UCSF

Arboviruses cause an overwhelming number of clinical cases of disease each year around the world. The kinetics of viral replication is of critical importance to the dissemination of virus within the mosquito and ultimately transmission between hosts. Arboviral replication and dissemination is dependent upon their ability to evade the immune response and modulate cell toxicity in two separate hosts. Although there is a tremendous amount of study on the replication of arboviruses in mammalian hosts, there is much less focus on their replication in insects. In mosquitos the siRNA pathway of RNA interference (RNAi) is an indispensible component of the antiviral immune system and a key repressor of viral replication.

In this work, we explore two poorly understood aspects of antiviral RNAi in mosquitoes: systemic dsRNA spread and piRNA mediated immunity. We show that the Aedes aegypti cells, Aag2, effectively take up long dsRNA from the extracellular medium to initiate RNAi. Pharmacological and genetic analyses reveal that dsRNA enters the cell via clathrin-mediated endocytosis. Uptake of exogenous dsRNA directed against Sindbis virus (SINV) inhibits viral replication. However, SINV inhibits RNAi initiated by dsRNA soaking after infection by inhibiting acidification of endosomes. Thus, Sindbis virus may control RNAi antiviral immunity in mosquitoes by suppressing exogenous dsRNA uptake. In addition to its biological role, from a technical point of view the observation that RNAi can be initiated by naked dsRNA in Aedes aegypti cells may facilitate studies encompassing a wide variety of biological processes in mosquitoes.

We also show that in infected cells and mosquitos, both virally derived siRNAs (v-siRNAs) and piRNAs (v-piRNAs) are detected in Aedes aegypti. Although the piRNA pathway is generally associated with germline defense against selfish genetic elements such as transposons, in Aedes aegypti the piRNA pathway mediates antiviral immunity in vivo in somatic tissues and characterize the mechanism of v-piRNA biogenesis. We show that both retro-transcription dependent synthesis of viral DNA and Piwi4 are essential for the biogenesis of virally derived small RNAs and that disruption of either causes an increase in viral replication. We propose that the synthesis of DNA from non-retroviral RNA viruses form loci termed Endogenous Viral Elements (EVEs) that are transcribed as piRNA precursors and feed into the ‘Ping-Pong’ mechanism of secondary piRNA synthesis using viral RNA as a target. Our results illustrate a novel somatic function for the piRNA pathway where retro-transcription of viral RNA produces DNA loci that initiate small RNA synthesis to target and degrade RNA viruses. These observations highlight additional complexity to RNAi mediated antiviral immunity in A. aegypti.