Dynamic events in HIV‑1 replication
We are interested in the biology of the human immunodeficiency virus (HIV‑1). With our work, we want to contribute to a detailed understanding of the intricate interactions of this important pathogen with its host cell during the viral replication cycle.
The interaction of a virus with its host cell is a highly dynamic process, involving ordered and regulated formation, transport, transformation and dissociation of (nucleo)protein complexes. Biochemical, electron microscopic and structural studies provide detailed images of HI virions and information about the composition of functionally important subviral complexes. However, these methods yield snapshots or ensemble data, which do not reflect the dynamics of individual events occurring in the infected cell. In contrast, modern fluorescence imaging techniques enable us to directly observe transport processes and protein interactions within living cells. Furthermore, super-resolution and correlative microscopy now bridge the gap between electron and light microscopy, allowing the visualization of subviral details via fluorescence microscopy. All these advanced imaging methods require attachment of fluorescent labels to the viral protein of interest.
Our aim is to analyze dynamic processes in HIV‑1 replication in a quantitative manner. For this, we develop fluorescently labeled HIV‑1 derivatives and fluorescent probes, which allow us to follow individual events in virus-cell interaction with high time resolution. These studies are combined with biochemical and virological analyses. In our current work, we particularly focus on the processes involved in the formation of the infectious HIV‑1 capsid by proteolytic maturation, and on the fate and role of this capsid structure upon entry of the virus into a new host cell.
Figure 1 | Fluorescent probes and fluorescently labeled virus particles are important tools for our research.
1 | HIV‑1 Assembly and Maturation
HIV‑1 particles are released from the host cell as immature non-infectious virions. They only become infectious after undergoing a complex series of proteolytic maturation steps, in which the viral polyproteins Gag and GagPol are cleaved at multiple sites in an ordered sequence by the viral protease. This proteolytic processing is accompanied by dramatic rearrangements of the virus architecture. It is well established that the process of proteolytic and structural maturation needs to be tightly regulated to achieve formation of infectious virus. Precise characterization of this regulation is crucial for our understanding of HIV‑1 morphogenesis.
Despite intense research on this topic, a number of important questions are still unanswered. This includes very basic questions as:
when, where and how is maturation initiated?
- what are the structural intermediates?
- what is the time course of proteolytic and structural maturation?
Our aim is to develop a better understanding of the complex and dynamic events occurring during HIV‑1 particle assembly, polyprotein processing and structural maturation. For this, we develop novel fluorescence-based readout systems to monitor protease activation or activity, and combine biochemical and virological approaches with microscopic analyses.
Figure 2 | HIV‑1 assembly and maturation. Ca. 2500 molecules of Gag polyprotein assemble at the plasma membrane to form a spherical virus bud. Immature virus particles are released by abscission of the viral lipid envelope from the plasma membrane. The viral protease, which is packaged into the particle, cleaves the polyprotein into five domains. Protein processing is followed by dramatic rearrangements of the virus architecture, resulting in the characteristic cone-shaped capsid, a hallmark of the infectious form of the virus. © Virology Heidelberg.
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?
Figure 3 | HIV‑1 post entry events. The HIV‑1 capsid is released into the cytosol of an infected cell by membrane fusion. Subsequently, the viral RNA genome is reverse transcribed into double stranded DNA, which is transported to and into the nucleus, where it is integrated into the host cell genome. The viral capsid plays a central role in this replication phase. It acts as a container for reverse transcription of the viral RNA, mediates contact with cellular proteins that promote intracellular trafficking and nuclear import of the genome and probably serves to protect the viral genome from cellular defense systems. The timing and site of capsid uncoating and the recruitment of capsid-interacting host cell factors are critical determinants of HIV‑1 replication. With our work, we want to contribute to a detailed understanding of these processes.
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