Mapping of the Functional Domains of the Newcastle Disease Virus Phosphoprotein (P).

The phosphoprotein (P) of the Newcastle disease virus (NDV) plays a central role in the transcription and the replication of the viral genome. Together with the large (L) protein, it forms and stabilizes the active RNA-dependent RNA polymerase complex. It also mediates the interaction between the L protein subunit of this complex with the viral nucelocapsid (NP:RNA) template comprising a nucleocapsid tightly bound RNA genome. The association of P and the unassembled nucleocapsid protein (NP˚) is crucial in ensuring the specific binding of NP to the RNA genome. Although much is known about the interactions between the proteins of the transcriptive complex in the Sendai and vesicular stomatitis viruses, very little work has been done on the transcriptive complex of NDV. Therefore in this study, the yeast two-hybrid system was employed to identify the domains of P protein involved in interaction with itself, NP˚ and L proteins. The interaction domain of P with NP:RNA was also localized. To identify the P protein self-association domain, a total of 11 mutants with successive deletions from either the N- or C-terminal of the P gene were generated. Full length P protein (cloned in fusion with the binding domain of the transcriptional activator, BD) as well as each deletion mutant (cloned in fusion with the activation domain, AD) were co-transformed into the yeast Saccharomyces cerevisiae strain L40, separately. To analyze positive interactions, ß-galactosidase (ß-gal) assay was performed. A high level of ß-gal activity was detected in the interaction of P with itself, indicating the ability of NDV P protein to oligomerize. The self-association domain was mapped to the C-terminal half of the protein between amino acid residues 247 to 291. To map the domains of P involved in the interaction with NP˚, the P protein and its derivatives along with full length NP were co-transformed into the yeast. A strong ß-gal activity was observed in the interaction of full length P and NP, indicating a specific interaction between P and NP proteins. Surprisingly, the same region in the C-terminal of P which is important for self-association (residues 247-291) was shown to be involved in the interaction with NP˚ as well. In order to map the regions of L that interact with P, the six functional domains of L, along with full length P were co-transformed into the yeast. However, due to the low expression level of the domains, no ß-gal activity was detected in the interactions of the domains with P. Therefore, an in vitro co-translation approach was employed to identify P:L interaction domain. The results showed a region located in the C-terminal half of P (amino acids 292-338), just beside the P protein oligomerization domain, is involved in the stabilization of L protein. Co-immunoprecipitation was performed to investigate the regions of P that interact with NP:RNA template. Purified NP:RNA template along with in vitro translated P or its derivatives were separately co-immunoprecipitated using anti-His antibody. The results showed that the immediate N-terminal of P protein is involved in its interaction with NP:RNA template. In summary, the current study had delineated the key functional regions of the P. This knowledge will be useful for structural and functional exploration of the NDV P protein.

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