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The anti-immune dengue subgenomic flaviviral RNA is present in vesicles in mosquito saliva and is associated with increased infectivity.

ABSTRACT: Mosquito transmission of dengue viruses to humans starts with infection of skin resident cells at the biting site. There is great interest in identifying transmission-enhancing factors in mosquito saliva in order to counteract them. Here we report the discovery of high levels of the anti-immune subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We established that sfRNA is present in saliva using three different methods: northern blot, RT-qPCR and RNA sequencing. We next show that salivary sfRNA is protected in detergent-sensitive compartments, likely extracellular vesicles. In support of this hypothesis, we visualized viral RNAs in vesicles in mosquito saliva and noted a marked enrichment of signal from 3'UTR sequences, which is consistent with the presence of sfRNA. Furthermore, we show that incubation with mosquito saliva containing higher sfRNA levels results in higher virus infectivity in a human hepatoma cell line and human primary dermal fibroblasts. Transfection of 3'UTR RNA prior to DENV2 infection inhibited type I and III interferon induction and signaling, and enhanced viral replication. Therefore, we posit that sfRNA present in salivary extracellular vesicles is delivered to cells at the biting site to inhibit innate immunity and enhance dengue virus transmission.

SUBMITTER: Yeh SC

PROVIDER: S-EPMC10062553 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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The anti-immune dengue subgenomic flaviviral RNA is present in vesicles in mosquito saliva and is associated with increased infectivity.

Yeh Shih-Chia SC Strilets Tania T Tan Wei-Lian WL Castillo David D Medkour Hacène H Rey-Cadilhac Félix F Serrato-Pomar Idalba M IM Rachenne Florian F Chowdhury Avisha A Chuo Vanessa V Azar Sasha R SR Singh Moirangthem Kiran MK Hamel Rodolphe R Missé Dorothée D Kini R Manjunatha RM Kenney Linda J LJ Vasilakis Nikos N Marti-Renom Marc A MA Nir Guy G Pompon Julien J Garcia-Blanco Mariano A MA

PLoS pathogens 20230330 3

Mosquito transmission of dengue viruses to humans starts with infection of skin resident cells at the biting site. There is great interest in identifying transmission-enhancing factors in mosquito saliva in order to counteract them. Here we report the discovery of high levels of the anti-immune subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We established that sfRNA is present in saliva using three different methods: northern blot, RT-qPCR and RNA sequencing. We ne ...[more]

PMID: 36996041

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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:During the life cycle of mosquito-borne flaviviruses, substantial subgenomic flaviviral RNA (sfRNA) is produced via incomplete degradation of viral genomic RNA by host XRN1. Zika virus (ZIKV) sfRNA has been detected in mosquito and mammalian somatic cells. Human neural progenitor cells (hNPCs) in the developing brain are the major target cells of ZIKV, and antiviral RNA interference (RNAi) plays a critical role in hNPCs. However, whether ZIKV sfRNA was produced in ZIKV-infected hNPCs as well as its function remains not known. In this study, we demonstrate that abundant sfRNA was produced in ZIKV-infected hNPCs. RNA pulldown and mass spectrum assays showed ZIKV sfRNA interacted with host proteins RHA and PACT, both of which are RNA-induced silencing complex (RISC) components. Functionally, ZIKV sfRNA can antagonize RNAi by outcompeting small interfering RNAs (siRNAs) in binding to RHA and PACT. Furthermore, the 3' stem loop (3'SL) of sfRNA was responsible for RISC components binding and RNAi inhibition, and 3'SL can enhance the replication of a viral suppressor of RNAi (VSR)-deficient virus in a RHA- and PACT-dependent manner. More importantly, the ability of binding to RISC components is conversed among multiple flaviviral 3'SLs. Together, our results identified flavivirus 3'SL as a potent VSR in RNA format, highlighting the complexity in virus-host interaction during flavivirus infection.IMPORTANCEZika virus (ZIKV) infection mainly targets human neural progenitor cells (hNPCs) and induces cell death and dysregulated cell-cycle progression, leading to microcephaly and other central nervous system abnormalities. RNA interference (RNAi) plays critical roles during ZIKV infections in hNPCs, and ZIKV has evolved to encode specific viral proteins to antagonize RNAi. Herein, we first show that abundant sfRNA was produced in ZIKV-infected hNPCs in a similar pattern to that in other cells. Importantly, ZIKV sfRNA acts as a potent viral suppressor of RNAi (VSR) by competing with siRNAs for binding RISC components, RHA and PACT. The 3'SL of sfRNA is responsible for binding RISC components, which is a conserved feature among mosquito-borne flaviviruses. As most known VSRs are viral proteins, our findings highlight the importance of viral non-coding RNAs during the antagonism of host RNAi-based antiviral innate immunity.

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Dataset Information

The anti-immune dengue subgenomic flaviviral RNA is present in vesicles in mosquito saliva and is associated with increased infectivity.

Publications

The anti-immune dengue subgenomic flaviviral RNA is present in vesicles in mosquito saliva and is associated with increased infectivity.

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