Viral RNA switch mediates the dynamic control of flavivirus replicase recruitment by genome cyclization

Liu, Zhong-Yu; Li, Xiaofeng; Jiang, Tao; Deng, Yong-Qiang; Ye, Qing; Zhao, Hui; Yu, Jiu-Yang; Qin, Cheng‐Feng

doi:10.7554/elife.17636

Cited by 70 publications

(105 citation statements)

References 76 publications

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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: 97%

“…It was also showed that the 5=-SLA and 5=-3= CS base-pairing restoration mutants consistently replicated moderately lower than the WT, suggesting that tertiary or quaternary interactions may be involved in the functionalization of these cis-acting RNA elements. Previously, studies suggested that the conserved UFS element is required for ZIKV replication (45) and that the SL-I/xrRNA1 structure is responsible for the production of ZIKV sfRNA (62). Thus, the RNA-based regulation mechanism of ZIKV should be highly similar to other well-studied mosquitoborne flaviviruses.…”

Section: Discussionmentioning

confidence: 99%

“…50% confluence at the time of transfection, which was performed using Lipofectamine 2000 (Thermo Fisher Scientific). At the indicated time points p.t., coverslips were fixed with acetone-methanol (3/7) at Ϫ20°C, and ZIKV E protein expression was detected by IFA as previously described (45). At day 3 p.t., culture supernatants were collected and used to infect C6/36 cells maintained in RPMI 1640 (Thermo Fisher Scientific) containing 2% FBS (Biowest).…”

Section: Fig 5 Legend (Continued)mentioning

confidence: 99%

“…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). The core sequence (5=, UCAAUAUGCU; 3=, AGCAUAUUGA) of the CS element is in consensus with the mosquito-borne flaviviruses (38,46), whereas the secondary structure of the SLA element is highly conserved in all known species of flaviviruses as shown by Mfold (47, 48) predictions (Fig.…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Section: Fig 5 Legend (Continued)mentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…99 Recently, Liu et al (2016) identified a conserved RNA switch, the 5′-UAR-flanking stem (UFS), which extends the SLB structure and enhances recruitment of NS5 and RNA synthesis. 101 …”

Section: Biochemistry and Molecular Biology Of Flavivirusesmentioning

confidence: 99%

Biochemistry and Molecular Biology of Flaviviruses

et al. 2018

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Flaviviruses, such as dengue, Japanese encephalitis, tick-borne encephalitis, West Nile, yellow fever, and Zika viruses, are critically important human pathogens that sicken a staggeringly high number of humans every year. Most of these pathogens are transmitted by mosquitos, and not surprisingly, as the earth warms and human populations grow and move, their geographic reach is increasing. Flaviviruses are simple RNA–protein machines that carry out protein synthesis, genome replication, and virion packaging in close association with cellular lipid membranes. In this review, we examine the molecular biology of flaviviruses touching on the structure and function of viral components and how these interact with host factors. The latter are functionally divided into pro-viral and antiviral factors, both of which, not surprisingly, include many RNA binding proteins. In the interface between the virus and the hosts we highlight the role of a noncoding RNA produced by flaviviruses to impair antiviral host immune responses. Throughout the review, we highlight areas of intense investigation, or a need for it, and potential targets and tools to consider in the important battle against pathogenic flaviviruses.

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Flavors of Flaviviral RNA Structure: towards an Integrated View of RNA Function from Translation through Encapsidation

et al. 2019

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For many viruses, RNA is the holder of genetic information and serves as the template for both replication and translation. While host and viral proteins play important roles in viral decision‐making, the extent to which viral RNA (vRNA) actively participates in translation and replication might be surprising. Here, the focus is on flaviviruses, which include common human scourges such as dengue, West Nile, and Zika viruses, from an RNA‐centric viewpoint. In reviewing more recent findings, an attempt is made to fill knowledge gaps and revisit some canonical views of vRNA structures involved in replication. In particular, alternative views are offered on the nature of the flaviviral promoter and genome cyclization, and the feasibility of refining in vitro‐derived models with modern RNA probing and sequencing methods is pointed out. By tracing vRNA structures from translation through encapsidation, a dynamic molecule closely involved in the self‐regulation of viral replication is revealed.

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Viral RNA switch mediates the dynamic control of flavivirus replicase recruitment by genome cyclization

Cited by 70 publications

References 76 publications

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

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

Biochemistry and Molecular Biology of Flaviviruses

Flavors of Flaviviral RNA Structure: towards an Integrated View of RNA Function from Translation through Encapsidation

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