Antiviral Wolbachia strains associate with Aedes aegypti endoplasmic reticulum membranes and disturb host cell lipid distribution to restrict dengue virus replication

The insect endosymbiotic bacterium Wolbachia pipientis is being utilised as a biocontrol tool to reduce the incidence of Aedes aegypti -transmitted viral diseases like dengue. However, the precise mechanisms underpinning Wolbachia ’s antiviral activity are not well defined. Here we generated a panel of Ae. aegypti -derived cell lines infected with antiviral strains w Mel and w AlbB or the non-antiviral strain w Pip to understand host cell morphological changes specifically induced by antiviral strains. Antiviral strains were frequently found to be entirely wrapped by the host endoplasmic reticulum (ER) membrane, while w Pip bacteria clustered separately in the host cell cytoplasm. ER-derived lipid droplets (LDs) increased in volume in w Mel-and w AlbB-infected cell lines and mosquito tissues compared to cells infected with w Pip or Wolbachia -free controls. Inhibition of fatty acid synthase (required for triacylglycerol biosynthesis) reduced LD formation and significantly restored ER-associated dengue virus replication in cells occupied by w Mel. Together, this suggests that antiviral Wolbachia strains may specifically alter the lipid composition of the ER to preclude the establishment of DENV replication complexes. Defining Wolbachia ’s antiviral mechanisms will support the application and longevity of this effective biocontrol tool that is already being used at scale. Importance Aedes aegypti transmits a range of important human pathogenic viruses like dengue. However, infection of Ae. aegypti with the insect endosymbiotic bacterium, Wolbachia , reduces the risk of mosquito to human viral transmission. Wolbachia is being utilized at field sites across more than 13 countries to reduce the incidence of viruses like dengue, but it is not well understood how Wolbachia induces its antiviral effects. To examine this at the subcellular level, we compared how different strains of Wolbachia with varying antiviral strengths, associate with and modify host cell structures. Strongly antiviral strains were found to specifically associate with the host endoplasmic reticulum and induce striking impacts on host cell lipid distribution. Inhibiting Wolbachia -induced lipid redistribution partially restored dengue virus replication demonstrating this is a contributing role for Wolbachia ’s antiviral activity. These findings provide new insights into how antiviral Wolbachia strains associate with and modify Ae. aegypti host cells. ### Competing Interest Statement The authors have declared no competing interest.