In this project, we want to elucidate the dynamic events in the early phase of HIV-1 replication, from entry of the viral capsid into the cell up to release of the viral genome („uncoating“) for integration into the host cell DNA. Towards this aim, we develop and apply fluorescent labeling strategies that allow us to visualize entering viral complexes by live cell imaging and super-resolution microscopy.
In order to follow the capsid on its journey through the infected cell, we have developed a minimally invasive strategy to tag the capsid protein CA with fluorescent dye molecules, based on genetic code expansion (GCE) and click-labelling (Schifferdecker et al.). We have also collaborated with the Kräusslich group to establish detection of newly synthesized HIV-1 DNA by live cell microscopy (T. Müller et al., Elife 2021).
Using these tools, together with correlative light and electron microscopy (CLEM) conducted by Kräusslich and Lusic/Beck in collaboration with us (Zila, Margiotta et al., Cell 2021) lead to a revised model of HIV-1 post-entry. While it was initially assumed that the role of the capsid is mainly to deliver the viral genome into the cytosol, results from us and others indicate that CA stays associated with the viral genome up to the nucleus and even during passage through the nuclear pore. Completion of reverse transcription of the genome and its release from the capsid apparently occur in the nucleus, when the HIV-1 has reached the site of integration. The capsid protects the genome throughout this passage and mediates interactions with crucial host factors. It can thus be considered to be the master organizer of the post entry replication phase (T. Müller et al., Ann Rev Virology 2022).