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

Abstract

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.The Flavivirus genus contains nearly 80 viruses distributed worldwide and includes important human pathogens such as dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and tickborne encephalitis virus (TBEV). Phylogenetic analysis clustered flaviviruses into the following three major groups, based on the vector of transmission: (i) mosquito-borne viruses, (ii) tick-borne viruses, and (iii) viruses with no known vector (NKV) (13,26).Flaviviruses are small enveloped viruses containing a positive-sense single-stranded RNA genome of approximately 11 kb in length, with a 5Ј cap structure and a 3Ј nonpolyadenylated terminus. The genomic RNA is flanked by 5Ј-and 3Ј-untranslated regions (UTRs) and encodes a single polyprotein that is co-and posttranslationally processed by viral and cellular proteases into three structural proteins (C, prM, and E) and seven nonstructural proteins (NSs) (reviewed in reference 30). Apart from the viral genomic RNA and the replicationrelated replicative-form and intermediate RNAs (11,12), an additional small flavivirus RNA (sfRNA) has been detected in mice and both mammalian and insect cells infected with flaviviruses belonging to the JEV serogroup (29,49,56). Recently, it was shown that production of sfRNA is not unique to JEV and closely related viruses but that all arthropod-borne flaviviruses generate an sfRNA upon infection of mammalian cells (31,44). The ...

A noncoding RNA produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease XRN1 and alters host mRNA stability

“…Formation of sfRNA by the cellular XRN1 exoribonuclease represses enzymatic activity Previous work using sh/siRNA-mediated Xrn1 knockdown in infected cells or incubation with a recombinant Xrn1 protein derived from yeast have strongly implicated a role for the cellular exoribonuclease in the formation of sfRNA from West Nile virus and Yellow Fever virus 39 UTRs (Pijlman et al 2008;Silva et al 2010). To extend these observations, we inserted the 39 UTR of Dengue virus type 2 (DenV2) into a reporter construct and prepared in vitrotranscribed RNAs bearing a 59 monophosphate so that they would be effective substrates for XRN1.…”

“…Based on previous work (Pijlman et al 2008;Funk et al 2010;Silva et al 2010) and our observations for Dengue and West Nile viruses reported here, the ability to generate sfRNA and repress XRN1 is likely to be conserved throughout the arthropod-borne flaviviruses. Since the formation of sfRNA inactivates XRN1 in both mosquito and human cell extracts (Figs.…”

“…RNAi-mediated knockdowns and applications of a recombinant yeast enzyme suggest that sfRNA is generated as a viral genomic RNA decay product by the action of the XRN1 exoribonuclease (Pijlman et al 2008). XRN1 appears to stall at a set of conserved pseudoknot-like structures at the 59 side of the flavivirus 39 UTR (Funk et al 2010;Silva et al 2010). Importantly, the ability of West Nile virus to generate sfRNA has been linked to cytopathology in infected cells and pathogenicity in mice (Pijlman et al 2008;Funk et al 2010;Schuessler et al 2012).…”

Learning from the Enemy: Innate RNA Interference Renders Mosquitos Asymptomatic to Arboviral Infection and Provides Researchers with New Approaches to Subdue its Effects

“…Silva et al detected the presence of an "RNA pseudoknot", (closely resembling a stress granule in form and function) in vitro upon 1) simulating stress conditions and 2) exposing the cell to yellow fever flaviviral RNA [20]. Although much research remains to be done in this area [19], studies in Drosophila suggest that they are active in arthropods and their conserved nature suggests that a detectable marker is plausible.…”

“…First, as they appear when an organism is under stress, their presence is generally indicative of a infected cell. Furthermore, they localize infectious molecules into a single cluster; infection-specific proteins and nucleotides tend to be bind TIA-1 and TIAR epitopes on the outside, allowing for easy detection and classification of the infection [20].…”