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RNA Synthesis and Capping by Non-segmented Negative Strand RNA Viral Polymerases: Lessons From a Prototypic Virus.

ABSTRACT: Non-segmented negative strand (NNS) RNA viruses belonging to the order Mononegavirales are highly diversified eukaryotic viruses including significant human pathogens, such as rabies, measles, Nipah, and Ebola. Elucidation of their unique strategies to replicate in eukaryotic cells is crucial to aid in developing anti-NNS RNA viral agents. Over the past 40 years, vesicular stomatitis virus (VSV), closely related to rabies virus, has served as a paradigm to study the fundamental molecular mechanisms of transcription and replication of NNS RNA viruses. These studies provided insights into how NNS RNA viruses synthesize 5'-capped mRNAs using their RNA-dependent RNA polymerase L proteins equipped with an unconventional mRNA capping enzyme, namely GDP polyribonucleotidyltransferase (PRNTase), domain. PRNTase or PRNTase-like domains are evolutionally conserved among L proteins of all known NNS RNA viruses and their related viruses belonging to Jingchuvirales, a newly established order, in the class Monjiviricetes, suggesting that they may have evolved from a common ancestor that acquired the unique capping system to replicate in a primitive eukaryotic host. This article reviews what has been learned from biochemical and structural studies on the VSV RNA biosynthesis machinery, and then focuses on recent advances in our understanding of regulatory and catalytic roles of the PRNTase domain in RNA synthesis and capping.

SUBMITTER: Ogino T

PROVIDER: S-EPMC6636387 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3´ end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming.

Project description:Negative-strand RNA viruses (NSVs) include some of the most pathogenic human viruses known. NSVs completely rely on the host cell for protein translation, but their codon usage bias is often different from that of the host. This discrepancy may have originated from the unique mechanism of NSV RNA synthesis in that the genomic RNA sequestered in the nucleocapsid serves as the template. The stability of the genomic RNA in the nucleocapsid appears to regulate its accessibility to the viral RNA polymerase, thus placing constraints on codon usage to balance viral RNA synthesis. By in situ analyses of vesicular stomatitis virus RNA synthesis, specific activities of viral RNA synthesis were correlated with the genomic RNA sequence. It was found that by simply altering the sequence and not the amino acid that it encoded, a significant reduction, up to an ∼750-fold reduction, in viral RNA transcripts occurred. Through subsequent sequence analysis and thermal shift assays, it was found that the purine/pyrimidine content modulates the overall stability of the polymerase complex, resulting in alteration of the activity of viral RNA synthesis. The codon usage is therefore constrained by the obligation of the NSV genome for viral RNA synthesis.IMPORTANCE Negative-strand RNA viruses (NSVs) include the most pathogenic viruses known. New methods to monitor their evolutionary trends are urgently needed for the development of antivirals and vaccines. The protein translation machinery of the host cell is currently recognized as a main genomic regulator of RNA virus evolution, which works especially well for positive-strand RNA viruses. However, this approach fails for NSVs because it does not consider the unique mechanism of their viral RNA synthesis. For NSVs, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the genome sequestered in the nucleocapsid. Our work suggests a paradigm shift that the interactions between the RNA genome and the nucleocapsid protein regulate the activity of vRdRp, which selects codon usage.

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RNA Synthesis and Capping by Non-segmented Negative Strand RNA Viral Polymerases: Lessons From a Prototypic Virus.

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