Donaldson, Callum; (2018) Investigating the relationship between core stability and early life cycle events in HIV-1. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

HIV-1 capsid (CA) plays a vital role in the early stages of HIV-1 infection. The CA lattice surrounding the viral core is predominantly assembled from CA hexamers and stabilised by intra-hexamer and inter-hexamer interactions. Optimal stability of the lattice is known to be critical for efficient infection; however, a comprehensive screen of the effects of stabilising all lattice interfaces has not been performed. Disulphide cross-linking of residues across lattice interfaces has been used in vitro to stabilise CA assemblies. In this study, putatively stabilising cysteine CA mutations were designed at each interface of the CA lattice and their effects on early life cycle events, including reverse transcription and nuclear entry, assessed. The introduction of cysteine mutations at intra-hexamer (both NTD-NTD and NTD-CTD) and inter-hexamer (dimeric CTD-CTD only) lattice interfaces resulted in cross-linking and hyperstable viral cores in infected cells. These cores were minimally infectious and encountered sequential blocks to infectivity at reverse transcription, nuclear entry and post-nuclear entry. The infectivity defect of hyper-stable core mutant, A14C/E45C, was partially compensated – without an observable decrease in stability – by addition of mutations reported to perturb interactions with CPSF6. In contrast, Nup153 and CypA mutations were unable to compensate the infectivity defect suggesting that this was a CPSF6-specific effect. Proximal ligation assays were performed to visualise and quantify interactions between CA and host factors, indicating that hyper-stable cores encountered a block to nuclear entry in G1/S arrested cells. Overall, the results of this study suggest that mutations at different lattice interfaces can result in global changes to the intrinsic stability of the viral core and results in fitness defects at multiple stages of the HIV-1 early life cycle.

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