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A glance at subgenomic flavivirus RNAs and microRNAs in flavivirus infections.

ABSTRACT: The family Flaviviridae comprises a wide variety of viruses that are distributed worldwide, some of which are associated with high rates of morbidity and mortality. There are neither vaccines nor antivirals for most flavivirus infections, reinforcing the importance of research on different aspects of the viral life cycle. During infection, cytoplasmic accumulation of RNA fragments mainly originating from the 3' UTRs, which have been designated subgenomic flavivirus RNAs (sfRNAs), has been detected. It has been shown that eukaryotic exoribonucleases are involved in viral sfRNA production. Additionally, viral and human small RNAs (sRNAs) have also been found in flavivirus-infected cells, especially microRNAs (miRNAs). miRNAs were first described in eukaryotic cells and in a mature and functional state present as single-stranded 18-24 nt RNA fragments. Their main function is the repression of translation through base pairing with cellular mRNAs, besides other functions, such as mRNA degradation. Canonical miRNA biogenesis involves Drosha and Dicer, however miRNA can also be generated by alternative pathways. In the case of flaviviruses, alternative pathways have been suggested. Both sfRNAs and miRNAs are involved in viral infection and host cell response modulation, representing interesting targets of antiviral strategies. In this review, we focus on the generation and function of viral sfRNAs, sRNAs and miRNAs in West Nile, dengue, Japanese encephalitis, Murray Valley encephalitis and yellow fever infections, as well as their roles in viral replication, translation and cell immune response evasion. We also give an overview regarding other flaviviruses and the generation of cellular miRNAs during infection.

SUBMITTER: Bavia L

PROVIDER: S-EPMC4884392 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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A glance at subgenomic flavivirus RNAs and microRNAs in flavivirus infections.

Bavia Lorena L Mosimann Ana Luiza Pamplona AL Aoki Mateus Nóbrega MN Duarte Dos Santos Claudia Nunes CN

Virology journal 20160528

The family Flaviviridae comprises a wide variety of viruses that are distributed worldwide, some of which are associated with high rates of morbidity and mortality. There are neither vaccines nor antivirals for most flavivirus infections, reinforcing the importance of research on different aspects of the viral life cycle. During infection, cytoplasmic accumulation of RNA fragments mainly originating from the 3' UTRs, which have been designated subgenomic flavivirus RNAs (sfRNAs), has been detect ...[more]

PMID: 27233361

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Project description:Many viruses produce protein-coding and noncoding subgenomic RNAs (sgRNAs) that are critical for infection. A recently discovered pathway for viral sgRNA production uses exoribonuclease-resistant RNAs (xrRNAs), discrete folded RNA elements that block the processive exoribonucleolytic degradation of RNA. xrRNAs are widespread in animal-infecting flaviviruses but had been found only in three members of the plant virus genus Dianthovirus Also, xrRNAs had been found only in the 3' untranslated regions (3'UTRs) of viral RNAs, where they produce noncoding sgRNAs. The degree to which xrRNA elements exist in other viruses, the conservation of their ring-like fold, and the ability of xrRNAs to operate in diverse contexts were unknown. Using computational tools and biochemical assays, we discovered xrRNA elements pervading two large families of plant-infecting RNA viruses, demonstrating their importance and widespread utility. Comparison of the sequences and functional requirements suggests that all adopt the characteristic ring-like fold. Unexpectedly, many of these newly discovered xrRNAs are located in intergenic regions rather than 3´UTRs, and some are associated with the 5' ends of subgenomic RNAs that encode viral proteins. This suggests that xrRNAs are involved in the production of both coding and noncoding subgenomic RNAs and can operate as part of broader mechanisms to regulate RNA levels and protein expression. These discoveries expand the potential roles for xrRNAs and suggest that xrRNAs may represent a more general strategy for RNA maturation and maintenance than previously known.IMPORTANCE During infection, viruses often produce subgenomic RNAs (sgRNAs) that either serve as the template for protein synthesis or act as "riboregulators" that interact with and influence the viral and cellular machinery. Recently, a mechanism for producing sgRNAs was found that depends on the presence of specifically structured RNA elements (xrRNAs). However, the degree to which this mechanism is used, where the elements are found, their structural diversity, and what types of sgRNAs are produced by this pathway were unclear. This article describes the discovery of these structured RNA elements in two large families of plant viruses and shows that they are used to produce both protein-coding sgRNAs and "riboregulatory" RNAs. These discoveries provide evidence that xrRNA-based RNA maturation pathways may be more widespread than previously anticipated and that they are involved in producing a variety of RNAs of diverse functions.

Project description:SARS-CoV-2 transcribes a set of subgenomic RNAs (sgRNAs) essential for the translation of structural and accessory proteins to sustain its life cycle. We applied RNA-seq on 375 respiratory samples from individual COVID-19 patients and revealed that the majority of the sgRNAs were canonical transcripts with N being the most abundant (36.2%), followed by S (11.6%), open reading frame 7a (ORF7a; 10.3%), M (8.4%), ORF3a (7.9%), ORF8 (6.0%), E (4.6%), ORF6 (2.5%), and ORF7b (0.3%); but ORF10 was not detected. The profile of most sgRNAs, except N, showed an independent association with viral load, time of specimen collection after onset, age of the patient, and S-614D/G variant with ORF7b and then ORF6 being the most sensitive to changes in these characteristics. Monitoring of 124 serial samples from 10 patients using sgRNA-specific real-time RT-PCR revealed a potential of adopting sgRNA as a marker of viral activity. Respiratory samples harboring a full set of canonical sgRNAs were mainly collected early within 1 to 2 weeks from onset, and most of the stool samples (90%) were negative for sgRNAs despite testing positive by diagnostic PCR targeting genomic RNA. ORF7b was the first to become undetectable and again being the most sensitive surrogate marker for a full set of canonical sgRNAs in clinical samples. The potential of using sgRNA to monitor viral activity and progression of SARS-CoV-2 infection, and hence as one of the objective indicators to triage patients for isolation and treatment should be considered. IMPORTANCE Attempts to use subgenomic RNAs (sgRNAs) of SARS-CoV-2 to identify active infection of COVID-19 have produced diverse results. In this work, we applied next-generation sequencing and RT-PCR to profile the full spectrum of SARS-CoV-2 sgRNAs in a large cohort of respiratory and stool samples collected throughout infection. Numerous known and novel discontinuous transcription events potentially encoding full-length, deleted and frameshift proteins were observed. In particular, the expression profile of canonical sgRNAs was associated with genomic RNA level and clinical characteristics. Our study found sgRNAs as potential biomarkers for monitoring infectivity and progression of SARS-CoV-2 infection, which provides an alternative target for the management and treatment of COVID-19 patients.

Project description:BackgroundSARS-CoV-2, a positive-sense RNA virus in the family Coronaviridae, has caused a worldwide pandemic of coronavirus disease 2019 or COVID-19. Coronaviruses generate a tiered series of subgenomic RNAs (sgRNAs) through a process involving homology between transcriptional regulatory sequences (TRS) located after the leader sequence in the 5' UTR (the TRS-L) and TRS located near the start of ORFs encoding structural and accessory proteins (TRS-B) near the 3' end of the genome. In addition to the canonical sgRNAs generated by SARS-CoV-2, non-canonical sgRNAs (nc-sgRNAs) have been reported. However, the consistency of these nc-sgRNAs across viral isolates and infection conditions is unknown. The comprehensive definition of SARS-CoV-2 RNA products is a key step in understanding SARS-CoV-2 pathogenesis.MethodsHere, we report an integrative analysis of eight independent SARS-CoV-2 transcriptomes generated using three sequencing strategies, five host systems, and seven viral isolates. Read-mapping to the SARS-CoV-2 genome was used to determine the 5' and 3' coordinates of all junctions in viral RNAs identified in these samples.ResultsUsing junctional abundances, we show nc-sgRNAs make up as much as 33% of total sgRNAs in cell culture models of infection, are largely consistent in abundance across independent transcriptomes, and increase in abundance over time during infection. By assessing the homology between sequences flanking the 5' and 3' junction points, we show that nc-sgRNAs are not associated with TRS-like homology. By incorporating read coverage information, we find strong evidence for subgenomic RNAs that contain only 5' regions of ORF1a. Finally, we show that non-canonical junctions change the landscape of viral open reading frames.ConclusionsWe identify canonical and non-canonical junctions in SARS-CoV-2 sgRNAs and show that these RNA products are consistently generated by many independent viral isolates and sequencing approaches. These analyses highlight the diverse transcriptional activity of SARS-CoV-2 and offer important insights into SARS-CoV-2 biology.

Dataset Information

A glance at subgenomic flavivirus RNAs and microRNAs in flavivirus infections.

Publications

A glance at subgenomic flavivirus RNAs and microRNAs in flavivirus infections.

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