Understanding the role of Dead box helicase 1 (DDX1) and its co-factors in alphavirus infection

Palmalux, Natasha (2025) Understanding the role of Dead box helicase 1 (DDX1) and its co-factors in alphavirus infection. PhD thesis, University of Glasgow.

Abstract

The tRNA ligase complex (tRNA LC) is essential in tRNA maturation, stress response pathways and viral regulation, among others. It comprises proteins with distinct roles, including a non-canonical GMP-driven RNA ligase, RTCB, a cap-binding protein CGI99, and an ATP-dependent RNA helicase DDX1. In Sindbis virus (SINV), the tRNA-LC relocalises to viral replication organelles and interacts directly with viral RNA (vRNA). However, the functional role of the tRNA-LC in SINV infection remained unclear. This thesis characterises tRNA-LC interaction dynamics and elucidates its antiviral mechanism during SINV infection. Co-precipitation and crosslinking mass spectrometry (XL-MS) demonstrated robust inter-protein interactions, with DDX1, RTCB, and CGI99 forming the core of a tightly coordinated complex. CGI99 emerged as a central component, corroborated by Alphafold3 (AF3) modelling and complex destabilisation following a siRNA-mediated knockdown. Novel interactions, such as those with RPL11 and MYH9, suggest broader functional implications. The tRNA-LC displayed potent antiviral activity, as depletion of DDX1 and CGI99 significantly increased viral protein production and downregulated over 11,000 host genes during SINV infection. These findings indicate that tRNA-LC plays a central role in restricting viral lifecycle, and in its absence, the cellular microenvironment is more favourable to host viral infection. To identify the antiviral mechanism exerted by the tRNA-LC, I assessed how its RNA and protein interaction landscapes were altered during infection. The interaction landscape of the tRNA-LC analysed via iCLIP2 and protein-protein interaction analysis revealed a transition from cellular mRNA to vRNA binding during infection, primarily targeting the 5’ UTR and the start of the coding sequence. Enhanced interactions with ribosomal factors suggested involvement in translation regulation. Using a SINV replicon system, DDX1 was identified as regulating both viral replication and translation. This study proposes that the tRNA-LC inhibits vRNA processes by blocking essential viral factors such as replicase or translation components accessing the vRNA, offering new insights into its antiviral mechanisms and potential applications against positive-strand RNA viruses.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QR Microbiology > QR180 Immunology; Q Science > QR Microbiology > QR355 Virology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Supervisor's Name: Castello, Professor Alfredo and da Silva Filipe, Dr. Ana
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-85147
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 02 Jun 2025 09:43
• Last Modified: 02 Jun 2025 09:45
• Thesis DOI: 10.5525/gla.thesis.85147
• URI: https://theses.gla.ac.uk/id/eprint/85147

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