Research topics

HIV-1 persistence and control of virus replication in the brain

 

Current Team:
 Dr. Martha Schneider, Stephanie Rebensburg, Christine Kammel

Selected past contributors:
Manja Meggendorfer, Ina Rothenaigner, Michelle Vincendeau, Susanne Kramer-Hämmerle, Markus Neumann, Francesca Silberstein, Eva Ludwig, Alexandra Ludvigsen, Andrea Kleinschmidt

Goals:
Our group has a long-standing record of studying HIV-1-persistence in brain cells. Our overall goals are to:
(1) elucidate mechanisms that affect HIV replication in brain reservoirs;
(2) explore the potential of brain reservoir cells to contribute to neuropathogenicity of HIV.
We expect the understanding of virus-host interactions in brain cells to contribute to the development of therapeutic strategies that specifically target HIV reservoirs in the brain. 

Past work:
Astrocytes are the most abundant cells of the central nervous system. Their role as reservoirs for HIV-1 in the brain is supported by studies with postmortem brain tissues from infected individuals. We established a cell model for persistently HIV-1-infected astrocytes (TH4-7-5 cell line). These cells produce only very limited amounts of virus due to specific inhibition of the expression of viral structural proteins. In contrast, HIV-1 regulatory factors like Tat, Rev and Nef are efficiently expressed in TH4-7-5 cells (Brack-Werner, 1992; Kleinschmidt et al., 1994; Neumann et al.1995; Ludvigsen et al.,1996). Efficient production of HIV-1 structural proteins requires the viral Rev regulatory factor. We demonstrated that Rev shows poor activity, abnormal localization and impeded transport into the nucleus in astrocytes (Ludwig et al., 1999; Neumann et al., 2001). These studies provided the first evidence linking the Rev-regulatory axis with host control of HIV replication.

In addition we contributed to studies addressing the influence of HIV-1 persistence on expression of various genes in astrocytes. Upregulated production of cytokines, chemokines and cellular activation markers suggests that persistently HIV-1 infected astrocytes have the potential to contribute to neuropathogenic events in the brain. (Werner et al., 1991; Kohleisen et al., 1992; Cota et al., 2000; Andersen et al., 2003; Kramer-Hämmerle et al., 2005).

We used the expertise gained from studying HIV-1-cell interactions in astrocytes to investigate HIV-1-cell interactions in neural progenitor cells (NPC), which are crucial for the plasticity and self-repair of the brain. Using a human neural progenitor cell line as experimental model, we demonstrated that HIV-1 can persist in NPC populations and may induce cellular changes (Rothenaigner et al., 2007).

Together our studies indicate that astrocytes and neural progenitor cells have the potential to form HIV-1 reservoirs in the brain and that HIV-1 persistence may contribute to neuropathogenicity of HIV. These concepts have been confirmed and expanded by other published studies, which we summarized in several invited reviews (e.g. Brack-Werner, 1999; Kramer-Hämmerle et al., 2005).

Recent research highlights: 
We are using astrocytes to identify cellular factors that interact with Rev and that may play a role in modulating HIV replication during persistent infection. These studies revealed for the first time that several hnRNPs can interact with Rev via an N-terminal motif in Rev and that these hnRNPs may promote Rev activity (Hadian et al., 2009). We also identified an interesting family of proteins called Risp/FAM21 that suppress Rev activity, (Kramer-Hämmerle et al., 2005; Vincendeau et al., 2010). Risp/FAM21 expression is inversely related to Rev activity and virus production in astrocytes. Importantly, increasing Risp/FAM21 expression reduces residual virus production by persistently infected astrocytes to undetectable levels.

Ongoing work:
To study HIV-1 control in NPCs we generated enriched populations of persistently infected human NPCs. Interestingly, in these cells HIV replication is blocked at the level of transcription.  We have identified several agents that are capable of activating HIV transcription in these cells. We are using these cells as a model to (1) elucidate mechanisms of transcriptional HIV latency in the brain and to  (2) identify compounds that may prevent activation of HIV-1 transcription in the brain.

With regard to the Risp/FAM21 family, the goals of our current studies are to:
(1) to explore whether the inhibitory effects of Risp/FAM21 can be exploited to achieve stable HIV suppression in persistently infected cells, and to:
(2) investigate the influence of Risp/FAM21 expression on HIV production by various cell types, including immune cells.

Identification and evaluation of new inhibitors of HIV-1 replication

 

Current team:
Dr. Markus Helfer, Stephanie Rebensburg

Selected past contributors:
Dr. Stephan Kremb,  Dr. Horst Wolff  

Goals:
The overall goal of this project is to identify new inhibitors of HIV-1 replication which promote the development of urgently needed, affordable anti-HIV drugs. Therefore we try to exploit all available resources. These include small molecule-libraries as well as natural resources like terrestrial plants and herbal medicines or marine algae.

Past work: 
We previously developed a plasmid with a reporter gene that is activated by Rev and Tat to produce a red fluorescent protein (DsRed1) (Wolff et al., 2003). We used this reporter gene to quantify Rev activity in various studies (Wolff et al., 2006, Kramer-Hämmerle et al., 2005) and to identify cell-permeable peptides that inhibit Rev activity (Hariton-Gazal et al, 2005). Furthermore, we generated a HeLa-derived cell line (LC5-CD4) that expresses high levels of the HIV receptor CD4 and the co-receptor CXCR4 on the cell surface (Wachinger et al., 1998) and sublines that express CCR5 in addition to CD4 and CXCR4.

Recent research highlights:
We recently established full HIV-replication technology, which we call EASY-HIT (Exploratory Assay SYstem for the discovery of HIV inhibiTors), for the identification and analysis of HIV-1 inhibitors (Kremb et al., 2010). This technology is based on the HIV-1 indicator cell line LC5-RIC, which was established by stable insertion of the DSRed-1 reporter gene into LC5-CD4 cells. HIV-1 infection and expression of early viral proteins in LC5-RIC results in the production of the red fluorescent protein, providing a convenient, quantitative fluorescent marker for infection. We established a two-step assay that monitors HIV infection by determining both reporter production and amounts of infectious virus released by test cultures. This assay yields high scores for robustness and reproducibility and was validated for screening of compound libraries. Furthermore, this assay can be used to identify biological extracts with anti-HIV-1 activities and for bio-assay guided fractionation procedures. Furthermore, LC5-RIC sublines have been established for testing of HIV-1 variants with different cellular tropisms.

Even more recently, we identified and described a commercial herbal medicine made from the roots of the medicinal plant Pelargonium sidoides as a very potent inhibitor of HIV infection. Ingrerdients in extracts of this plant inactivate the virus thus hindering it to proper attach to its target cell (Helfer et al., 2014). 

Ongoing work: 
Our objective is to identify new hits for inhibition of HIV-1 replication and to evaluate their potential as leads for new anti-HIV drugs. For this purpose we are using the EASY-HIT technology to identify compounds that inhibit HIV at different steps of the HIV replication cycle. Libraries for screening include various small molecule libraries available at the HMGU and provided by industrial collaborators (Sanofi Aventis) as well as collections of biological extracts from different sources (plants, algae, etc.), either available at the HMGU or provided by collaborators from industry (e.g. Sanofi Aventis) and academia. These screening efforts have led to a number of interesting hits with novel modes-of-action or directed against novel targets. Among the latter is a peptoid that that binds CXCR4 (collaboration with Prof. Horst Kessler, TUM) and prevents entry of X4 viruses into cells (Demmer et al., 2012).

As part of the lead development efforts, we are studying the structure-activity-relationship (SAR) of several promising hit candidates in collaboration with Prof. Thorsten Bach, TUM.

Collaborations:
The highly interdisciplinary approach of our project depends on fruitful collaborations with scientists from various fields. Within the HMGU we collaborate with members of the Institute of Biochemical Plant Pathology (Prof. Jörg Durner) for analysis of potential anti-HIV activity of plant secondary metabolites. The chemical nature of anti-HIV-1 activity in biological extracts is analyzed in collaboration with the Analytical BioGeoChemistry unit (PD Dr. Philippe Schmitt-Kopplin). Libraries for screening are curated and stored by the Assay Development Unit, Institute of Toxicology (Dr. Kamyar Hadian). 

External collaborators include various members of the Department of Chemistry of the Technische Universität München (Prof. Thorsten Bach, Organic chemistry I; SAR of small molecules; Prof. Horst Kessler; CXCR4 antagonists). Close collaborations with experts of marine biology and marine biogeochemistry (Dr. Stephan Kremb, King Abdullah University of Science and Technology, Saudi-Arabia) allow us to address marine organisms for anti-HIV-1 activity.