Phosphoprotein of the rinderpest virus forms a tetramer through a coiled coil region important for biological function: A structural insight

Abstract

Phosphoprotein (P) of negative sense RNA viruses functions as a transcriptional transactivator of the viral polymerase (L). We report here the characterization of oligomeric P protein of rinderpest virus (RPV) and provide a structural basis for its multimerization. By size exclusion chromatography and dynamic light scattering analyses we show that bacterially expressed P protein exists as an oligomer, thus excluding the role of phosphorylation in P protein oligomerization. Gel filtration analyses of various parts of the P protein, also expressed in Escherichia coli, revealed that the predicted coiled coil region in the C-terminal domain is responsible for P protein oligomerization. Dynamic light scattering analysis confirmed the oligomeric nature of the coiled coil region of P. Chemical cross-linking analysis suggested that the C-terminal coiled coil region exists as a tetramer. The tetramer is formed by coiled coil interaction as shown by circular dichroism spectral analysis. Based on sequence homology, we propose a three-dimensional structure of the multimerization domain of RPV P using the crystal structure for multimerization domain of sendai virus (SeV) P as a template. Four-stranded coiled coil structure of the model is stabilized by a series of interactions predominantly between short nonpolar side chains emerging from different strands. In an in vivo replication/transcription system using a synthetic minigenome of RPV, we show that multimerization is essential for P protein function(s), and the multimerization domain is highly conserved between two morbilli-viruses namely RPV and peste de petits ruminants virus. These results are discussed in the context of biological functions of P protein among various negative-stranded RNA viruses.