Early cytoplasmic uncoating is associated with infectivity of HIV-1

Significance After fusion, HIV delivers its capsid formed of CA proteins into the cytoplasm. The regulated disassembly of the capsid (uncoating) is critical to the timely exposure of the viral genome and infection. The timing of this process is controversial. Utilizing an intravirion GFP fluid phase marker with a live-cell imaging approach, where we use less than one virion per cell, we demonstrate that the uncoating of HIV-1 particles is an early cytoplasmic process taking place ∼30 minutes after fusion, and dependent on ongoing reverse transcription. Early uncoating is observed in culture cells, primary T cells, and macrophages with either HIV or pseudotyped envelope. Partial CA retention with the viral complex facilitates late CA functions, including nuclear import and integration site selection. After fusion, HIV delivers its conical capsid into the cytoplasm. To release the contained reverse-transcribing viral genome, the capsid must disassemble in a process termed uncoating. Defining the kinetics, dynamics, and cellular location of uncoating of virions leading to infection has been confounded by defective, noninfectious particles and the stochastic minefield blocking access to host DNA. We used live-cell fluorescent imaging of intravirion fluid phase markers to monitor HIV-1 uncoating at the individual particle level. We find that HIV-1 uncoating of particles leading to infection is a cytoplasmic process that occurs ∼30 min postfusion. Most, but not all, of the capsid protein is rapidly shed in tissue culture and primary target cells, independent of entry pathway. Extended time-lapse imaging with less than one virion per cell allows identification of infected cells by Gag-GFP expression and directly links individual particle behavior to infectivity, providing unprecedented insights into the biology of HIV infection.