Molecular characterization studies of the porcine reproductive and respiratory syndrome virus
To facilitate a detailed study of the role of the nucleocapsid (N) protein in the porcine reproductive and respiratory syndrome virus (PRRSV) life cycle, as well as to acquire the necessary genetic information to study PRRSV, overlapping segments of a pathogenic Type II PRRSV were cloned and sequenced. The exact 5' terminus was determined and fragments comprising the entire genome were combined to generate a full-length cDNA clone. This molecular clone is the first step toward the development of a reverse genetics system that will enable the detailed study of the molecular determinants of PRRSV biology. Based on the immunoreactivity of a panel of N specific monoclonal antibodies (MAbs) with a series of N protein deletion mutants, five antigenic domains were identified. The majority of MAbs specifically recognized the local protein conformation formed in part by amino acid residues 52 to 69 and the carboxy terminus was shown to be essential for maintaining the antigenic structure of the N protein. Phosphorylation is a versatile mechanism utilized to modulate various protein functions. Here, we demonstrated that the N protein is a serine phosphoprotein both in virus-infected cells and the virus particle. Analysis of phosphoprotein distribution indicated that phosphorylation does not affect N protein subcellular localization. Furthermore, only one species of the N protein was detected in virus-infected cells suggesting that multiple phosphorylated isoforms of N do not exist. Investigation of the homotypic interactive capacity of the N protein revealed that it forms homodimers in the cytoplasm, at least initially, via non-covalent interactions that are stabilized through the involvement of RNA. While disulfide bond formation was not the initial driving force mediating N protein dimerization, disulfide-linked dimers did form when virus particles entered the oxidizing environments of the ER and extracellular milieu. Mutational analysis revealed that cysteine at position 23 was responsible for the formation of disulfide-linkages between N proteins. In summary, analysis of the antigenic, post-translational and oligomeric properties of the PRRSV N protein provided insight into the structure of the N protein, revealed a possible mode of functional regulation and delineated the various mechanisms involved in N-N interactions.