Structure-function of the nucleocapsid \nprotein of PRRSV North American genotype

\nThe open reading frame 7 (ORF7) of porcine reproductive and respiratory syndrome \nvirus (PRRSV) codes for the nucleocapsid protein (N) of 123 amino acids in size. \nThe N protein is multimerized to form an isomeric structure which appears to be\n unique to the arterivirus\n family among the order Nidovirales. In previous studies \nusing a collection of monoclonal antibodies raised against the N protein of various\n North American genotypes and a series of deletion mutants of the N protein expressed\n in HeLa cells, we demonstrated that the 11 most C-terminal amino acids play a critical \nrole in the formation of conformational epitopes specific for those monoclonal antibodies\n (Wootton et al., Clin. Diag. Lab. Immunol. 5 (1998) 773-779). Thus, the current research \nwas designed to study the specific role of the individual amino acids within the C-terminal \n11 amino acids in folding and multimerization of the N protein. The computer analysis \npredicts that the stretch of C-terminal amino acids is able to form a strong beta-sheet \nfollowed by the coil structure. Based on this prediction, a total of eight mutants was\n constructed to substitute the individual amino acids at the C-terminal region: valine\n at position 112 to proline (V112P), arginine at position 113 to aspartic acid (R113D)\n or to proline (R113P), leucine at position 114 to proline (L114P), isoleucine at position \n115 to proline (I115P), arginine at position 116 to proline (R116P), threonine at position\n 118 to serine (T118S), and proline at position 121 to alanine (P121A). The mutant proteins,\n transiently expressed in HeLa cells using a T7 recombinant vaccinia virus, were used for \nradioimmunoprecipitation to determine their immunoreactivities with the \nconformation-dependent\n monoclonal antibodies. Mutations in the beta-sheet region including L114P, I115P, R116P, \ntotally abolished the reactivities for all conformational monoclonal antibodies. The\n mutation in the coil region (P121A) only affected the binding of group III monoclonal \nantibodies. Since more than 80% of the monoclonal antibodies examined represented group\n III, these monoclonal antibodies were further studied using our single amino acid \nmutations. Based on the reactivities, group III monoclonal antibodies were further \ndivided into 5 different subclasses. The bead-binding assays using the glutathione\n S-transferase (GST)-N fusion protein expressed in \nE. coli and the co-immunoprecipitation assays expressed in HeLa cells demonstrated \nthat the N protein interacts with the N protein in vitro. To further demonstrate \nthat these N-N interactions actually occur in vivo, a mammalian two hybrid assay \nwas employed. L114P, I115P, R116P, and the deletion of C-terminal 11 amino acids \nabolished the activities for chloramphenicol acetyltransferase (CAT) whereas the\n wild-type N protein induced a significant level of CAT activity, indicating that\n N-N interactions also occur in vivo and are mediated by the C-terminal end. It \nappears that the PRRSV N protein possesses 6 putative beta-sheets between an\n unstructured N-terminal arm and an ordered C-terminus, forming a basic core \nstructure of a jelly roll barrel. Taken together, our studies indicate that\n the non-covalent interaction between the C-terminal regions of N proteins is\n an important intermolecular interaction, and that this interaction may be \ncritical for isomeric formation of the N protein during PRRSV maturation.\n

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