Investigations into the antiviral activities and functions of the Aedes aegypti Ago2 protein

Dummunee, Krittika (2026) Investigations into the antiviral activities and functions of the Aedes aegypti Ago2 protein. PhD thesis, University of Glasgow.

Abstract

Arboviruses represent a significant and growing threat to global public health, causing diseases such as dengue, chikungunya, and Zika. These viruses are transmitted by arthropod vectors, primarily mosquitoes, which support viral replication while remaining largely asymptomatic. A key determinant of this ability is the insect antiviral immune system, particularly RNA interference (RNAi). The exogenous small interfering RNA (exo-siRNA) pathway represents the primary antiviral defence mechanism in insects, recognising viral double-stranded RNA intermediates generated during replication and directing sequence-specific degradation of viral RNA. Central to this process is Argonaute-2 (Ago2), the catalytic component of the RNA-induced silencing complex (RISC), which uses virus-derived small interfering RNAs (vsiRNAs) to guide cleavage of complementary viral RNA targets. While the antiviral role of RNAi has been extensively characterised in Drosophila melanogaster, the molecular mechanisms governing Ago2-mediated antiviral defence in mosquito vectors remain incompletely understood.

This thesis aimed to investigate the role of Ago2 catalytic activity in antiviral RNAi and to identify host proteins that interact with Ago2 and may influence antiviral responses in mosquito cells. Using Aedes aegypti–derived cell lines, including an Ago2 knockout line (AF525) and RNAi-competent AF5 cells, a series of molecular and virological approaches were employed to dissect the contribution of Ago2 to antiviral defence against Semliki Forest virus (SFV), a model alphavirus. Recombinant cell lines expressing either wild-type Ago2 or catalytic mutants targeting the conserved PIWI-domain catalytic tetrad were generated to assess the requirement of Ago2 slicing activity during infection.

Functional analyses demonstrated that mutation of the Ago2 catalytic tetrad significantly impaired antiviral RNAi activity, resulting in enhanced viral replication compared with cells expressing wild-type Ago2. These findings confirm that Ago2 endonucleolytic activity is essential for efficient suppression of SFV replication and highlight the importance of RISC-mediated cleavage of viral RNA in antiviral defence. The results further support a model in which Dicer-2-mediated production of vsiRNAs alone is insufficient to control viral infection, and that effective antiviral activity requires functional Ago2-mediated target cleavage.

To further explore the regulation of Ago2 activity, immunoprecipitation coupled with mass spectrometry was used to identify proteins associated with Ago2 in mosquito cells under both infected and uninfected conditions. This analysis revealed a diverse Ago2 interactome containing RNA-binding proteins, RNA processing factors, and proteins of previously unknown function. Functional screening of selected candidate interactors identified the DEAD-box RNA helicase AAEL014414-PA (AAIP12) as a potential antiviral factor. Knockdown of AAIP12 resulted in increased viral replication, suggesting that this helicase contributes to host antiviral defence, potentially through modulation of RNA metabolism or RNAi-related processes.

Together, the findings presented in this thesis provide new insight into the molecular mechanisms underlying Ago2-mediated antiviral immunity in mosquito cells. By demonstrating the importance of Ago2 catalytic activity and identifying candidate host factors associated with Ago2 function, this work advances current understanding of RNAi-based antiviral defence in mosquito vectors. These findings contribute to a broader understanding of virus–vector interactions and provide a foundation for future studies exploring how host RNA regulatory pathways influence arbovirus replication and transmission.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported by funding from the Ministry of Higher Education, Science, Research and Innovation (Royal Thai Government Scholarship).
• Subjects: Q Science > QR Microbiology > QR355 Virology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Supervisor's Name: Brennan, Dr. Ben
• Date of Award: 2026
• Depositing User: Theses Team
• Unique ID: glathesis:2026-85996
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 12 Jun 2026 08:15
• Last Modified: 12 Jun 2026 08:16
• Thesis DOI: 10.5525/gla.thesis.85996
• URI: https://theses.gla.ac.uk/id/eprint/85996

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