Reading Between the Strands: Deciphering Double Stranded RNA-Based Immune Responses in Mosquito-Virus Interactions
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Authors
Prince, Brian
Issue Date
2025
Type
Dissertation
Language
en_US
Keywords
Arboviruses , Dicer-2 , dsRNA , Msoquitoes , RNAi , Toll receptors
Alternative Title
Abstract
Mosquitoes are responsible for transmitting several medically important viruses that cause widespread illness and pose a major public health concern. Among them, Culex quinquefasciatus is a globally distributed vector species that contributes to the spread of multiple arthropod-borne viruses. Despite their significance, the molecular mechanisms by which Culex species mosquitoes recognize and respond to viral infection remain poorly understood. Antiviral immunity in mosquitoes is initiated by the recognition of viral pathogen-associated molecular patterns, with double-stranded RNA (dsRNA), a replication intermediate of many viruses, serving as a key immunostimulatory signal. While RNA interference (RNAi) remains the most well-characterized antiviral pathway triggered by dsRNA, accumulating evidence suggests that mosquitoes also employ RNAi-independent mechanisms that detect and respond to viral dsRNA.This dissertation examines these alternative responses in Cx. quinquefasciatus, focusing on immune pathways and proteins operating outside of RNAi. We first provide initial evidence for an RNAi-independent, dsRNA-triggered antiviral response. We also characterize Cx. quinquefasciatus Toll6, a previously unstudied Toll-like receptor in this species, as an antiviral gene and potential dsRNA sensor, alongside a broader survey of Toll gene phylogeny and inducibility.
We then further characterized non-canonical functions of Dicer-2 (Dcr-2), a key RNAi component known to bind viral dsRNA. Using a CRISPR-generated Dcr-2 knockout cell line, coupled with poly(I:C)-based proteomics and transcriptomic profiling, we demonstrate that Dcr-2 regulates immune gene expression and restricts virus replication independently of its RNAi activity. These analyses also revealed additional dsRNA-binding proteins and candidate Dcr-2 binding partners, helping to refine our understanding of dsRNA sensing and Dcr-2’s downstream signaling network.
Finally, we investigated adenosine deaminase acting on RNA (ADAR), a conserved dsRNA-binding protein. Using both knockdown and overexpression approaches, we found that each reduced virus replication and dampened immune gene expression, suggesting that ADAR levels must be tightly regulated to balance antiviral defense and host immune activity. These effects occurred independently of Dcr-2, indicating that ADAR modulates virus infection and immune signaling through a Dcr-2-independent mechanism, potentially involving its RNA editing activity.
Together, this work advances our understanding of dsRNA-based immunity in Cx. quinquefasciatus, highlights novel roles for conserved immune factors, and provides tools and insights for probing antiviral defenses in mosquitoes. These findings may ultimately inform strategies for controlling mosquito-borne diseases by targeting immune sensing pathways.