Localization and Fine Mapping of Antigenic Sites on the Nucleocapsid Protein N of Porcine Reproductive and Respiratory Syndrome Virus with Monoclonal Antibodies

Abstract

The purpose of this study was to analyze the antigenic structure of the nucleocapsid protein N of the Lelystad virus isolate of porcine reproductive and respiratory syndrome virus (PRRSV) and to identify antigenic differences between this prototype European isolate and other North American isolates. To do this, we generated a panel of monoclonal antibodies (mAbs) directed against the N protein of Lelystad virus and tested them in competition assays with other N-specific mAbs described previously (Drew et al., 1995; Nelson et al., 1993; van Nieuwstadt et al., 1996). Four different competition groups of mAbs were identified. Pepscan analysis with solid-phase dodecapeptides was used to identify specific antigenic regions in the N protein that were bound by the mAbs. In this pepscan analysis, we found that the mAb of the first competition group reacted with linear peptides whose core sequences consisted of amino acids 2-12 (site A), the mAbs of the second group reacted with peptides whose core sequences consisted of amino acids 25-30 (site B), and the mAb of the third group reacted with peptides whose core sequences consisted of amino acids 40-46 (site C). However, the fourth group of mAbs binding to an antigenic region, provisionally designated as domain D, reacted very weakly or did not react at all with solid-phase dodecapeptides. To further characterize the structure of the epitopes in domain D, we produced chimeric constructs composed of the N protein sequences of Lelystad virus and another arterivirus lactate dehydrogenase-elevating virus, which was used because its N protein has similarity in amino acid sequence and hydropathicity profile but does not react with our mAbs. When the mAbs specific to domain D were tested for binding to the chimeric N proteins expressed by Semliki Forest virus, we found that the regions between amino acids 51-67 and amino acids 80-90 are involved in the formation or are part of the epitopes in domain D. Therefore, we conclude that the N protein contains four distinct antigenic regions. The epitopes mapped to sites A-C are linear, whereas the epitopes mapped to domain D are more conformation dependent or discontinuous. Sites A and C contain epitopes that are conserved in European but not in North American isolates; site B contains epitopes that are conserved in European and North American isolates; and site D contains epitopes that are either conserved or not conserved in European and North American isolates. The antigenic regions identified here might be important for the development of diagnostic test for PRRSV in particular tests that discriminate between different antigenic types of PRRSV.

Antigenic Importance of the Carboxy-Terminal Beta-Strand of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein

“…The C terminus has also been recognized as being an important determinant of discontinuous epitope formation in the N protein of the European lineage of PRRSV. In this case, deletion of either 23 or 31 amino acids from the C terminus of N proteins derived from European PRRSV isolates destroyed binding of conformation-dependent MAbs (17,21). These findings support the notion that the C terminus of the N protein is critical for the generation of discontinuous epitopes.…”

“…In this case, to circumvent the structural changes associated with deleting C-terminal amino acids, a chimera between the N protein of LV and lactate dehydrogenase elevating virus (LDV), another member of the family Arteriviridae, was constructed (17). In the chimeric protein, C-terminal amino acids 111 to 128 of the LV N protein (the European N protein is 128 amino acids long, whereas the North American N protein is 123 amino acids) were replaced by the corresponding amino acids of the LDV N protein.…”

Colocalization and Interaction of the Porcine Arterivirus Nucleocapsid Protein with the Small Nucleolar RNA-Associated Protein Fibrillarin

“…It is highly immunogenic (18) and is a basic protein with an isoelectric point of 10.4 that has been reported to be a serine phosphoprotein (36). Upon translation, the N protein interacts with itself by noncovalent interactions.…”

Disulfide Linkages Mediating Nucleocapsid Protein Dimerization Are Not Required for Porcine Arterivirus Infectivity

“…It has been detected not only in the cytoplasm but also in the nucleus and nucleolus, and a nuclear localization signal has been identified (42,43). The N-terminal half of the N protein is presumed to be mostly disordered and involved in RNA binding (12,62), while the C terminus is believed to be the conformation-determining region of the protein (37,55,57). As the sole protein component of the viral nucleocapsid, the N protein has been reported to interact with itself through covalent and noncovalent mechanisms (59).…”