2 | Dynamics of HIV‑1 post-entry events
Early post-entry events, from cytoplasmic entry of the viral core to integration of the viral genome into the host DNA, represent the most enigmatic steps in the HIV‑1 replication cycle. Fusion of the viral envelope with the cell membrane releases the capsid, which encases the ssRNA genome, into the cytoplasm. The viral RNA genome is converted into dsDNA by the viral reverse transcriptase and transported through the nuclear pore into the host nucleus, where it is covalently integrated into the host genome. Reverse transcription, nuclear import and integration occur within ill-characterised nucleoprotein complexes (functionally designated as reverse transcriptase complex and pre-integration complex).
Uncoating of the HIV‑1 capsid is apparently functionally linked to reverse transcription and nuclear import. Therefore, events in the post-entry phase need to be very tightly controlled in time and space. Results from many labs indicate that the viral capsid plays a central role in regulating post-entry steps. An increasing number of capsid binding host cell proteins that either promote or restrict viral replication is also implicated in these events.
While these facts are well established, the sequence and intracellular localisation of molecular events, temporal and functional correlation between subsequent steps and the roles of viral and cellular proteins involved are a matter of intense debate. The study of post-entry events is complicated by the facts that (i) the subviral complexes undergo a series of dynamic transitions, (ii) events are not synchronized and a cell may contain many viral complexes in different stages, and (iii) not all entry events are productive and some — or many — lead into a dead end. Furthermore, alternative pathways appear to exist for distinct steps; these may differ between different cell types, may be used alternatively, or even occur in parallel
Live-cell microscopy can help to overcome these obstacles, since it allows focusing on individual subviral complexes and can quantitatively describe a dynamic sequence of events with high time resolution. We are therefore developing and applying improved replication competent labeled HIV variants and novel, minimally invasive labelling strategies, in order to visualize individual events in the early stages of the HIV‑1 replication cycle using advanced microscopic methods.
Important questions we try to address in our work are:
- What is the role of viral proteins in post entry steps?
- Where and when does capsid uncoating happen?
- What is the function of specific host cell factors?
- What is the mechanism of PIC nuclear import in different host cell types?