Ward, Shannon Leigh
(2024)
An integrated approach to understanding the architecture of DDX3-mediated HIV translation pre-initiation complex.
PhD thesis, University of Nottingham.
Abstract
HIV transcripts contain highly structured 5′ untranslated regions (5′ UTR), necessitating a non-canonical translation pre-initiation complex (PIC), to allow ribosomes to bind and carry out translation. The PIC uniquely comprises the human DEAD-box RNA helicase, DDX3, bound directly to the trans-activation response (TAR) RNA stem loop of the 5′ UTR, with the addition of eukaryotic initiation factor 4G and poly(A) binding protein. Given its unique involvement in translation of HIV transcripts, the PIC makes an attractive drug target. This research aimed to resolve the high-resolution three-dimensional (3D) structure of the HIV translation PIC, to enhance understanding of its function and aid structure-based drug design for HIV therapeutics. In addition, the hypothesis that HIV Tat protein is a co-factor in PIC formation was investigated, because of its known interactions with DDX3 and TAR RNA.
A bottom-up approach was employed for recombinant protein expression and purification, to study the binding dynamics of DDX3 and TAR RNA in the presence of Tat, and to reconstitute a tripartite complex for structural analysis. Docking predictions and molecular dynamics simulations, using known structures from the Protein Data Bank , were used to investigate the potential interaction of Tats with DDX3 and TAR RNA in silico. Structure prediction tools, AlphaFold multimer and RoseTTAFoldNA, were used to predict the protein complex structure and TAR RNA interaction with DDX3. Additionally, a top-down strategy was used to purify the PIC in its native state, from lysates of HIV-infected cells. This involved partial purification using molecular weight cut-off centrifugal filters, followed by on-grid antibody affinity purification to enrich the PIC on electron microscopy (EM) grids.
Results show that cryo-EM may not be the optimal method for analysing the tripartite complex because of sample heterogeneity and complex size being too small as DDX3 appeared to bind TAR RNA as a monomer. The effect of Tat on the DDX3 – TAR RNA interaction could not be determined experimentally, prompting in silico examination, which indicated enhanced affinity between TAR RNA and DDX3 in the presence of Tats. Native gel electrophoresis revealed concentration-dependent oligomerisation of DDX3, which was enhanced in the presence of TAR RNA, indicating that PIC formation may be concentration dependent. Computational predictions propose a doughnut-shaped assembly of the PIC, containing an open centre that RNA could move through. In addition, RoseTTAFoldNA predicts that TAR RNA binds to DDX3 in a linear single stranded RNA conformation, supporting the proposed helicase unwinding mechanism of action. Native cryo-EM structure analysis resulted in a low-resolution structure of the PIC, revealing a globular structure with gaps.
Alternative methods are required to confirm the effect of Tat on the TAR RNA – DDX3 interaction. A top-down native purification approach appears the most suitable method of purification of the PIC for structural studies. Further work to obtain higher resolution structure information, combined with structure prediction fitting is required to elucidate the accurate high-resolution structure of this complex.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Borkar, Aditi
Scott, David |
| Keywords: |
HIV Tat protein; Pre-initiation complex formation; DDX3; Trans-activation response RNA |
| Subjects: |
Q Science > QR Microbiology > QR355 Virology |
| Faculties/Schools: |
UK Campuses > Faculty of Medicine and Health Sciences > School of Veterinary Medicine and Science |
| Item ID: |
78378 |
| Depositing User: |
Ward, Shannon
|
| Date Deposited: |
17 Jul 2024 04:40 |
| Last Modified: |
02 Sep 2024 13:01 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/78378 |
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