Research interests

All research projects in our group address different aspects of human immunodeficiency virus (HIV) biology. A major focus in the lab is the formation and architecture of the infectious HIV particle. These studies include the development of assembly inhibitors as a potential new antiviral strategy. A related project involves the investigation of HIV protease as a target for antiviral drugs, as well as the investigation of resistance development against inhibitors of protease and reverse transcriptase. Further projects address the role of host factors for HIV entry and release. More recently, we have been employing fluorescently labelled HIV derivatives to study the dynamics of HIV-cell interactions.

 

Assembly and release of HIV particles. Architecture of immature and mature HIV.

Assembly and budding of human immunodeficiency virus (HIV) is directed by the main structural polyprotein Gag and occurs at the plasma membrane of the infected cell. The virus is initially released as immature non-infectious particle consisting of mostly uncleaved Gag polyproteins. The immature virion consists of approximately 2500 Gag molecules arranged as spherical protein shell. Particle maturation occurs after proteolytic processing of Gag at five different cleavage sites, which is mediated by the virus-encoded, virion-associated protease (PR). Maturation leads to disassembly of the immature Gag layer, followed by a second assembly stage forming the mature cone-shaped capsid that incorporates the condensed viral genome associated with replication proteins.

Assembly of immature particles requires only the viral Gag polyprotein and can be mimicked in vitro using bacterially expressed purified Gag-derived proteins. We have developed in vitro assembly systems to analyze the molecular architecture of capsid assemblies, to study the effect of mutations on mature and immature particle formation and identify inhibitors of HIV assembly. These studies are complemented by analyses of HIV particle formation in tissue culture.
We employ structural analyses including transmission EM, cryo-EM and tomography to obtain detailed three-dimensional images of mature and immature virions, as well as viral budding sites, of wild-type and mutant HIV. These provide a better understanding of the mechanisms and protein interfaces governing assembly and maturation.

The assembly process also represents an attractive target for novel antiviral drugs. Using phage display we have identified the capsid assembly inhibitor peptide CAI as an inhibitor of HIV assembly in vitro. Structural analyses revealed that CAI binds to a previously unidentified assembly interface in the capsid protein CA. Further studies are aimed at identifying the role of this interface in assembly and maturation and at identifying small molecule analogs of CAI which can serve as lead compounds for antiviral drug development.

Structure of the CAI peptide (yellow) bound to the C-terminal domain of CA.

 

The functional role of cellular ESCRT proteins, as well as of protein ubiquitinylation and phosphorylation, for virion formation is investigated using cell biological and biochemical approaches. Furthermore, a random siRNA screening approach is directed towards identifying other cellular proteins required for HIV assembly and budding.

In collaboration with John Briggs (EMBL), Kay Grünewald (MPI for Biochemistry, Munich), Felix Rey (Institut Pasteur, Paris); EU consortium ‘HIV-ACE’ FP7

 

HIV Protease

The virus encoded protease (PR) induces HIV maturation by cleaving the viral polyproteins Gag and Gag-Pol into their functional subunits. Peptidomimetic inhibitors of PR prevent the formation of mature infectious HIV and are used as antiviral drugs for the treatment of HIV infected patients. However, resistance development limits their use. Thus, the development of alternative, structurally unrelated PR inhibitors would increase the therapeutic options. We are developing and using in vitro screening systems for the identification of novel PR inhibitors and have - together with our collaborators in Prague - described the non-peptidic carboranes as novel antiviral agents. Related studies are aimed at understanding the role of individual cleavage sites within Gag and the molecular mechanisms of resistance development against HIV PR inhibitors. Together with international collaborators from the EU project „HIV PI resistance“, we have characterized novel, substrate based resistance mechanisms, which will be the subject of further analyses.

Carborane inhibitors bound to the active site of PR

In collaboration with Jan Konvalinka (IOCHB, Prague),Francois Clavel (INSERM, Paris),  Monique Nijhuis  (UMC, Utrecht) - EU consortium „HIV PI resistance“ FP6. 

 

HIV-cell interactions

While numerous studies have yielded information on the interactions between viral proteins and cellular plasma membrane factors involved in viral entry, the dynamics of this process as well as the role of the host cell cytoskeleton are still not well understood. We have recently generated infectious fluorescent HIV derivatives, which allow the direct observation of virus-cell interactions in real time. These virus derivatives are also used to study dynamics of HIV particle formation. Furthermore, we have begun to explore high resolution fluorescence microscopy (STED, STORM) for the detailed analysis of HIV-cell interactions. A random siRNA screening approach (druggable genome), as well as several biochemical and cell biological approaches (e.g. TAP-tag purification, SILAC, automated confocal microscopy) are used to identify cellular proteins with functional roles in HIV replication.

A) Interaction of double fluorescent VLPs with HeLaP4 cells. Trajectories of double-labeled particles are shown in yellow. A bright-field image of the cell is underlaid in grey.
B) HI-virions (red) interacting with a living T-Lymphocyte (green).

 

In collaboration with Barbara Müller, Department of Virology Heidelberg ; Don Lamb and Christoph Bräuchle (LMU Munich) ; Karl Rohr and Roland Eils (DKFZ and BIOQUANT Heidelberg) ; Stefan Hell (BIOQUANT Heidelberg) ; Mike Heilemann (Bielefeld University) ; Holger Erfle, Rainer Pepperkok, Lars Kaderali. Viroquant ; SPP1175, SFB638.

 

Role of lipids in HIV replication

HIV is an enveloped virus, i.e. its protein capsid is surrounded by a lipid envelope, which is derived from the host cell membrane in the viral budding process. During infection of new host cells, this lipid envelope fuses to the cellular plasma membrane. Thus, lipids are involved in virus entry and egress, and they may play functional roles in viral protein composition, particle stability and infectivity. However, unlike viral proteins, viral lipids and their functional role a poorly understood. In collaboration with Britta Brügger and Felix Wieland, we have determined the lipidome of  a prototype HIV using mass spectrosopy and biochemical approaches. We are currently analyzing the influence of host cells and viral proteins on the lipid compostion of the envelope and study the influence of lipid composition on the properties of the virus. Using synthetic lipid vesicles, we also want to investigate the interactions of of virions and  viral proteins with lipid membranes.

In collaboration with Britta Brügger, Felix Wieland and Thomas Söllner (BZH Heidelberg), Petra Schwille (TU Dresden), SFB638, SPP1175.

 

Patient- and drug-specific models of HIV-1 entry

HIV-1 entry is a multistep process, involving interactions between the viral Envelope protein (Env) on the one side and the cellular receptor CD4 and a co-receptor molecule (the chemokine receptors CXCR4 or CCR5, respectively) on the other. The preference for one of these co-receptors (viral co-receptor tropism) and the entry efficiency into different host cells is largely determined by the amino acid sequence of the viral Env protein. The goal of this project is to develop a better understanding of the dependence of viral entry efficiency - as well as its sensitivity towards various entry inhibitors - on the viral Env sequence in conjunction with various receptor and co-receptor densities on the cell surface. In an interdisciplinary collaborative approach, this multi-dimensional data set will be evaluated to obtain computational models of the viral entry process. This will lead to a better understanding of the entry mechanism and the development of resistance against HIV entry inhibitors. Furthermore, improved algorithms predicting the efficacy of these drugs against virus variants detected in individual patients can be derived.

In collaboration with Barbara Müller, Department of Virology Heidelberg; Thomas Lengauer (MPI, Saarbrücken); Rolf Kaiser (University of Cologne); MedSys consortium.

 

HIV inhibitors and resistance – From bench to bedside:

Cooperations with CRSN Nouna to study the viral, immunological and public-health associated factors in transmission and therapy of HIV infection in Burkina Faso

The largely unabated HIV pandemic in sub-saharan Africa necessitates interdisciplinary projects linking virology, immunology and public health to address HIV transmission and therapy. In the framework of SFB 544 “Control of Tropical Infectious Diseases” we have launched a prevention of mother-to-child transmission service in Nouna. In collaboration with colleagues from Nouna and Ouagadougou we alsodetermine seroprevalence of HIV and other pathogens, analyze the genomic structure and resistance pattern of HIV, and determine normal ranges of lymphocyte subsets and response to antigen stimulation. While the majority of studies on antiviral resistance has focussed on HIV-1 subtype B, which is prevalent in Europe and the US, the vast majority of infections in sub-saharan Africa are caused by other HIV subtypes. In collaboration with researchers from Cameroon, we have generated prototype A/G-recombinant and O-type viruses, which we use to investigate potential subtype specific patterns of resistance development. Based on these results and the increasing availability of antiretroviral therapy (ART), we determine subtype-specific resistance, also addressing drug adherence, and study immune reconstitution and activation under ART.

 

Methods applied

The majority of our experiments are performed in tissue culture using non-infectious variants or fully infectious HIV and other retroviruses. We also work with purified components in vitro including structural analysis. Main methods in the lab include common technology in molecular cell biology with a focus on imaging, EM and tomography, high resolution light microscopy and live microscopy including photoconversion, FRET, FRAP.