Abstract
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.
Highlights
Flaviviruses (FVs) are single-stranded, (+)-sense RNA viruses that include West Nile (WNV), Yellow Fever (YFV), Japanese Encephalitis (JEV) and other human disease-causing viruses (Fields et al, 2013)
We probed the structure of these RNAs using dimethyl sulfate (DMS) and N-methylisatoic anhydride (NMIA, SHAPE chemistry) (Tijerina et al, 2007; Weeks, 2010; McGinnis et al, 2012), and used the resultant data to produce an experimentally-supported secondary structure of the complete 3′untranslated regions (UTRs) (Figure 2A,B)
During infections caused by FV’s, the formation of small flaviviral RNA (sfRNA) through incomplete degradation of the viral genome is directly linked to disease (Pijlman et al, 2008; Liu et al, 2014)
Summary
Flaviviruses (FVs) are single-stranded, (+)-sense RNA viruses that include West Nile (WNV), Yellow Fever (YFV), Japanese Encephalitis (JEV) and other human disease-causing viruses (Fields et al, 2013). DENV infection can lead to severe hemorrhagic fever, dengue shock syndrome, and death (Simmons and Farrar, 2012; The World Health Organization, 2014; United States Centers for Disease Control and Prevention, 2014). Translation of the FV ORF produces a single polypeptide that is processed by both viral and cellular proteases to yield ten viral proteins (Fields et al, 2013) Both FV UTRs fulfill critical roles in the viral lifecycle, (Iglesias and Gamarnik, 2011) including forming base pairing interactions during (−) strand synthesis of the viral RNA (Filomatori et al, 2006; Villordo and Gamarnik, 2009; Friebe and Harris, 2010; Villordo et al, 2010; Gebhard et al, 2011)
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