The large protein \u2018L\u2019 of Peste-des-petits-ruminants virus exhibits RNA triphosphatase activity, the first enzyme in mRNA capping pathway

Mohammad Yunus Ansari,Piyush Kumar Singh,Asutosh Bellur,Melkote Subbarao Shaila,Deepa Rajagopalan,Purnima Shanmugam

doi:10.1007/s11262-018-1617-5

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.

Highlights

Mononegavirales order consists of eight families of highly divergent viruses, which possess a linear, negatively-polar, single-stranded and mostly non-segmented RNA genome.Edited by Keizo Tomonaga.The gene organization in the viral order is conserved and the genes are transcribed from a single promoter at the 3′ terminal of genome resulting in multiple capped and polyadenylated mRNAs
We had earlier demonstrated that Rinderpest virus (RPV), an important member of Paramyxoviridae family follows conventional mRNA capping pathway and the carboxy-terminal region of the L protein of RPV carries the RNA triphosphatase (RTPase) domain [3]
In order to conclusively show that the L proteins of other members in the Morbillivirus genus exhibit RTPase activity, we investigated if Peste-des-petits-ruminants virus (PPRV) L protein has associated RTPase activity

Summary

Introduction

Mononegavirales order consists of eight families of highly divergent viruses, which possess a linear, negatively-polar, single-stranded and mostly non-segmented RNA genome. The gene organization in the viral order is conserved and the genes are transcribed from a single promoter at the 3′ terminal of genome resulting in multiple capped and polyadenylated mRNAs. The replication of the virus invariably proceeds via the synthesis of an antigenome and is performed by viral encoded polymerase complex. MRNA capping in the Rhabdoviridae family of viruses is thought to proceed via a novel unconventional mRNA capping pathway due to establishment of such pathway in the Lyssavirus and Vesiculovirus genera of Rhabdoviruses [1]. The finding that the Rinderpest virus (RPV) L protein follows the conventional mRNA capping pathway clearly demonstrated that members of Morbillivirus genus in Paramyxoviridae family need not necessarily follow the unconventional capping pathway [2].

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Results

Conclusion