Signature motifs of GDP polyribonucleotidyltransferase, a non-segmented negative strand RNA viral mRNA capping enzyme, domain in the L protein are required for covalent enzyme-pRNA intermediate formation.

Julie Neubauer,Todd J Green,Tomoaki Ogino,Minako Ogino

doi:10.1093/nar/gkv1286

Abstract

The unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase; block V) domain in RNA polymerase L proteins of non-segmented negative strand (NNS) RNA viruses (e.g. rabies, measles, Ebola) contains five collinear sequence elements, Rx(3)Wx(3–8)ΦxGxζx(P/A) (motif A; Φ, hydrophobic; ζ, hydrophilic), (Y/W)ΦGSxT (motif B), W (motif C), HR (motif D) and ζxxΦx(F/Y)QxxΦ (motif E). We performed site-directed mutagenesis of the L protein of vesicular stomatitis virus (VSV, a prototypic NNS RNA virus) to examine participation of these motifs in mRNA capping. Similar to the catalytic residues in motif D, G1100 in motif A, T1157 in motif B, W1188 in motif C, and F1269 and Q1270 in motif E were found to be essential or important for the PRNTase activity in the step of the covalent L-pRNA intermediate formation, but not for the GTPase activity that generates GDP (pRNA acceptor). Cap defective mutations in these residues induced termination of mRNA synthesis at position +40 followed by aberrant stop–start transcription, and abolished virus gene expression in host cells. These results suggest that the conserved motifs constitute the active site of the PRNTase domain and the L-pRNA intermediate formation followed by the cap formation is essential for successful synthesis of full-length mRNAs.

Highlights

The 5 -terminal cap structure of eukaryotic mRNAs is composed of N7-methylguanosine (m7G) linked to the 5 -end of mRNA through the 5 5 triphosphate bridge, and is essential for various steps of mRNA metabolism including stability and translation [Reviewed in [1,2,3,4]]
Using our newly developed in vitro RNA capping assay with an oligo-RNA as a substrate, we have shown that the multifunctional large (L) protein (2109 amino acids) of vesicular stomatitis virus (VSV), a prototypic nonsegmented negative strand (NNS) RNA virus belonging to the Vesiculovirus genus of the Rhabdoviridae family in the order Mononegavirales, catalyzes unconventional mRNA capping reactions with a novel set of enzymatic activities [Reviewed in [13,14]]
We confirmed that these motifs are strikingly conserved in L proteins of more than 220 known NNS RNA viruses. To analyze whether these conserved and some semiconserved amino acid residues of the VSV L protein participate in mRNA capping reactions, we mutated them to alanine and/or closely related amino acids

Summary

Introduction

The 5 -terminal cap structure (cap 0, m7G[5 ]ppp[5 ]N-) of eukaryotic mRNAs is composed of N7-methylguanosine (m7G) linked to the 5 -end of mRNA through the 5 5 triphosphate (ppp) bridge, and is essential for various steps of mRNA metabolism including stability and translation [Reviewed in [1,2,3,4]]. The cap 0 structure is formed on pre-mRNAs by nuclear mRNA capping enzyme with the RNA 5 -triphosphatase (RTPase) and mRNA guanylyltransferase (GTase) activities followed by mRNA (guanineN7)-methyltransferase (MTase). Many eukaryotic viruses need to possess the fully methylated cap 1 structure on their mRNAs to efficiently produce viral proteins in infected cells. The molecular mechanisms of the cap formation by some RNA viral enzymes are significantly different from those by eukaryotic nuclear enzymes [10,11,12]

Methods

Results

Conclusion