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

Abstract

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.

RNA Synthesis and Capping by Non-segmented Negative Strand RNA Viral Polymerases: Lessons From a Prototypic Virus

“…However, the inability to establish efficient in vitro transcription or capping systems for other NNS RNA viruses has hampered the progress in our understanding of their precise mechanisms of mRNA capping. Our hypothesis is challenged by studies on some paramyxoviral L proteins that suggest that their C-terminal portions may carry out conventional mRNA capping with their RTPase and GTase activities (Gopinath and Shaila, 2009; Nishio et al, 2011; Singh et al, 2015; Ansari et al, 2019), although there is no evidence that these activities are directly involved in paramyxoviral mRNA capping. Therefore, it would be particularly interesting to solve the mechanisms of mRNA capping used by paramyxoviruses.…”