sfRNA Production Research Articles

RNase L-induced bodies sequester subgenomic flavivirus RNAs to promote viral RNA decay

Subgenomic flavivirus RNAs (sfRNAs) are structured RNAs encoded by flaviviruses that promote viral infection by inhibiting cellular RNA decay machinery. Herein, we analyze sfRNA production and localization using single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) throughout West Nile virus, Zika virus, or dengue virus serotype 2 infection. We observe that sfRNAs are generated during the RNA replication phase of viral infection in the cytosol and accumulate in processing bodies (P-bodies), which contain RNA decay machinery such as XRN1 and Dcp1b. However, upon activation of the host antiviral endoribonuclease, ribonuclease L (RNase L), sfRNAs re-localize to ribonucleoprotein complexes known as RNase L-induced bodies (RLBs). RLB-mediated sequestration of sfRNAs reduces sfRNA association with RNA decay machinery inP-bodies, which coincides with increased viral RNA decay. These findings establish a functional role forRLBs in enhancing the cell-mediated decay of viral RNA by sequestering functional viral RNA decay products.

Zika virus noncoding sfRNAs sequester multiple host-derived RNA-binding proteins and modulate mRNA decay and splicing during infection

Insect-borne flaviviruses produce a 300-500-base long noncoding RNA, termed subgenomic flavivirus RNA (sfRNA), by stalling the cellular 5'-3'-exoribonuclease 1 (XRN1) via structures located in their 3' UTRs. In this study, we demonstrate that sfRNA production by Zika virus represses XRN1 analogous to what we have previously shown for other flaviviruses. Using protein-RNA reconstitution and a stringent RNA pulldown assay with human choriocarcinoma (JAR) cells, we demonstrate that the sfRNAs from both dengue type 2 and Zika viruses interact with a common set of 21 RNA-binding proteins that contribute to the regulation of post-transcriptional processes in the cell, including splicing, RNA stability, and translation. We found that four of these sfRNA-interacting host proteins, DEAD-box helicase 6 (DDX6) and enhancer of mRNA decapping 3 (EDC3) (two RNA decay factors), phosphorylated adaptor for RNA export (a regulator of the biogenesis of the splicing machinery), and apolipoprotein B mRNA-editing enzyme catalytic subunit 3C (APOBEC3C, a nucleic acid-editing deaminase), inherently restrict Zika virus infection. Furthermore, we demonstrate that the regulations of cellular mRNA decay and RNA splicing are compromised by Zika virus infection as well as by sfRNA alone. Collectively, these results reveal the large extent to which Zika virus-derived sfRNAs interact with cellular RNA-binding proteins and highlight the potential for widespread dysregulation of post-transcriptional control that likely limits the effective response of these cells to viral infection.

Fragile X mental retardation protein is a Zika virus restriction factor that is antagonized by subgenomic flaviviral RNA

Subgenomic flaviviral RNA (sfRNA) accumulates during infection due to incomplete degradation of viral genomes and interacts with cellular proteins to promote infection. Here we identify host proteins that bind the Zika virus (ZIKV) sfRNA. We identified fragile X mental retardation protein (FMRP) as a ZIKV sfRNA-binding protein and confirmed this interaction in cultured cells and mouse testes. Depletion of FMRP elevated viral translation and enhanced ZIKV infection, indicating that FMRP is a ZIKV restriction factor. We further observed that an attenuated ZIKV strain compromised for sfRNA production was disproportionately stimulated by FMRP knockdown, suggesting that ZIKV sfRNA antagonizes FMRP activity. Importantly, ZIKV infection and expression of ZIKV sfRNA upregulated endogenous FMRP target genes in cell culture and ZIKV-infected mice. Together, our observations identify FMRP as a ZIKV restriction factor whose activity is antagonized by the sfRNA. Interaction between ZIKV and FMRP has significant implications for the pathogenesis of ZIKV infections.

Homologous RNA secondary structure duplications in 3′ untranslated region influence subgenomic RNA production and replication of dengue virus

Subgenomic flavivirus RNA (sfRNA) is essential for flavivirus-induced cytopathicity. sfRNA-deficient mutant dengue virus (DENV) M8 harboring a deleted domain II in the 3′ untranslated region (UTR) has severely decreased cytopathicity and replication efficiency. Here, recovered viruses acquired from M8 by serial passages replicated similarly to the wild type (WT) but produced two types of sfRNA, whereas only one was detected in WT infected cells, resulting in mildly decreased cytopathicity. Sequencing and structure prediction showed that recovered viruses acquired a novel additional partial domain I sequence duplication and formed analogous RNA secondary structures. This was the first experimental evidence verifying that a longer precursor may exist in the 3′ UTR of DENV. Additionally, the duplicated structures could be compensated by the homologous structures from other flaviviruses, rather than heterologous viruses, implying that homologous secondary structures or higher tertiary structures played more important roles in sfRNA production and viral replication.

Flaviviruses Produce a Subgenomic Flaviviral RNA That Enhances Mosquito Transmission.

Mosquito-borne flaviviruses (MBFVs) are a global public health burden. MBFVs have several unique 3'UTR structures that inhibit the host RNA decay machinery to produce subgenomic flaviviral RNAs (sfRNAs). Number of sfRNA species and their relative quantities are dependent on the 3'UTR tertiary structures and can vary between tissues. Two recent in vivo studies demonstrated that sfRNA enhances mosquito transmission, resulting in increased infection rate of saliva. Transmission efficiency is determined by the immune response. First evidence points to sfRNA interference with the Toll and RNAi immune pathways. However, a more complex picture that includes flexibility in sfRNA production and interaction with immune-related proteins remains to be explored.

Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes.

Understanding the flavivirus transmission cycle is important to identify novel targets to interfere with disease and to aid development of virus control strategies. Flaviviruses produce an abundant noncoding viral RNA called sfRNA in both arthropod and mammalian cells. To evaluate the role of sfRNA in flavivirus transmission, we infected mosquitoes with the flavivirus West Nile virus and an sfRNA-deficient mutant West Nile virus. We demonstrate that sfRNA determines the infection and transmission rates of West Nile virus in Culex pipiens mosquitoes. Comparison of infection via the blood meal versus intrathoracic injection, which bypasses the midgut, revealed that sfRNA is important to overcome the mosquito midgut barrier. We also show that sfRNA is processed by the antiviral RNA interference machinery in mosquitoes. This is the first report to describe a pivotal biological function of sfRNA in arthropods. The results explain why sfRNA production is evolutionarily conserved.

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

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.

New hypotheses derived from the structure of a flaviviral Xrn1-resistant RNA: Conservation, folding, and host adaptation

Arthropod-borne flaviviruses (FVs) are a growing world-wide health threat whose incidence and range are increasing. The pathogenicity and cytopathicity of these single-stranded RNA viruses are influenced by viral subgenomic non-protein-coding RNAs (sfRNAs) that the viruses produce to high levels during infection. To generate sfRNAs the virus co-opts the action of the abundant cellular exonuclease Xrn1, which is part of the cell's normal RNA turnover machinery. This exploitation of the cellular machinery is enabled by discrete, highly structured, Xrn1-resistant RNA elements (xrRNAs) in the 3′UTR that interact with Xrn1 to halt processive 5′ to 3′ decay of the viral genomic RNA. We recently solved the crystal structure of a functional xrRNA, revealing a novel fold that provides a mechanistic model for Xrn1 resistance. Continued analysis and interpretation of the structure reveals that the tertiary contacts that knit the xrRNA fold together are shared by a wide variety of arthropod-borne FVs, conferring robust Xrn1 resistance in all tested. However, there is some variability in the structures that correlates with unexplained patterns in the viral 3′ UTRs. Finally, examination of these structures and their behavior in the context of viral infection leads to a new hypothesis linking RNA tertiary structure, overall 3′ UTR architecture, sfRNA production, and host adaptation.

Functional non-coding RNAs derived from the flavivirus 3′ untranslated region

Flaviviruses are single-stranded positive sense RNA enveloped viruses. The flavivirus genus includes important human pathogens such as dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), and Murray Valley encephalitis virus (MVEV). In addition to the viral proteins and viral genomic RNA, flaviviruses produce at least two functional non-coding RNAs derived from the 3′ untranslated region (3′UTR), the subgenomic flavivirus RNA (sfRNA) and a putative WNV miRNA (KUN-miR-1). In this review we summarize published data from studies with WNV, YFV, DENV, JEV, and MVEV on sfRNA production following incomplete degradation of the viral genomic RNA by the cellular 5′–3′ exoribonuclease 1 (XRN1), RNA structural elements involved in stalling XRN1 to generate sfRNA, and functions of sfRNA in modulating cellular mRNA decay and RNAi pathways as well as in modulating anti-viral type I interferon response. In addition, we also summarize data on the mechanisms of biogenesis of 3′UTR-derived KUN-miR-1 and its function in WNV replication in mosquito host, along with recent findings on a discovery of a second potential flaviviral miRNA vsRNA5, derived from the 3′UTR of DENV. This review thus summarizes the known mechanisms of generation and the functions of flaviviral 3′UTR-derived non-coding RNAs.

RNA structures that resist degradation by Xrn1 produce a pathogenic Dengue virus RNA

Dengue virus is a growing global health threat. Dengue and other flaviviruses commandeer the host cell's RNA degradation machinery to generate the small flaviviral RNA (sfRNA), a noncoding RNA that induces cytopathicity and pathogenesis. Host cell exonuclease Xrn1 likely loads on the 5' end of viral genomic RNA and degrades processively through ∼10 kB of RNA, halting near the 3' end of the viral RNA. The surviving RNA is the sfRNA. We interrogated the architecture of the complete Dengue 2 sfRNA, identifying five independently-folded RNA structures, two of which quantitatively confer Xrn1 resistance. We developed an assay for real-time monitoring of Xrn1 resistance that we used with mutagenesis and RNA folding experiments to show that Xrn1-resistant RNAs adopt a specific fold organized around a three-way junction. Disrupting the junction's fold eliminates the buildup of disease-related sfRNAs in human cells infected with a flavivirus, directly linking RNA structure to sfRNA production. DOI: http://dx.doi.org/10.7554/eLife.01892.001.

West Nile Virus Noncoding Subgenomic RNA Contributes to Viral Evasion of the Type I Interferon-Mediated Antiviral Response

We previously showed that a noncoding subgenomic flavivirus RNA (sfRNA) is required for viral pathogenicity, as a mutant West Nile virus (WNV) deficient in sfRNA production replicated poorly in wild-type mice. To investigate the possible immunomodulatory or immune evasive functions of sfRNA, we utilized mice and cells deficient in elements of the type I interferon (IFN) response. Replication of the sfRNA mutant WNV was rescued in mice and cells lacking interferon regulatory factor 3 (IRF-3) and IRF-7 and in mice lacking the type I alpha/beta interferon receptor (IFNAR), suggesting a contribution for sfRNA in overcoming the antiviral response mediated by type I IFN. This was confirmed by demonstrating rescue of mutant virus replication in the presence of IFNAR neutralizing antibodies, greater sensitivity of mutant virus replication to IFN-α pretreatment, partial rescue of its infectivity in cells deficient in RNase L, and direct effects of transfected sfRNA on rescuing replication of unrelated Semliki Forest virus in cells pretreated with IFN-α. The results define a novel function of sfRNA in flavivirus pathogenesis via its contribution to viral evasion of the type I interferon response.

An RNA Pseudoknot Is Required for Production of Yellow Fever Virus Subgenomic RNA by the Host Nuclease XRN1

Cells and mice infected with arthropod-borne flaviviruses produce a small subgenomic RNA that is colinear with the distal part of the viral 3'-untranslated region (UTR). This small subgenomic flavivirus RNA (sfRNA) results from the incomplete degradation of the viral genome by the host 5'-3' exonuclease XRN1. Production of the sfRNA is important for the pathogenicity of the virus. This study not only presents a detailed description of the yellow fever virus (YFV) sfRNA but, more importantly, describes for the first time the molecular characteristics of the stalling site for XRN1 in the flavivirus genome. Similar to the case for West Nile virus, the YFV sfRNA was produced by XRN1. However, in contrast to the case for other arthropod-borne flaviviruses, not one but two sfRNAs were detected in YFV-infected mammalian cells. The smaller of these two sfRNAs was not observed in infected mosquito cells. The larger sfRNA could also be produced in vitro by incubation with purified XRN1. These two YFV sfRNAs formed a 5'-nested set. The 5' ends of the YFV sfRNAs were found to be just upstream of the previously predicted RNA pseudoknot PSK3. RNA structure probing and mutagenesis studies provided strong evidence that this pseudoknot structure was formed and served as the molecular signal to stall XRN1. The sequence involved in PSK3 formation was cloned into the Sinrep5 expression vector and shown to direct the production of an sfRNA-like RNA. These results underscore the importance of the RNA pseudoknot in stalling XRN1 and also demonstrate that it is the sole viral requirement for sfRNA production.