRRSV N protein to the nucleolus of infected MARC-145 cells and porcine alveolar macrophages (PAMs). We also describe the use of enhanced green fluorescent protein as a tool for mapping peptide regions within the N protein responsible for nucleolar localization.