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Determination of the vRNA and cRNA promoter activity by M segment-specific non-coding nucleotides of influenza A virus.

ABSTRACT: Eight-segmented, negative-sense, single-stranded genomic RNAs of influenza A virus are terminated with 5' and 3' untranslated regions (UTRs). All segments have highly conserved extremities of 13 and 12 nucleotides at the 5' and 3' UTRs, respectively, constructing the viral RNA (vRNA) promoter. Adjacent to the duplex stem of 3 base pairs (bps) between the two conserved strands, additional 1-4 bps are existing in a segment-specific manner. We investigated the roles of the matrix (M) segment-specific base pair between the 14^th nucleotide uridine (U14') of the 5' UTR and the 13^th nucleotide adenosine (A13) of the 3' UTR by preparing possible vRNA promoters, named vXY, as well as cRNA promoters, named cYX. We analysed their RNA-dependent RNA replication efficiency using the minigenome replicon system and an enzyme assay system in vitro with synthetic RNA promoters. Notably, in contrast to vAC(s) that is a synthetic vRNA promoter with A14' and C13, base-pair disruption at the complementary RNA (cRNA) promoter in cAC(s), which has A13' and C14, not only reduced viral RNA replication in cells but also impaired de novo initiation of unprimed vRNA synthesis. Reverse genetics experiments confirmatively exhibited that this breakage in the cRNA promoter affected the rescue of infectious virus. The present study suggests that the first segment-specific base pair plays an essential role in generating infectious viruses by regulating the promoter activity of cRNA rather than vRNA. It could provide insights into the role of the segment-specific nucleotides in viral genome replication for sustainable infection.

SUBMITTER: Shin H

PROVIDER: S-EPMC8078515 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Influenza A virus (IAV) is a segmented negative-sense RNA virus and is the cause of major epidemics and pandemics. The replication of IAV is complex, involving the production of three distinct RNA species; mRNA, cRNA, and vRNA for all eight genome segments. While understanding IAV replication kinetics is important for drug development and improving vaccine production, current methods for studying IAV kinetics has been limited by the ability to detect all three different RNA species in a scalable manner. Here we report the development of a novel pipeline using total stranded RNA-Seq, which we named Influenza Virus Enumerator of RNA Transcripts (InVERT), that allows for the simultaneous quantification of all three RNA species produced by IAV. Using InVERT, we provide a full landscape of the IAV replication kinetics and found that different groups of viral genes follow different kinetics. The segments coding for RNA-dependent RNA Polymerase (RdRP) produced more vRNA than mRNA while some other segments (NP, NS, HA) consistently made more mRNA than vRNA. vRNA expression levels did not correlate with cRNA expression, suggesting complex regulation of vRNA synthesis. Furthermore, by studying the kinetics of a virus lacking the capacity to generate new polymerase complexes, we found evidence that further supports the model that cRNA synthesis requires newly synthesized RdRP and that incoming RdRP can only generate mRNA. Overall, InVERT is a powerful tool for quantifying IAV RNA species to elucidate key features of IAV replication.ImportanceInfluenza A virus (IAV) is a respiratory pathogen that has caused significant mortality throughout history and remains a global threat to human health. Although much is known about IAV replication, the regulation of IAV replication dynamics is not completely understood. This is due in part to both technical limitations and the complexity of the virus replication, which has a segmented genome and produces three distinct RNA species for each gene segment. We developed a new approach that allows the methodical study of IAV replication kinetics, shedding light on many interesting features of IAV replication biology. This study advances our understanding of the kinetics of IAV replication and will help to facilitate future research in the field.

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Determination of the vRNA and cRNA promoter activity by M segment-specific non-coding nucleotides of influenza A virus.

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