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RNA Structure Duplication in the Dengue Virus 3' UTR: Redundancy or Host Specificity?

ABSTRACT: Flaviviruses include a diverse group of medically important viruses that cycle between mosquitoes and humans. During this natural process of switching hosts, each species imposes different selective forces on the viral population. Using dengue virus (DENV) as model, we found that paralogous RNA structures originating from duplications in the viral 3' untranslated region (UTR) are under different selective pressures in the two hosts. These RNA structures, known as dumbbells (DB1 and DB2), were originally proposed to be enhancers of viral replication. Analysis of viruses obtained from infected mosquitoes showed selection of mutations that mapped in DB2. Recombinant viruses carrying the identified variations confirmed that these mutations greatly increase viral replication in mosquito cells, with low or no impact in human cells. Use of viruses lacking each of the DB structures revealed opposite viral phenotypes. While deletion of DB1 reduced viral replication about 10-fold, viruses lacking DB2 displayed a great increase of fitness in mosquitoes, confirming a functional diversification of these similar RNA elements. Mechanistic analysis indicated that DB1 and DB2 differentially modulate viral genome cyclization and RNA replication. We found that a pseudoknot formed within DB2 competes with long-range RNA-RNA interactions that are necessary for minus-strand RNA synthesis. Our results support a model in which a functional diversification of duplicated RNA elements in the viral 3' UTR is driven by host-specific requirements. This study provides new ideas for understanding molecular aspects of the evolution of RNA viruses that naturally jump between different species.IMPORTANCE Flaviviruses constitute the most relevant group of arthropod-transmitted viruses, including important human pathogens such as the dengue, Zika, yellow fever, and West Nile viruses. The natural alternation of these viruses between vertebrate and invertebrate hosts shapes the viral genome population, which leads to selection of different viral variants with potential implications for epidemiological fitness and pathogenesis. However, the selective forces and mechanisms acting on the viral RNA during host adaptation are still largely unknown. Here, we found that two almost identical tandem RNA structures present at the viral 3' untranslated region are under different selective pressures in the two hosts. Mechanistic studies indicated that the two RNA elements, known as dumbbells, contain sequences that overlap essential RNA cyclization elements involved in viral RNA synthesis. The data support a model in which the duplicated RNA structures differentially evolved to accommodate distinct functions for viral replication in the two hosts.

SUBMITTER: de Borba L

PROVIDER: S-EPMC6325252 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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RNA Structure Duplication in the Dengue Virus 3' UTR: Redundancy or Host Specificity?

de Borba Luana L Villordo Sergio M SM Marsico Franco L FL Carballeda Juan M JM Filomatori Claudia V CV Gebhard Leopoldo G LG Pallarés Horacio M HM Lequime Sebastian S Lambrechts Louis L Sánchez Vargas Irma I Blair Carol D CD Gamarnik Andrea V AV

mBio 20190108 1

Flaviviruses include a diverse group of medically important viruses that cycle between mosquitoes and humans. During this natural process of switching hosts, each species imposes different selective forces on the viral population. Using dengue virus (DENV) as model, we found that paralogous RNA structures originating from duplications in the viral 3' untranslated region (UTR) are under different selective pressures in the two hosts. These RNA structures, known as dumbbells (DB1 and DB2), were or ...[more]

PMID: 30622191

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Project description:Dengue viruses (DENV) are expanding global pathogens that are transmitted through the bite of mosquitoes, mostly Aedes aegypti. As RNA viruses, DENV rely on RNA-binding proteins (RBPs) to complete their life cycle. Alternatively, RBPs can act as restriction factors that prevent DENV multiplication. While the importance of RBPs is well-supported in humans, there is a dearth of information about their influence on DENV transmission by mosquitoes. Such knowledge could be harnessed to design novel, effective interventions against DENV. Here, we successfully adapted RNA-affinity chromatography coupled with mass spectrometry-a technique initially developed in mammalian cells-to identify RBPs in Ae. aegypti cells. We identified fourteen RBPs interacting with DENV serotype 2 3'UTR, which is involved in the viral multiplication and produces subgenomic flaviviral RNA (sfRNA). We validated the RNA affinity results for two RBPs by confirming that AePur binds the 3'UTR, whereas AeStaufen interacts with both 3'UTR and sfRNA. Using in vivo functional evaluation, we determined that RBPs like AeRan, AeExoRNase, and AeRNase have pro-viral functions, whereas AeGTPase, AeAtu, and AePur have anti-viral functions in mosquitoes. Furthermore, we showed that human and mosquito Pur homologs have a shared affinity to DENV2 RNA, although the anti-viral effect is specific to the mosquito protein. Importantly, we revealed that AeStaufen mediates a reduction of gRNA and sfRNA copies in several mosquito tissues, including the salivary glands and that AeStaufen-mediated sfRNA reduction diminishes the concentration of transmission-enhancing sfRNA in saliva, thereby revealing AeStaufen's role in DENV transmission. By characterizing the first RBPs that associate with DENV2 3'UTR in mosquitoes, our study unravels new pro- and anti-viral targets for the design of novel therapeutic interventions as well as provides foundation for studying the role of RBPs in virus-vector interactions.

Project description:Dengue virus (DENV) utilizes host factors throughout its life cycle. In this study, we identified RNA helicase A (RHA), a member of the DEAD/H helicase family, as an important host factor of DENV. In response to DENV2 infection, nuclear RHA protein was partially redistributed into the cytoplasm. The short interfering RNA-mediated knockdown of RHA significantly reduced the amounts of infectious viral particles in various cells. The RHA knockdown reduced the multistep viral growth of DENV2 and Japanese encephalitis virus but not Zika virus. Further study showed that the absence of RHA resulted in a reduction of both viral RNA and protein levels, and the data obtained from the reporter replicon assay indicated that RHA does not directly promote viral protein synthesis. RHA bound to the DENV RNA and associated with three nonstructural proteins, including NS1, NS2B3, and NS4B. Further study showed that different domains of RHA mediated its interaction with these viral proteins. The expression of RHA or RHA-K417R mutant protein lacking ATPase/helicase activity in RHA-knockdown cells successfully restored DENV2 replication levels, suggesting that the helicase activity of RHA is dispensable for its proviral effect. Overall, our work reveals that RHA is an important factor of DENV and might serve as a target for antiviral agents.IMPORTANCE Dengue, caused by dengue virus, is a rapidly spreading disease, and currently there are no treatments available. Host factors involved in the viral replication of dengue virus are potential antiviral therapeutic targets. Although RHA has been shown to promote the multiplication of several viruses, such as HIV and adenovirus, its role in the flavivirus family, including dengue virus, Japanese encephalitis virus, and emerging Zika virus, remains elusive. The current study revealed that RHA relocalized into the cytoplasm upon DENV infection and associated with viral RNA and nonstructural proteins, implying that RHA was actively engaged in the viral life cycle. We further provide evidence that RHA promoted the viral yields of DENV2 independent of its helicase activity. These findings demonstrated that RHA is a new host factor required for DENV replication and might serve as a target for antiviral drugs.

Dataset Information

RNA Structure Duplication in the Dengue Virus 3' UTR: Redundancy or Host Specificity?

Publications

RNA Structure Duplication in the Dengue Virus 3' UTR: Redundancy or Host Specificity?

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