The large protein 'L' of Peste-des-petits-ruminants virus exhibits RNA triphosphatase activity, the first enzyme in mRNA capping pathway.

Peste-des-petits-ruminants is a highly contagious and fatal disease of goats and sheep caused by non-segmented, negative strand RNA virus belonging to the Morbillivirus genus-Peste-des-petits-ruminants virus (PPRV) which is evolutionarily closely related to Rinderpest virus (RPV). The large protein 'L' of the members of this genus is a multifunctional catalytic protein, which transcribes and replicates the viral genomic RNA as well as possesses mRNA capping, methylation and polyadenylation activities; however, the detailed mechanism of mRNA capping by PPRV L protein has not been studied. We have found earlier that the L protein of RPV has RNA triphosphatase (RTPase), guanylyltransferase (GTase) and methyltransferase activities, and unlike vesicular stomatitis virus (VSV), follows the conventional pathway of mRNA capping. In the present work, using a 5'-end labelled viral RNA as substrate, we demonstrate that PPRV L protein has RTPase activity when present in the ribonucleoprotein complex of purified virus as well as recombinant L-P complex expressed in insect cells. Further, a minimal domain in the C-terminal region (aa1640-1840) of the L protein has been expressed in E. coli and shown to exhibit RTPase activity. The RTPase activity of PPRV L protein is metal-dependent and functions with a divalent cation, either magnesium or manganese. In addition, RTPase associated nucleotide triphosphatase activity (NTPase) of PPRV L protein is also demonstrated. This work provides the first detailed study of RTPase activity and identifies the RTPase domain of PPRV L protein.

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

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 morbilliviruses 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.