Unconventional Mechanism of mRNA Capping by the RNA-Dependent RNA Polymerase of Vesicular Stomatitis Virus

Abstract

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.

RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

“…However, in non-segmented negative-sense RNA viruses, GT activity has only been demonstrated for VSV L protein (Ogino & Banerjee, 2007). In the present work, we have demonstrated, for the first time for any paramyxovirus, that L protein is able to cap viral mRNA.…”

“…RTPase activity of L protein, the first step in viral mRNA capping, was tested in vitro using synthetic c-32 P-labelled RNA substrate containing sequences corresponding to viral genes. Viral mRNA 59 cis sequences were used as the substrate because in many negative-sense RNA viruses, the capping machinery of these viruses requires specific cis-acting signals in the RNA (Ogino et al, 2005;Ogino & Banerjee, 2007;Wang et al, 2007). Under optimal conditions, L protein in the RNP complex, as well as L protein co-expressed with P protein in insect cells, efficiently catalyses the removal of cphosphate from the triphosphate-ended RNA (Fig.…”

“…VSV, a prototype model for negative-sense RNA viruses, forms a covalent complex with the substrate RNA rather than the nucleotide and uses GDP instead of GMP for capping by a polyribo nucleotidyl transferase activity (Ogino & Banerjee, 2007). In addition, it also forms an unusual tetra-phosphate cap structure on viral mRNA (GppppG) (Ogino & Banerjee, 2008).…”

“…In negative-sense RNA viruses, L protein is believed to possess activities required for capping, methylation and polyadenylation of mRNAs (Banerjee, 1987). Purified ribonucleoprotein (RNP) complexes from vesicular stomatitis virus (VSV) and Sendai virus containing the N, L and P proteins along with the genomic RNA have been shown to catalyse capping as well as cap methylation activities in vitro (Galloway et al, 2008;Grdzelishvili et al, 2006;Li et al, 2006;Ogino et al, 2005;Ogino & Banerjee, 2007. However, there is no direct demonstration of RTPase or GT activity associated with the RNP complex or L protein of any paramyxovirus.…”

Histidine-mediated RNA transfer to GDP for unique mRNA capping by vesicular stomatitis virus RNA polymerase

“…In direct contrast, we have recently shown that the multifunctional RNA-dependent RNA polymerase (RdRp) L protein (2,109 amino acids) of vesicular stomatitis virus (VSV), a prototype nonsegmented negative-strand (NNS) RNA virus belonging to the genus Vesiculovirus of the family Rhabdoviridae in the order Mononegavirales, carries out an unconventional mRNA capping mechanism that involves stepwise action of guanosine 5′-triphosphatase (GTPase) followed by RNA:GDP polyribonucleotidyltransferase (PRNTase) (4,5), as proposed below:…”