Abstract

The acquired immune deficiency syndrome (AIDS) has caused over 60 million deaths since the etiological agent, human immunodeficiency virus type 1 (HIV-1), was first discovered in 1981. Over 6000 new HIV-1 infections are reported every day, predominantly in economically deprived regions of sub-Saharan Africa. Despite impressive developments in antiretroviral therapy, current medical intervention is unable to prevent or cure HIV-1 infection, necessitating expensive life-long treatment. Difficulties in establishing a vaccine or cure, arise from its capacity to cause life-long latent infection, and its extraordinary ability to evolve resistance to therapeutic intervention. Capsid uncoating is the process by which p24CA proteins (CA) disassemble from the viral ribonucleoprotein during the early phase of the HIV-1 lifecycle. Despite intensive investigation, much is yet unknown about the spatial and temporal occurrence of this process within the cell, and the viral or host cellular factors involved. However, studies investigating p24CA mutations which alter the stability, and consequently the kinetics of capsid uncoating, have shown that timely capsid uncoating is crucial for efficient HIV-1 infection. Recent advancements in microscopic techniques have enabled high resolution analysis of cellular protein interactions, including the in situ localisation and dynamics of these events. We have developed three imaging techniques for the analysis of different aspects of HIV-1 capsid uncoating: 1) a dual-fluorescently labelled virus 2) a fluorescently labelled antibody targeting a capsid internalised repeat peptide array, and 3) a split-luciferase system tagging an internalised component of the capsid core. Fluorescent labelling of both the CA and IN proteins enabled the sensitive and specific analysis of uncoating in response to both restriction factors and CA mutations, and the visualisation of CA colocalised pre-integration complexes (PICs) within the nucleus. This system is ideally suited for studying the longer-term kinetics of uncoating, and the in-situ visualisation of protein interactions. The use of the repeat peptide array in conjunction with fluorescently labelled antibodies, reinforced reports of an initial uncoating event early after viral fusion. This system enabled the rapid and reproducible imaging of uncoating events in real-time, within the same cell sample population. Finally, the split-luciferase system added further weight to a primary early uncoating stage, and showed capsid disassembly responses specific to mutations within the p24CA that affect the stability of the viral core. Put together, these three assays support a model of uncoating involving an initial early phase of uncoating, followed by a more gradual disassembly of CA from the PICs during cytoplasmic trafficking towards the nucleus. The colocalisation of these components within the nucleus suggests incomplete uncoating at the time of nuclear docking. The user-friendliness of the split-luciferase system, along with its capacity for high-throughput, real-time analysis, support great potential for its use as a screening assay for testing antiviral compounds targeting capsid uncoating events.