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Regulation of flavivirus RNA synthesis and capping

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RNA viruses, such as flaviviruses, are able to efficiently replicate and cap their RNA genomes in vertebrate and invertebrate cells. Flaviviruses use several specialized proteins to first make an uncapped negative strand copy of the viral genome that is used as a template for the synthesis of large numbers of capped genomic RNAs. Despite using relatively simple mechanisms to replicate their RNA genomes, there are significant gaps in our understanding of how flaviviruses switch between negative and positive strand RNA synthesis and how RNA capping is regulated. Recent work has begun to provide a conceptual framework for flavivirus RNA replication and capping and shown some surprising roles for genomic RNA during replication and pathogenesis. WIREs RNA 2013, 4:723–735. doi: 10.1002/wrna.1191 This article is categorized under: RNA Processing > Capping and 5' End Modifications RNA in Disease and Development > RNA in Disease

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Model for NS3/NS5 interaction based on known structures and enzymatic active sites. In this model, the NS3 RNA triphosphatase/helicase domain interacts with NS5 via a flexible linker found between the capping enzyme and RdRP domains (dashed line). The NS3 protease domain is not included in this model for clarity. During positive strand RNA synthesis, the single‐stranded negative strand RNA template enters the RdRP active site, and the polymerase catalyzes the elongation of a triphosphorylated positive strand RNA. The positive strand RNA is initially duplexed with the negative strand RNA, and this dsRNA is unwound by the helicase activity present in NS3. The positive strand RNA interacts with the RNA triphosphatase active site, which removes the γ‐phosphate from the triphosphorylated RNA, resulting in a di‐phosphorylated RNA substrate. The di‐phosphorylated RNA is then fed into the NS5 capping enzyme where the guanylyltransferase function caps the RNA and the methyltransferase function methylates the RNA. The model was developed using the following PDB files [NS3 helicase/RNA triphosphatase domain (PDB codes: 2 JLR/2JLU), NS5 capping enzyme (PDB code: 3EVG), NS5 RdRP (PDB code: 2J7U)].
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Schematic of flavivirus RNA replication. 44S positive strand viral RNAs (vRNA) are trafficked from incoming viral particles to the endoplasmic reticulum where viral polyproteins are translated. The newly synthesized NS5 RdRP generates a negative strand RNA (colored green) using the positive strand RNA as a template. It is unknown if RNA capping activity or RNA helicase activity occurs during negative strand RNA synthesis. The replicative form (RF) RNA is a duplex of negative and positive strand RNA thought to act as a template for additional capped positive strand RNA synthesis via the action of NS3 and NS5. The 28S replicative intermediate (RI) form is comprised of newly synthesized capped positive strand RNA (colored red) and displaced original capped positive strand RNA. The NS3 RNA triphosphatase and NS5 guanylyltransferase/ methyltransferase enzymes generate a new RNA cap on the 5′ end of the nascent RNA strand. NS3 RNA helicase and NTPase activities are necessary for unwinding dsRNA during positive strand RNA synthesis. Released positive strand RNAs can be used for additional protein translation, interference with RNAi or RNA decay pathways, packaging into viral particles, or generate additional RF forms.
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Flavivirus genomic RNA structures. (a) Linear structure of a generic flavivirus RNA genome showing the positions of critical RNA structures and protein coding regions. (b) Cyclization of flavivirus positive strand genome promotes negative strand RNA synthesis. The 5′ end of the positive strand genomic RNA interacts with the 3′ end of the positive strand RNA via interactions between the CS and UAR/DAR regions. Hybridization between the 5′ UAR and 3′ DAR causes a reorganization of the 3′ SL structure, exposing the 3′ end of the positive strand RNA. The RdRP domain of NS5 binds to SLA on the 5′ end of the positive strand genome and utilizes the exposed 3′ end of the positive strand RNA as a template for negative strand RNA synthesis.
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Potential model for positive strand RNA replication within the replication compartment. Flavivirus RF RNA is entirely contained within replication compartments, which protects the negative strand RNA (colored green) from host antiviral factors such as RISC and PKR. dsRNA may initially interact with a separate NS3 helicase molecule (shown without the protease domain for clarity) that unwinds the RF form dsRNA and directs the original capped positive strand RNA out of the replication compartment for translation, interference with the miRNA and RNA decay pathways, and virion packaging. The 3′ end of unwound negative strand RNA enters the NS5 RdRP domain within the NS3:NS5 replication complex and results in the synthesis of a new capped positive strand RNAs (colored red) as described in Figure . The negative strand RNA likely forms a new dsRNA duplex with the nascent positive strand RNA to regenerate the RF form within the replication compartment, and the newly synthesized positive strand RNA would be released from the replication compartment during the next round of positive strand RNA synthesis.
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Proposed mechanism of flavivirus RNA capping. NS3 RNA triphosphatase binds to and cleaves the γ‐phosphate from newly synthesized positive strand RNAs, generating a di‐phosphorylated RNA substrate. The NS5 capping enzyme (CE) binds GTP and forms the guanylated intermediate in a Mg2+‐dependent reaction. The di‐phosphorylated RNA substrate interacts with the guanylated NS5 protein, which transfers the GMP moiety to the di‐phosphorylated RNA to form the base cap structure (GpppAGUAn). The base cap structure is first methylated at the guanine N7 position by the methyltransferase function within the capping enzyme, presumably by the action of a second NS5 capping enzyme protein. The cap 0 structure is then 2′O methylated to form the cap 1 structure (m7GpppAm2GUAn). This model would allow the cap to be fully formed without repositioning.
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RNA Processing > Capping and 5′ End Modifications
RNA in Disease and Development > RNA in Disease

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