The 5′ and 3′ Downstream AUG Region Elements Are Required for Mosquito-Borne Flavivirus RNA Replication

Friebe, Peter; Shi, Pei Yong; Harris, Eva

doi:10.1128/jvi.02037-10

Cited by 69 publications

(76 citation statements)

References 22 publications

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“…Also in the related members of the Flaviviridae family, which include West Nile virus (WNV), yellow fever virus (YFV) and dengue virus (DENV), such a genome circularization has been described to be essential for RNA replication (You et al 2001;Corver et al 2003;Alvarez et al 2005;Zhang et al 2008;Friebe et al 2011). Moreover, in the case of DENV this RNA circularization has been observed by atomic force microscopy in the absence of viral or cellular proteins, demonstrating that such a RNA circularization can be formed solely by RNA base-pairing (Alvarez et al 2005).…”

Section: Possible Circularization Of Hcvmentioning

confidence: 99%

Conserved RNA secondary structures and long-range interactions in hepatitis C viruses

Fricke

Dünnes

Zayas

et al. 2015

RNA

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Hepatitis C virus (HCV) is a hepatotropic virus with a plus-strand RNA genome of ∼9.600 nt. Due to error-prone replication by its RNA-dependent RNA polymerase (RdRp) residing in nonstructural protein 5B (NS5B), HCV isolates are grouped into seven genotypes with several subtypes. By using whole-genome sequences of 106 HCV isolates and secondary structure alignments of the plus-strand genome and its minus-strand replication intermediate, we established refined secondary structures of the 5 ′ untranslated region (UTR), the cis-acting replication element (CRE) in NS5B, and the 3 ′ UTR. We propose an alternative structure in the 5 ′ UTR, conserved secondary structures of 5B stem-loop (SL)1 and 5BSL2, and four possible structures of the X-tail at the very 3 ′ end of the HCV genome. We predict several previously unknown long-range interactions, most importantly a possible circularization interaction between distinct elements in the 5 ′ and 3 ′ UTR, reminiscent of the cyclization elements of the related flaviviruses. Based on analogy to these viruses, we propose that the 5 ′ -3 ′ UTR base-pairing in the HCV genome might play an important role in viral RNA replication. These results may have important implications for our understanding of the nature of the cis-acting RNA elements in the HCV genome and their possible role in regulating the mutually exclusive processes of viral RNA translation and replication.

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Section: Possible Circularization Of Hcvmentioning

confidence: 99%

Conserved RNA secondary structures and long-range interactions in hepatitis C viruses

Fricke

Dünnes

Zayas

et al. 2015

RNA

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“…cis-Acting RNA elements are involved not only in viral replication (35,36,41,(44)(45)(46)54) and translation (55,56) of flaviviruses but also in host adaption (57), neurovirulence (58), and pathogenicity (59)(60)(61). Using the ZIKV replicon and the newly developed infectious clone of ZIKV, we have shown that 5=-SLA and 5=-3= CS interaction are indispensable for ZIKV replication.…”

Section: Discussionmentioning

confidence: 99%

“…Flavivirus vRNA replication requires the participation of various cis-acting RNA elements in genomic 5= and 3= ends. The 5= stem-loop A (SLA) structure binds to viral NS5, and the latter is translocated to the 3= end to initiate minus-strand RNA synthesis though 5=-3= terminal interactions (35), which include the 5=-3= upstream AUG region (UAR), 5=-3= downstream AUG region (DAR) and 5=-3= cyclization sequence (CS) base-pairing interactions (38,(41)(42)(43)(44). The dynamic modulation of viral NS5 recruitment to the 5= end by the 5=-UAR-flanking stem (UFS) is also critical for efficient vRNA replication (45).…”

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confidence: 99%

Characterization of cis -Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone

Liu

Huang

et al. 2017

J Virol

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Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis-acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5=-SLA promoter and 5=-3= cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses.IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable infectious clone of a 2016 ZIKV strain using a novel self-splicing ribozyme-based technology that abolished the potential toxicity of ZIKV cDNA clones to the E. coli host. Moreover, two crucial cis-acting replication elements (5=-SLA and 5=-CS) of ZIKV were first identified using this novel reverse genetics system. This novel self-splicing ribozyme-based reverse genetics platform will be widely utilized in future ZIKV studies and provide insight for the development of infectious clones of other emerging viruses.

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“…cisActing elements at the 5= end include the 5=-end stem-loop (SLA), which binds viral NS5 RdRp and promotes vRNA replication (8-10); the 5= upstream of AUG region (UAR); the 5= downstream of AUG region (DAR); and the 5= cyclization sequence (CS) (11)(12)(13)(14). The last three are required for genome cyclization by base-pairing with corresponding complementary sequences in the 3=UTR.…”

Section: T He Flavivirus Genus Contains Numerous Important Agents Ofmentioning

confidence: 99%

Novel cis -Acting Element within the Capsid-Coding Region Enhances Flavivirus Viral-RNA Replication by Regulating Genome Cyclization

Liu

Jiang

et al. 2013

J Virol

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c cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5= cyclization sequence (5=CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5=CS, and the presence of DCS-PK facilitates the formation of 5=-3= RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution. T he flavivirus genus contains numerous important agents of human infectious diseases, including dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV). Human infection by flaviviruses can result in symptoms ranging from mild fever to severe encephalitis and hemorrhagic fever. Due to lack of effective vaccines and specific medicines against many flaviviruses, they pose a significant threat to human health around the world.Flaviviruses are enveloped RNA viruses with single-stranded, positive-sense genomes. Their 5= capped viral genome RNA (vRNA) is approximately 10 to 11 kb and contains a single open reading frame (ORF) flanked by 5= and 3= untranslated regions (UTRs). The ORF encodes a polyprotein with more than 3,000 residues, which is cotranslationally and/or posttranslationally proteolytically processed into three structural proteins (capsid, pre-membrane/membrane, and envelope) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by viral and cellular proteases. NS3 and NS5 perform essential enzyme activities required for vRNA replication. The C-terminal domain of NS3 has helicase/NTPase (1) and RNA triphosphatase activities (2, 3). The N-terminal domain of NS5 encodes guanylyltransferase (4) and methyltransferase activities (5, 6), whereas the C-terminal domain of NS5 has RNA-dependent RNA polymerase (RdRp) activity. These enzyme activities are involved in vRNA synthesis, 5= capping, and internal adenosine methylation (7).The 5=UTR, 3=UTR, and capsid-coding sequence are involved in vRNA replication, translation, and perhaps encapsidation, and many cis-acting elements have ...

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The 5′ and 3′ Downstream AUG Region Elements Are Required for Mosquito-Borne Flavivirus RNA Replication

Cited by 69 publications

References 22 publications

Conserved RNA secondary structures and long-range interactions in hepatitis C viruses

Conserved RNA secondary structures and long-range interactions in hepatitis C viruses

Characterization of cis -Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone

Novel cis -Acting Element within the Capsid-Coding Region Enhances Flavivirus Viral-RNA Replication by Regulating Genome Cyclization

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