Viral RNA is a target for Wolbachia-mediated pathogen blocking

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. AUTHORS SUMMARY Viruses transmitted by arthropod vectors pose a significant global health risk. Incidence of diseases caused by these viruses can thus be reduced by implementing effective vector control strategies. This need is further exacerbated due to the lack of commercially available vaccines and antivirals. Presence of the intracellular bacteria Wolbachia pipientis is associated with virus inhibition in multiple mosquito vectors. Furthermore, Wolbachia is inherited transovarially and spreads across the vector population like a natural gene drive, making it an attractive vector control agent. In this study, we examine how the presence of the bacterium in arthropod cells prevents initial establishment of vertebrate cell derived viruses. Our results indicate rapid turnover of incoming viral RNA very early during infection in Wolbachia-colonized cells, thus establishing it as a cellular target for pathogen blocking. Additionally, upon evaluating how these events might further limit virus spread, we find that infectivity of progeny viruses belonging to multiple RNA virus families are reduced on a per-particle basis. This aspect of virus inhibition is independent of any particular Wolbachia-host association and affects how these viruses replicate in naïve mosquito and vertebrate cells, thus providing a collective basis of reduced virus dissemination and transmission in Wolbachia-colonized mosquitoes.