Burgess, Ailidh Jean Owen; (2025) Structural and functional studies of DARPin interactions with HIV-1 CA. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The HIV-1 capsid core, comprising ~250 hexamers and 12 pentamers of the capsid protein, primarily functions to encapsidate and protect the viral genome and the cadre of non-structural proteins required for replication and integration. In addition to this passive function, it is now apparent that ordered capsid assemblies are required for the spatiotemporal coordination of reverse transcription with viral uncoating. Moreover, capsid assemblies mediate interactions with various host cell factors required for viral cytoplasmic trafficking, entry into the nucleus, and targeting of viral genomes to sites of integration. Indeed, the structure, conformation, and degree of stability of capsid assemblies are all critical determinants of successful viral replication. Further, the multifunctionality of capsid renders it genetically vulnerable and therefore a good target for restriction factors and new therapeutics. To probe how interactions with capsid can modulate its function, this study employed Designed Ankyrin Repeat Protein (DARPin) technology. DARPins are antibody mimetics that offer diverse libraries of high affinity and high specificity binders. With a simple rigid architecture, high stability, and high expression levels, DARPins are attractive reagents for both in vitro and in cell-based studies. Solution NMR and SEC-MALLS were used to screen a panel of capsidbinding DARPins, finding differential capsid hexamer/monomer specificity and revealing five distinct classes of capsid-binding DARPins. In complementary structural studies, DARPin interactions with capsid were characterised using a combination of X-ray crystallography and cryoEM. These structural and biophysical studies provide the molecular details of capsid-recognition by these targeting molecules, and in combination with cell-based studies, identified a DARPin capable of inhibiting infection, acting as an artificial restriction factor with therapeutic potential. Additionally, DARPins that preferentially bind different oligomeric states of capsid, without impacting virus replication, have potential as molecular tools to probe the trajectory of the capsid core and the uncoating process within infected cells.

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