After cell fusion, HIV delivers its conical capsid into the cytoplasm. The disassembly of the capsid is termed uncoating and is critical to infection. The understanding of the kinetics, dynamics, and localization of uncoating of infectious particles has been eluded by the unavoidable presence of non-infectious particles. The timing of uncoating remains under discussion with some models proposing that uncoating happens early and other models suggest that the intact capsid docks at the nuclear pore. These different hypotheses formed from diverse assays, lack the information for the kinetics and localization of uncoating of productively infectious viral particles.
We used live-cell fluorescent imaging of intravirion fluid phase markers to determine the integrity of the HIV conical capsid core. To visualize dynamic changes in capsid integrity and composition, we utilized the HIV-iGFP construct. During viral maturation of HIV-iGFP, the GFP is liberated from Gag. A minority population of the free GFP is trapped in the capsid, while the remaining free GFP is located outside of the capsid. With this technique, the loss of the fluid phase GFP occurs in two steps: with fusion and upon the loss of capsid core integrity. Live-cell microscopy of HIV-iGFP virions with a viral complex marker such as Vpr or Integrase allows for the timing of these two steps. Through viral challenge with less than one virion per cell we are able to connect viral particle phenotype to infection.
The time between fusion and capsid integrity loss, for both HIV and VSV-G mediated fusion, in tissue culture and primary cells (macrophages and T cells), is approximately 30 minutes. Also, capsid integrity loss occurs entirely in the cytoplasm and co-relates to a big loss of p24CA. With our low MOI approach, we were able to image individual particle uncoating that produces a viable infection and differentiate between different rates of uncoating. This analysis revealed that all particles associated with cellular infection showed changes in capsid integrity ~29 minutes. We were also able to halt uncoating by blocking specific steps of reverse transcription, linking uncoating to the occurrence of the first-strand transfer step.
Together, these observations validate the early cytoplasmic uncoating model. Our live-imaging assay has the ability to follow uncoating at the single infectious particle level providing unprecedented insights into the early steps of HIV infection.