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Journal of Virology, April 2003, p. 4546-4557, Vol. 77, No. 8
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.8.4546-4557.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Homo-Oligomerization of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein and the Role of Disulfide Linkages

Sarah K. Wootton and Dongwan Yoo*

Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1

Received 28 October 2002/ Accepted 17 January 2003

As a step toward understanding the assembly pathway of the porcine reproductive and respiratory syndrome virus (PRRSV), the oligomeric properties of the nucleocapsid (N) protein were investigated. In this study, we have demonstrated that under nonreducing conditions the N protein forms disulfide-linked homodimers. However, inclusion of an alkylating agent (N-ethylmaleimide [NEM]) prevented disulfide bond formation, suggesting that these intermolecular disulfide linkages were formed as a result of spurious oxidation during cell lysis. In contrast, N protein homodimers isolated from extracellular virions were shown to have formed NEM-resistant intermolecular disulfide linkages, the function of which is probably to impart stability to the virion. Pulse-chase analysis revealed that N protein homodimers become specifically disulfide linked within the virus-infected cell, albeit at the later stages of infection, conceivably when the virus particle buds into the oxidizing environment of the endoplasmic reticulum. Moreover, NEM-resistant disulfide linkages were shown to occur only during productive PRRSV infection, since expression of recombinant N protein did not result in the formation of NEM-resistant disulfide-linked homodimers. Mutational analysis indicated that of the three conserved cysteine residues in the N protein, only the cysteine at position 23 was involved in the formation of disulfide linkages. The N protein dimer was shown to be stable both in the presence and absence of intermolecular disulfide linkages, indicating that noncovalent interactions also play a role in dimerization. Non-disulfide-mediated N protein interactions were subsequently demonstrated both in vitro by the glutathione S-transferase (GST) pull-down assay and in vivo by the mammalian two-hybrid assay. Using a series of N protein deletion mutants fused to GST, amino acids 30 to 37 were shown to be essential for N-N interactions. Furthermore, since RNase A treatment markedly decreased N protein-binding affinity, it appears that at least in vitro, RNA may be involved in bridging N-N interactions. In cross-linking experiments, the N protein was shown to assemble into higher-order structures, including dimers, trimers, tetramers, and pentamers. Together, these findings demonstrate that the N protein possesses self-associative properties, and these likely provide the basis for PRRSV nucleocapsid assembly.

* Corresponding author. Mailing address: Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Phone: (519) 824-4120, ext. 54729. Fax: (519) 767-0809. E-mail: dyoo{at}uoguelph.ca.

Journal of Virology, April 2003, p. 4546-4557, Vol. 77, No. 8
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.8.4546-4557.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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