Globally re-emerging dengue viruses are transmitted from human-to-human by Aedes mosquitoes. While viral determinants of human pathogenicity have been defined, there is a lack of knowledge of how dengue viruses influence mosquito transmission. Identification of viral determinants of transmission can help identify isolates with high epidemiological potential. Additionally, mechanistic understanding of transmission will lead to better understanding of how dengue viruses harness evolution to cycle between the two hosts. Here, we identified viral determinants of transmission and characterized mechanisms that enhance production of infectious saliva by inhibiting immunity specifically in salivary glands. Combining oral infection of Aedes aegypti mosquitoes and reverse genetics, we identified two 3' UTR substitutions in epidemic isolates that increased subgenomic flaviviral RNA (sfRNA) quantity, infectious particles in salivary glands and infection rate of saliva, which represents a measure of transmission. We also demonstrated that various 3'UTR modifications similarly affect sfRNA quantity in both whole mosquitoes and human cells, suggesting a shared determinism of sfRNA quantity. Furthermore, higher relative quantity of sfRNA in salivary glands compared to midgut and carcass pointed to sfRNA function in salivary glands. We showed that the Toll innate immune response was preferentially inhibited in salivary glands by viruses with the 3'UTR substitutions associated to high epidemiological fitness and high sfRNA quantity, pointing to a mechanism for higher saliva infection rate. By determining that sfRNA is an immune suppressor in a tissue relevant to mosquito transmission, we propose that 3'UTR/sfRNA sequence evolution shapes dengue epidemiology not only by influencing human pathogenicity but also by increasing mosquito transmission, thereby revealing a viral determinant of epidemiological fitness that is shared between the two hosts.

Viral RNA-host protein interactions are critical for replication of flaviviruses, a genus of positive-strand RNA viruses comprising major vector-borne human pathogens including dengue viruses (DENV). We examined three conserved host RNA-binding proteins (RBPs) G3BP1, G3BP2 and CAPRIN1 in dengue virus (DENV-2) infection and found them to be novel regulators of the interferon (IFN) response against DENV-2. The three RBPs were required for the accumulation of the protein products of several interferon stimulated genes (ISGs), and for efficient translation of PKR and IFITM2 mRNAs. This identifies G3BP1, G3BP2 and CAPRIN1 as novel regulators of the antiviral state. Their antiviral activity was antagonized by the abundant DENV-2 non-coding subgenomic flaviviral RNA (sfRNA), which bound to G3BP1, G3BP2 and CAPRIN1, inhibited their activity and lead to profound inhibition of ISG mRNA translation. This work describes a new and unexpected level of regulation for interferon stimulated gene expression and presents the first mechanism of action for an sfRNA as a molecular sponge of anti-viral effectors in human cells.

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.