Volume 31, Number 1, January-February 2000
|Page(s)||24 - 25|
|How to cite this article||Vet. Res. (2000) 24-25|
Structure-function of the nucleocapsid protein of PRRSV North American genotypeD. Yoo and S. Wootton
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph Ontario, Canada N1G 2W1
Abstract - The open reading frame 7 (ORF7) of porcine reproductive and respiratory syndrome virus (PRRSV) codes for the nucleocapsid protein (N) of 123 amino acids in size. The N protein is multimerized to form an isomeric structure which appears to be unique to the arterivirus family among the order Nidovirales. In previous studies using a collection of monoclonal antibodies raised against the N protein of various North American genotypes and a series of deletion mutants of the N protein expressed in HeLa cells, we demonstrated that the 11 most C-terminal amino acids play a critical role in the formation of conformational epitopes specific for those monoclonal antibodies (Wootton et al., Clin. Diag. Lab. Immunol. 5 (1998) 773-779). Thus, the current research was designed to study the specific role of the individual amino acids within the C-terminal 11 amino acids in folding and multimerization of the N protein. The computer analysis predicts that the stretch of C-terminal amino acids is able to form a strong beta-sheet followed by the coil structure. Based on this prediction, a total of eight mutants was constructed to substitute the individual amino acids at the C-terminal region: valine at position 112 to proline (V112P), arginine at position 113 to aspartic acid (R113D) or to proline (R113P), leucine at position 114 to proline (L114P), isoleucine at position 115 to proline (I115P), arginine at position 116 to proline (R116P), threonine at position 118 to serine (T118S), and proline at position 121 to alanine (P121A). The mutant proteins, transiently expressed in HeLa cells using a T7 recombinant vaccinia virus, were used for radioimmunoprecipitation to determine their immunoreactivities with the conformation-dependent monoclonal antibodies. Mutations in the beta-sheet region including L114P, I115P, R116P, totally abolished the reactivities for all conformational monoclonal antibodies. The mutation in the coil region (P121A) only affected the binding of group III monoclonal antibodies. Since more than 80% of the monoclonal antibodies examined represented group III, these monoclonal antibodies were further studied using our single amino acid mutations. Based on the reactivities, group III monoclonal antibodies were further divided into 5 different subclasses. The bead-binding assays using the glutathione S-transferase (GST)-N fusion protein expressed in E. coli and the co-immunoprecipitation assays expressed in HeLa cells demonstrated that the N protein interacts with the N protein in vitro. To further demonstrate that these N-N interactions actually occur in vivo, a mammalian two hybrid assay was employed. L114P, I115P, R116P, and the deletion of C-terminal 11 amino acids abolished the activities for chloramphenicol acetyltransferase (CAT) whereas the wild-type N protein induced a significant level of CAT activity, indicating that N-N interactions also occur in vivo and are mediated by the C-terminal end. It appears that the PRRSV N protein possesses 6 putative beta-sheets between an unstructured N-terminal arm and an ordered C-terminus, forming a basic core structure of a jelly roll barrel. Taken together, our studies indicate that the non-covalent interaction between the C-terminal regions of N proteins is an important intermolecular interaction, and that this interaction may be critical for isomeric formation of the N protein during PRRSV maturation.
Corresponding author: D. Yoo Tel.: (1) (519) 824 4120, ext. 4729, fax: (1) (519) 767 0809,
© INRA, EDP Sciences 2000