The HIV-1 capsid has been recognised as an attractive antiviral target due to its central role during infection. It acts as a shield to protect the viral genome, facilitates reverse transcription, engages with the nuclear pore complex, and directs integration site targeting. To achieve all of these functions, the capsid disassembles, in a process referred to as uncoating, with temporal and spatial precision. HIV has evolved to exploit a network of host cofactors to regulate capsid stability

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... e the cell has developed restriction factors that recognise the CA lattice to block infection. Despite of that, only few host binders have been identified, and their precise role is not well understood. The fragility of the capsid has so far hindered the development of methods sensitive enough to identify different uncoating behaviours in a high throughput way to allow detailed kinetic studies of how molecules alter capsid stability. Here, we have developed a single-particle fluorescence microscopy method to follow the real-time uncoating kinetics of authentic HIV capsids in vitro. This novel assay allows us to visualise the dynamic interactions of molecules with hundreds of individual capsids and determine their effect on capsid stability. We found that spontaneous uncoating is initiated shortly after the viral membrane is compromised. The opening of the first defect in the lattice is the rate-limiting step of uncoating, which is followed by a rapid, catastrophic collapse. Interestingly, we found that the two phases of uncoating can be modulated differentially. Binding of factors to the CANTD-CTD hexameric interface, such as PF74 and CPSF6p, accelerate or do not alter capsid opening but strongly stabilise the remaining lattice. In contrast, binding of polyanions to the central capsid pore, such as hexacarboxybenzene and the newly discovered CA-binders IP6 and ATP, strongly delay initiation of uncoating but do not prevent subsequent lattice disassembly. We also determined that CPSF6 binds the HIV-1 capsid with higher affin [...]