Abstract
The RNA synthesis machinery of vesicular stomatitis virus (VSV) comprises the genomic RNA encapsidated by the viral nucleocapsid protein (N) and associated with the RNA dependent RNA polymerase, the viral components of which are a large protein (L) and an accessory phosphoprotein (P). The 241 kDa L protein contains all the enzymatic activities necessary for synthesis of the viral mRNAs, including capping, cap methylation and polyadenylation. Those RNA processing reactions are intimately coordinated with nucleotide polymerization such that failure to cap results in termination of transcription and failure to methylate can result in hyper polyadenylation. The mRNA processing reactions thus serve as a critical check point in viral RNA synthesis which may control the synthesis of incorrectly modified RNAs. Here, we report the length at which viral transcripts first gain access to the capping machinery during synthesis. By reconstitution of transcription in vitro with highly purified recombinant polymerase and engineered templates in which we omitted sites for incorporation of UTP, we found that transcripts that were 30-nucleotides in length were uncapped, whereas those that were 31-nucleotides in length contained a cap structure. The minimal RNA length required for mRNA cap addition was also sufficient for methylation since the 31-nucleotide long transcripts were methylated at both ribose-2′-O and guanine-N-7 positions. This work provides insights into the spatial relationship between the active sites for the RNA dependent RNA polymerase and polyribonucleotidyltransferase responsible for capping of the viral RNA. We combine the present findings with our recently described electron microscopic structure of the VSV polymerase and propose a model of how the spatial arrangement of the capping activities of L may influence nucleotide polymerization.
Highlights
The RNA synthesis machinery of the non-segmented negativestrand (NNS) RNA viruses contains at its core a large polymerase protein (L) that possesses all the enzymatic activities for genome transcription and replication
Using a prototype of the nonsegmented negative strand RNA viruses, vesicular stomatitis virus, we probed the spatial relationship between the RNA dependent RNA polymerase and 59 mRNA capping and methylation activities of the large polymerase protein
Because the 59 mRNA processing reactions dramatically impact the nucleotide polymerization activity of the protein, they may function as a quality control step in viral transcription
Summary
The RNA synthesis machinery of the non-segmented negativestrand (NNS) RNA viruses contains at its core a large polymerase protein (L) that possesses all the enzymatic activities for genome transcription and replication. The RNA dependent RNA polymerase (RdRP) was readily identified by the presence of a set of motifs in CRIII [1] Consistent with this assignment, substitution of an aspartic acid residue predicted to coordinate a catalytically essential magnesium ion ablates nucleotide polymerization in vitro. L protein was known to possess the enzymatic activities for mRNA cap addition, their identity proved difficult to pin down unambiguously This is because the enzymatic activities themselves are unusual, in that the cap is added by the action of a polyribonucleotidyltransferase (PRNTase) that transfers pRNA onto a GDP acceptor through a covalent L-pRNA intermediate [7,9,17] This contrasts with all other known capping reactions which involve an RNA guanylyltransferase that transfers GMP onto a diphosphate RNA acceptor [18]. Polymerases that were defective in mRNA cap addition terminated transcription prematurely, further underscoring the link between correct 59 mRNA processing and nucleotide polymerization [7]
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