Dynamic RNA structures in the dengue virus genome

Iglesias, Néstor Gabriel; Gamarnik, Andrea V.

doi:10.4161/rna.8.2.14992

Cited by 57 publications

(62 citation statements)

References 96 publications

(144 reference statements)

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“…Additionally, mutagenesis of viral sequences may result in disruption of the various long-range interactions required for establishment of a productive replication complex (8)(9)(10). Thus, strategies to generate infectious viruses expressing heterologous sequences have to contend with both suboptimal growth conditions in Escherichia coli and the complex secondary RNA structural requirements of the viral genome.…”

Section: Engue Virus (Denv) Is An Arthropod-borne Pathogen Of Thementioning

confidence: 99%

Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

Schoggins

Dorner

Feulner

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

167

175

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Dengue virus (DENV) is a global disease threat for which there are no approved antivirals or vaccines. Establishing state-of-the-art screening systems that rely on fluorescent or luminescent reporters may accelerate the development of anti-DENV therapeutics. However, relatively few reporter DENV platforms exist. Here, we show that DENV can be genetically engineered to express a green fluorescent protein or firefly luciferase. Reporter viruses are infectious in vitro and in vivo and are sensitive to antiviral compounds, neutralizing antibodies, and interferons. Bioluminescence imaging was used to follow the dynamics of DENV infection in mice and revealed that the virus localized predominantly to lymphoid and gut-associated tissues. The high-throughput potential of reporter DENV was demonstrated by screening a library of more than 350 IFN-stimulated genes for antiviral activity. Several antiviral effectors were identified, and they targeted DENV at two distinct life cycle steps. These viruses provide a powerful platform for applications ranging from validation of vaccine candidates to antiviral discovery. Flaviviridae family that causes significant morbidity and mortality worldwide. Each year, over 50 million people are affected by dengue fever, and ∼500,000 are hospitalized with the more severe dengue hemorrhagic fever (1). The virus is endemic to tropical environments in Southeast Asia, the Pacific, and the Americas, and has recently reemerged as far north as southern Florida (2). Currently, no vaccines or antivirals have been approved for prevention or treatment of DENV infection.Four genetically and antigenically distinct DENV serotypes circulate, and each can be isolated from infected human sera and propagated in cell culture. The pathogenesis and immune response to patient-derived virus can be studied in vitro and in vivo by quantifying viral genomes or antigens. These detection methods are also used to study the molecular virology of DENV with reagents such as virus-like particles (VLPs) and subgenomic replicons. In some cases, VLPs and subgenomic replicons have been engineered to take advantage of reporter proteins, such as luciferase, which can be used in high-throughput screening platforms for discovery of inhibitors of viral entry or replication (3). A caveat to these tools is the inability to fully recapitulate the entire viral life cycle. This can be overcome by launching virus production from full-length infectious molecular clones, which have been generated for DENV serotypes 1, 2, and 4 (4-6).Full-length flavivirus infectious clones are often difficult to work with, largely due to instability in various bacterial cell lines and the inability to rescue sufficient quantities of DNA for downstream applications (7). Additionally, mutagenesis of viral sequences may result in disruption of the various long-range interactions required for establishment of a productive replication complex (8-10). Thus, strategies to generate infectious viruses expressing heterologous sequences have to contend with both sub...

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Section: Engue Virus (Denv) Is An Arthropod-borne Pathogen Of Thementioning

confidence: 99%

Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

Schoggins

Dorner

Feulner

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

167

175

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“…Considering their location in the viral genomes, the yet best characterized circularization elements were termed as CS, UAR, and DAR, respectively (see Fig. 1; for review, see Iglesias and Gamarnik 2011;Bidet and Garcia-Blanco 2014;Brinton and Basu 2015). Although circularization of the viral genome is essential for the initiation of RNA synthesis, the replication process nevertheless depends on a fine balance of the levels of the circular and linear forms of the flaviviral genome (Villordo et al ′ -terminal region of the core (C) protein coding region are depicted according to the experimental data reported by Dong et al (2008).…”

Section: Introductionmentioning

confidence: 99%

Arginine methylation enhances the RNA chaperone activity of the West Nile virus host factor AUF1 p45

Friedrich

Schmidt

Schierhorn

et al. 2016

RNA

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A prerequisite for the intracellular replication process of the Flavivirus West Nile virus (WNV) is the cyclization of the viral RNA genome, which enables the viral replicase to initiate RNA synthesis. Our earlier studies indicated that the p45 isoform of the cellular AU-rich element binding protein 1 (AUF1) has an RNA chaperone activity, which supports RNA cyclization and viral RNA synthesis by destabilizing a stem structure at the WNV RNA's 3 ′ -end. Here we show that in mammalian cells, AUF1 p45 is consistently modified by arginine methylation of its C terminus. By a combination of different experimental approaches, we can demonstrate that the methyltransferase PRMT1 is necessary and sufficient for AUF1 p45 methylation and that PRMT1 is required for efficient WNV replication. Interestingly, in comparison to the nonmethylated AUF1 p45, the methylated AUF1 p45 aDMA exhibits a significantly increased affinity to the WNV RNA termini. Further data also revealed that the RNA chaperone activity of AUF1 p45 aDMA is improved and the methylated protein stimulates viral RNA synthesis considerably more efficiently than the nonmethylated AUF1 p45. In addition to its destabilizing RNA chaperone activity, we identified an RNA annealing activity of AUF1 p45, which is not affected by methylation. Arginine methylation of AUF1 p45 thus represents a specific determinant of its RNA chaperone activity while functioning as a WNV host factor. Our data suggest that the methylation modifies the conformation of AUF1 p45 and in this way affects its RNA binding and restructuring activities.

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“…The DENV genome is a plus-stranded RNA molecule that contains a single open reading frame flanked by highly structured 5= and 3= untranslated regions (UTRs) (1)(2)(3). RNA elements located within these regions are responsible for translation initiation and genome replication (4)(5)(6)(7). The 5= UTR is about 100 nucleotides (nt) long and includes three different elements: (i) stem-loop A (SLA), which is the promoter for viral polymerase binding and activation (8)(9)(10); (ii) stem-loop B (SLB), which contains a sequence known as 5= upstream of the AUG region (5= UAR) that is complementary to a sequence present at the 3= UTR (3= UAR) and mediates longrange RNA-RNA interactions between the ends of the genome (11); and (iii) a spacer sequence between SLA and SLB rich in U's, which functions as an enhancer of viral replication (10).…”

mentioning

confidence: 99%

Overlapping Local and Long-Range RNA-RNA Interactions Modulate Dengue Virus Genome Cyclization and Replication

Borba

Villordo

Iglesias

et al. 2015

J Virol

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The dengue virus genome is a dynamic molecule that adopts different conformations in the infected cell. Here, using RNA folding predictions, chemical probing analysis, RNA binding assays, and functional studies, we identified new cis-acting elements present in the capsid coding sequence that facilitate cyclization of the viral RNA by hybridization with a sequence involved in a local dumbbell structure at the viral 3= untranslated region (UTR). The identified interaction differentially enhances viral replication in mosquito and mammalian cells. Dengue virus (DENV) is a member of the Flaviviridae family that includes other important pathogens such as yellow fever virus (YFV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). The DENV genome is a plus-stranded RNA molecule that contains a single open reading frame flanked by highly structured 5= and 3= untranslated regions (UTRs) (1-3). RNA elements located within these regions are responsible for translation initiation and genome replication (4-7). The 5= UTR is about 100 nucleotides (nt) long and includes three different elements: (i) stem-loop A (SLA), which is the promoter for viral polymerase binding and activation (8-10); (ii) stem-loop B (SLB), which contains a sequence known as 5= upstream of the AUG region (5= UAR) that is complementary to a sequence present at the 3= UTR (3= UAR) and mediates longrange RNA-RNA interactions between the ends of the genome (11); and (iii) a spacer sequence between SLA and SLB rich in U's, which functions as an enhancer of viral replication (10). The viral 3= UTR is about 450 nucleotides long and comprises four defined domains: domain A1, which features a variable region (VR) (12); domains A2 and A3, which present two almost-identical dumbbell-like secondary structures (DB1 and DB2), which appear to work as enhancers for viral RNA replication (13-15); and domain A4, which contains a small hairpin (sHP) and the 3= stem-loop (3= SL), which are essential elements for viral replication (3,16). In addition to RNA structures defined in the UTRs that play different roles during infection, important RNA elements have been described in the protein coding region. In this regard, essential sequences that mediate long-range RNA-RNA interactions known as 5= cyclization sequence (5= CS) and 5= downstream of AUG region (5= DAR) are located within the capsid coding sequence (11,13,(17)(18)(19)(20)(21). Also, a hairpin known as cHP, located between 5= CS and 5= DAR, has been shown to be necessary for efficient RNA replication (22). The current model for viral RNA synthesis includes the interaction of the viral polymerase NS5 with the 5=-end SLA promoter and its transfer to the 3=-end initiation site by cyclization of the viral genome (9). Despite great advances in knowledge of cis-acting RNA elements in the flavivirus genomes, the molecular details and mechanisms by which many of them function during viral replication are still not well understood.Intrigued by dual roles of RNA sequences in v...

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Dynamic RNA structures in the dengue virus genome

Cited by 57 publications

References 96 publications

Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

Arginine methylation enhances the RNA chaperone activity of the West Nile virus host factor AUF1 p45

Overlapping Local and Long-Range RNA-RNA Interactions Modulate Dengue Virus Genome Cyclization and Replication

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