Abstract

The unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase) domain of the vesicular stomatitis virus (VSV) L protein possesses a dual-functional "priming-capping loop" that governs terminal de novo initiation for leader RNA synthesis and capping of monocistronic mRNAs during the unique stop-start transcription cycle. Here, we investigated the roles of basic amino acid residues on a helix structure directly connected to the priming-capping loop in viral RNA synthesis and identified single point mutations that cause previously unreported defective phenotypes at different steps of stop-start transcription. Mutations of residue R1183 (R1183A and R1183K) dramatically reduced the leader RNA synthesis activity by hampering early elongation, but not terminal de novo initiation or productive elongation, suggesting that the mutations negatively affect escape from the leader promoter. On the other hand, mutations of residue R1178 (R1178A and R1178K) decreased the efficiency of polyadenylation-coupled termination of mRNA synthesis at the gene junctions, but not termination of leader RNA synthesis at the leader-to-N-gene junction, resulting in the generation of larger amounts of aberrant polycistronic mRNAs. In contrast, both the R1183 and R1178 residues are not essential for cap-forming activities. The R1183K mutation was lethal to VSV, whereas the R1178K mutation attenuated VSV and triggered the production of the polycistronic mRNAs in infected cells. These observations suggest that the PRNTase domain plays multiple roles in conducting accurate stop-start transcription beyond its known role in pre-mRNA capping.

Highlights

  • Vesicular stomatitis virus (VSV), a non-segmented negative strand (NNS) RNA virus belonging to the Rhabdoviridae family in the order Mononegavirales, has been used as a prototypic virus to study the molecular mechanisms of transcription and replication in this order of viruses (Reviewed in [1])

  • Vesicular stomatitis virus (VSV), an animal rhabdovirus closely related to rabies virus, has served as a paradigm for understanding the basic molecular mechanisms of transcription

  • We biochemically demonstrated that the loop of the VSV PRNTase domain governs terminal de novo initiation to synthesize leader RNA (LeRNA) and pre-mRNA capping [29]

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Summary

Introduction

Vesicular stomatitis virus (VSV), a non-segmented negative strand (NNS) RNA virus belonging to the Rhabdoviridae family in the order Mononegavirales, has been used as a prototypic virus to study the molecular mechanisms of transcription and replication in this order of viruses (Reviewed in [1]). The VSV RNA-dependent RNA polymerase (RdRp) complex comprising the large (L) protein and its co-factor phospho- (P) protein enters from the 30-end of the genomic RNA template encapsidated with the nucleocapsid (N) protein (called N-RNA template) to synthesize the uncapped leader RNA (LeRNA) with ~47 nucleotides (nt) [2,3,4,5]. After synthesis of LeRNA, the RdRp sequentially transcribes five internal genes (N, P, M, G, and L) into monocistronic mRNAs with a 50-cap 1 structure [m7G(50)ppp (50)Am-, m7G, N7-methylguanosine; Am, 20-O-methyladenosine] and 30-poly(A) tail via a stop-start transcription mechanism [4,6,7,8]. During replication, these signals for mRNA synthesis on the genome are ignored by the RdRp to synthesize a full-length anti-genome, which is known to be co-replicationally encapsidated with the N proteins [12,13,14,15]

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