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Wolbachia-mediated virus blocking in mosquito cells is dependent on XRN1-mediated viral RNA degradation and influenced by viral replication rate.

ABSTRACT: Wolbachia is currently being developed as a novel tool to block the transmission of dengue viruses (DENV) by Aedes aegypti. A number of mechanisms have been proposed to explain the DENV-blocking phenotype in mosquitoes, including competition for fatty acids like cholesterol, manipulation of host miRNAs and upregulation of innate immune pathways in the mosquito. We examined the various stages in the DENV infection process to better understand the mechanism of Wolbachia-mediated virus blocking (WMVB). Our results suggest that infection with Wolbachia does not inhibit DENV binding or cell entry, but reduces virus replication. In contrast to a previous report, we also observed a similar reduction in replication of West Nile virus (WNV). This reduced replication is associated with rapid viral RNA degradation in the cytoplasm. We didn't find a role for host miRNAs in WMVB. Further analysis showed that the 3' end of the virus subgenomic RNA was protected and accumulated over time suggesting that the degradation is XRN1-mediated. We also found that sub genomic flavivirus RNA accumulation inactivated XRN1 in mosquito cells in the absence of Wolbachia and led to enhancement of RNA degradation in its presence. Depletion of XRN1 decreased WMVB which was associated with a significant increase in DENV RNA. We also observed that WMVB is influenced by virus MOI and rate of virus replication. A comparatively elevated blocking was observed for slowly replicating DENV, compared to WNV. Similar results were obtained while analysing different DENV serotypes.

SUBMITTER: Thomas S

PROVIDER: S-EPMC5833283 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Wolbachia-mediated virus blocking in mosquito cells is dependent on XRN1-mediated viral RNA degradation and influenced by viral replication rate.

Thomas Saijo S Verma Jiyoti J Woolfit Megan M O'Neill Scott L SL

PLoS pathogens 20180301 3

Wolbachia is currently being developed as a novel tool to block the transmission of dengue viruses (DENV) by Aedes aegypti. A number of mechanisms have been proposed to explain the DENV-blocking phenotype in mosquitoes, including competition for fatty acids like cholesterol, manipulation of host miRNAs and upregulation of innate immune pathways in the mosquito. We examined the various stages in the DENV infection process to better understand the mechanism of Wolbachia-mediated virus blocking (WM ...[more]

PMID: 29494679

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Project description:The ability of the endosymbiont Wolbachia pipientis to restrict RNA viruses is presently being leveraged to curb global transmission of arbovirus-induced diseases. Past studies have shown that virus replication is limited early in arthropod cells colonized by the bacterium, although it is unclear if this phenomenon is replicated in mosquito cells that first encounter viruses obtained through a vertebrate blood meal. Furthermore, these cellular events neither explain how Wolbachia limits dissemination of viruses between mosquito tissues, nor how it prevents transmission of infectious viruses from mosquitoes to vertebrate host. In this study, we try to address these issues using an array of mosquito cell culture models, with an additional goal being to identify a common viral target for pathogen blocking. Our results establish the viral RNA as a cellular target for Wolbachia-mediated inhibition, with the incoming viral RNA experiencing rapid turnover following internalization in cells. This early block in replication in mosquito cells initially infected by the virus thus consequently reduces the production of progeny viruses from these same cells. However, this is not the only contributor to pathogen blocking. We show that the presence of Wolbachia reduces the per-particle infectivity of progeny viruses on naïve mosquito and vertebrate cells, consequently limiting virus dissemination and transmission, respectively. Importantly, we demonstrate that this aspect of pathogen blocking is independent of any particular Wolbachia-host association and affects viruses belonging to Togaviridae and Flaviviridae families of RNA viruses. Finally, consistent with the idea of the viral RNA as a target, we find that the encapsidated virion RNA is less infectious for viruses produced from Wolbachia-colonized cells. Collectively, our findings present a common mechanism of pathogen blocking in mosquitoes that establish a link between virus inhibition in the cell to virus dissemination and transmission.

Project description:Mosquito-borne arboviruses are a major source of human disease. One strategy to reduce arbovirus disease is to reduce the mosquito's ability to transmit virus. Mosquito infection with the bacterial endosymbiont Wolbachia pipientis wMel is a novel strategy to reduce Aedes mosquito competency for flavivirus infection. However, experiments investigating cyclic environmental temperatures have shown a reduction in maternal transmission of wMel, potentially weakening the integration of this strain into a mosquito population relative to that of other Wolbachia strains. Consequently, it is important to investigate additional Wolbachia strains. All Zika virus (ZIKV) suppression studies are limited to the wMel Wolbachia strain. Here we show ZIKV inhibition by two different Wolbachia strains: wAlbB (isolated from Aedes albopictus mosquitoes) and wStri (isolated from the planthopper Laodelphax striatellus) in mosquito cells. Wolbachia strain wStri inhibited ZIKV most effectively. Single-cycle infection experiments showed that ZIKV RNA replication and nonstructural protein 5 translation were reduced below the limits of detection in wStri-containing cells, demonstrating early inhibition of virus replication. ZIKV replication was rescued when Wolbachia was inhibited with a bacteriostatic antibiotic. We observed a partial rescue of ZIKV growth when Wolbachia-infected cells were supplemented with cholesterol-lipid concentrate, suggesting competition for nutrients as one of the possible mechanisms of Wolbachia inhibition of ZIKV. Our data show that wAlbB and wStri infection causes inhibition of ZIKV, making them attractive candidates for further in vitro mechanistic and in vivo studies and future vector-centered approaches to limit ZIKV infection and spread.IMPORTANCE Zika virus (ZIKV) has swiftly spread throughout most of the Western Hemisphere. This is due in large part to its replication in and spread by a mosquito vector host. There is an urgent need for approaches that limit ZIKV replication in mosquitoes. One exciting approach for this is to use a bacterial endosymbiont called Wolbachia that can populate mosquito cells and inhibit ZIKV replication. Here we show that two different strains of Wolbachia, wAlbB and wStri, are effective at repressing ZIKV in mosquito cell lines. Repression of virus growth is through the inhibition of an early stage of infection and requires actively replicating Wolbachia Our findings further the understanding of Wolbachia viral inhibition and provide novel tools that can be used in an effort to limit ZIKV replication in the mosquito vector, thereby interrupting the transmission and spread of the virus.

Project description:Dengue virus (DENV) is a mosquito-borne virus that infects upward of 300 million people annually and has the potential to cause fatal hemorrhagic fever and shock. While the parameters contributing to dengue immunopathogenesis remain unclear, the collapse of redox homeostasis and the damage induced by oxidative stress have been correlated with the development of inflammation and progression toward the more severe forms of disease. In the present study, we demonstrate that the accumulation of reactive oxygen species (ROS) late after DENV infection (>24 hpi) resulted from a disruption in the balance between oxidative stress and the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant response. The DENV NS2B3 protease complex strategically targeted Nrf2 for degradation in a proteolysis-independent manner; NS2B3 licensed Nrf2 for lysosomal degradation. Impairment of the Nrf2 regulator by the NS2B3 complex inhibited the antioxidant gene network and contributed to the progressive increase in ROS levels, along with increased virus replication and inflammatory or apoptotic gene expression. By 24 hpi, when increased levels of ROS and antiviral proteins were observed, it appeared that the proviral effect of ROS overcame the antiviral effects of the interferon (IFN) response. Overall, these studies demonstrate that DENV infection disrupts the regulatory interplay between DENV-induced stress responses, Nrf2 antioxidant signaling, and the host antiviral immune response, thus exacerbating oxidative stress and inflammation in DENV infection.IMPORTANCE Dengue virus (DENV) is a mosquito-borne pathogen that threatens 2.5 billion people in more than 100 countries annually. Dengue infection induces a spectrum of clinical symptoms, ranging from classical dengue fever to severe dengue hemorrhagic fever or dengue shock syndrome; however, the complexities of DENV immunopathogenesis remain controversial. Previous studies have reported the importance of the transcription factor Nrf2 in the control of redox homeostasis and antiviral/inflammatory or death responses to DENV. Importantly, the production of reactive oxygen species and the subsequent stress response have been linked to the development of inflammation and progression toward the more severe forms of the disease. Here, we demonstrate that DENV uses the NS2B3 protease complex to strategically target Nrf2 for degradation, leading to a progressive increase in oxidative stress, inflammation, and cell death in infected cells. This study underlines the pivotal role of the Nrf2 regulatory network in the context of DENV infection.

Project description:Mosquito-transmitted viruses are spread globally and present a great risk to human health. Among the many approaches investigated to limit the diseases caused by these viruses are attempts to make mosquitos resistant to virus infection. Coinfection of mosquitos with the bacterium Wolbachia pipientis from supergroup A is a recent strategy employed to reduce the capacity for major vectors in the Aedes mosquito genus to transmit viruses, including dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV). Recently, a supergroup B Wolbachia wStri, isolated from Laodelphax striatellus, was shown to inhibit multiple lineages of ZIKV in Aedes albopictus cells. Here, we show that wStri blocks the growth of positive-sense RNA viruses DENV, CHIKV, ZIKV, and yellow fever virus by greater than 99.9%. wStri presence did not affect the growth of the negative-sense RNA viruses LaCrosse virus or vesicular stomatitis virus. Investigation of the stages of the ZIKV life cycle inhibited by wStri identified two distinct blocks in viral replication. We found a reduction of ZIKV entry into wStri-infected cells. This was partially rescued by the addition of a cholesterol-lipid supplement. Independent of entry, transfected viral genome was unable to replicate in Wolbachia-infected cells. RNA transfection and metabolic labeling studies suggested that this replication defect is at the level of RNA translation, where we saw a 66% reduction in mosquito protein synthesis in wStri-infected cells. This study's findings increase the potential for application of wStri to block additional arboviruses and also identify specific blocks in viral infection caused by Wolbachia coinfection.IMPORTANCE Dengue, Zika, and yellow fever viruses are mosquito-transmitted diseases that have spread throughout the world, causing millions of infections and thousands of deaths each year. Existing programs that seek to contain these diseases through elimination of the mosquito population have so far failed, making it crucial to explore new ways of limiting the spread of these viruses. Here, we show that introduction of an insect symbiont, Wolbachia wStri, into mosquito cells is highly effective at reducing yellow fever virus, dengue virus, Zika virus, and Chikungunya virus production. Reduction of virus replication was attributable to decreases in entry and a strong block of virus gene expression at the translational level. These findings expand the potential use of Wolbachia wStri to block viruses and identify two separate steps for limiting virus replication in mosquitos that could be targeted via microbes or other means as an antiviral strategy.

Dataset Information

Wolbachia-mediated virus blocking in mosquito cells is dependent on XRN1-mediated viral RNA degradation and influenced by viral replication rate.

Publications

Wolbachia-mediated virus blocking in mosquito cells is dependent on XRN1-mediated viral RNA degradation and influenced by viral replication rate.

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