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Research group Müller

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We have moved! You can find us now on the 3rd floor of the CIID (INF344).


Fluorescently labelled HIV derivatives for studying virus-cell interactions

The interaction of viruses with their host cell is a highly dynamic process, involving the ordered and regulated formation, transport, transformation and decay of protein complexes. Biochemical, electron microscopical and structural studies revealed detailed images of mature and immature HI virions and provide information about the composition of functionally important subviral complexes. However, these methods yield static images or ensemble data, which do not reflect the dynamics of individual events occurring in the infected cell. In contrast, modern imaging techniques enable us to observe transport processes and protein interactions within living cells. Furthermore, super-resolution fluorescence microscopy and correlative microscopy have started to bridge the gap between electron and light microscopy, allowing the visualization of subviral details by fluorescence microscopy. All these advanced imaging methods require attachment of fluorescent labels to the viral protein of interest.

For a quantitative investigation of dynamic processes in HIV-1 replication we have developed fluorescently labeled HIV-1 derivatives that allow us to follow individual events in virus-cell interaction with high time resolution (~1s). We found that a fluorescent protein can be inserted between the matrix and capsid domains of the main viral structural protein Gag in the complete viral context. Co-expression with unlabeled virus allows us to produce labeled virus with wild-type protein composition and morphology. While this system does not permit the analysis of HIV-1 spread over multiple rounds of replication, we can use it to visualize attachment and uptake of individual virus particles or to analyze formation of individual particles at the plasma membrane of HIV-1 producing cells. In collaboration with the group of D. Lamb (LMU Munich) we have used this system to determine the kinetics of Gag assembly at the plasma membrane. We also apply it to characterize the transient recruitment of cellular proteins involved in HIV-1 release to nascent viral assembly sites and to study the effect of F-actin on the assembly process.

In the past years we have expanded our panel of labeled HIV-1 derivatives, including variants suitable for super-resolution or correlative microscopy. Currently, we are exploring novel labeling strategies that should yield labeled HIV-1 variants with improved replication capacity and permit the detailed observation of other steps in HIV-1 replication.

Dynamics of HIV-1 maturation

HIV-1 particles are released from the host cell as immature virions, that only become infectious after undergoing a complex series of proteolytic maturation steps. The viral polyproteins Gag and GagPol are cleaved at multiple sites by the viral protease. This proteolytic processing is accompanied by dramatic rearrangements of the virion architecture.

Despite intense research on HIV-1 maturation, a number of important questions are still unanswered. This includes very basic questions as:

…when and where is maturation initiated?
…how is it triggered?
…what are the structural intermediates?
…how long does maturation take?

It is known from the work of many groups, including ours, that the process of maturation needs to be tightly regulated to ensure formation of infectious virus. Thus, answers to all these basic questions are crucial for our understanding of HIV-1 morphogenesis.

Our aim is to develop a better understanding of the complex , dynamic events occurring during HIV-1 maturation. For this, we combine biochemical and virological approaches with novel fluorescence-based readout systems and microscopic analyses.

This project is currently funded by a grant from the Deutsche Forschungsgemeinschaft. We collaborate with the groups of Jan Konvalinka (IOCHB Prague; protease enzymology and chemical biology), John Briggs (EMBL, Heidelberg; virus structure) and Don Lamb (LMU Munich; biophysics).

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. Env glycoprotein mediated fusion of the viral envelope with the cell membrane releases the capsid, containing a nucleoprotein complex comprising the RNA genome, into the cytoplasm. There, the viral RNA genome is converted into DNA by the viral reverse transcriptase. The DNA copy is transported, as part of a nucleoprotein complex to the nucleus and through the nuclear pore to the host cell genome, to which it is then covalently linked by the viral integrase. Reverse transcription and nuclear transport occur within ill-characterised nucleoprotein assemblies, functionally designated reverse transcriptase complex (RTC) and pre-integration complex (PIC).

Uncoating of the viral capisid appears to be functionally linked to reverse transcription and nuclear import. Thus, it can be assumed that events in the post-entry phase need to be very tightly controlled in time and space. Recent results from different labs indicate that the viral CA protein plays a key role. An increasing number of host cell proteins is also implicated in these events, reported to either promote or restrict viral replication.

While these key features are universally accepted, 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 subject of intense debate.

Important questions we try to address in our work are:

What is the role of viral proteins?
Where and when does capsid uncoating happen?
What is the function of specific host cell factors?
What is the mechanism of PIC nuclear import?

The study of post-enty events is complicated by the facts that:
* the subviral compexes undergo a series of dynamic transitions
*events are not synchronized; a cell may contain several complexes in different stages
* not all entry events are productive; some - or many - may lead into a dead end
* several pathways appear to exist for distinct steps; these may differ in different cell types, may be used alternatively, or may 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.

Therefore we are currently developing and applying novel, minimally invasive labelling strategies with the aim of generating multi-labeled HIV-1 derivatives. These should allow us to visualize individual events in the early stages of the HIV-1 replication cycle using advanced microscopic methods.

This project is currently funded by the Deutsche Forschungsgemeinschaft through a project within SFB1129 and the EcTop programme from the the CellNetworks Cluster of Excellence.
SFB1129 - Project 6
We collaborate with the groups of Edward Lemke (EMBL, Heidelberg; novel labelling strategies), Carsten Schultz (EMBL, Heidelberg; chemical biology) and Hans-Georg Kräusslich (Virology Heidelberg; architecture of HIV-1 post-entry complexes)

Group members

Barbara Müller, apl. Prof., Dr. rer. nat.
phone: +49-6221-561325
fax: +49-6221-567888
e-mail: barbara_mueller@med.uni-heidelberg.de

Maria Anders-Össwein, Technician (MTA)
phone: +49-6221-561324
fax: +49-6221-567888
e-mail: maria.anders@med.uni-heidelberg.de

Annica Flemming, PhD student
phone: +49-6221-561323
fax: +49-6221-567888
e-mail: annica.flemming@med.uni-heidelberg.de
Rène Sahm, PhD student
phone: +49-6221-561323
fax: +49-6221-567888
e-mail: rene.sahm@med.uni-heidelberg.de
Sandra Schifferdecker, Master student
phone: +49-6221-561324
fax: +49-6221-567888

Former lab members

Jessica Dunder, Masters student
Manon Eckhardt, PhD student, postdoc
Anke-Mareil Heuser, technician
Christa Kuhn, guest scientist
Marko Lampe, PhD student
Liridona Maliqi, Bachelor student 
Anja Meier, Diploma student
Denise Müller, Technician
Oliver Meub, Diploma student
Sheikh Abdul Rahman, PhD Student
Anna Carydis Ramirez, guest scientist (CIGB Havanna)
Dr. Volkan Sakin, postdoc
Anna-Lena Schäfer, Bachelor student
Christina Schulte-Huxel, bachelor student

Selected publications

***co-corresponding authors

Mattei S., Tan A, Glass B, Müller B., Kräusslich H.G., and Briggs J.A.G. (2018) High-resolution structures of HIV-1 Gag cleavage mutants determine structural switch for virus maturation. Proc Natl Acad Sci USA. https://doi.org/10.1073/pnas.181123711.5
Sakin V, Hanne J, Dunder J, Anders-Össwein M, Laketa V, Nikić I, Kräusslich HG, Lemke EA, Müller B. (2017) A Versatile Tool for Live-Cell Imaging and Super-Resolution Nanoscopy Studies of HIV-1 Env Distribution and Mobility. Cell Chem Biol. 24: 635-645.e5
see also: https://doi.org/10.1016/j.chembiol.2017.05.006
Mücksch F, Laketa V, Müller B, Schultz C, Kräusslich HG (2017) Synchronized HIV assembly by tunable PIP2 changes reveals PIP2 requirement for stable Gag anchoring. Elife. pii: e25287. doi: 10.7554/eLife.25287
Hanne J,  Göttfert F, Schimer J, Anders-Össwein M, Konvalinka J, Engelhardt J, Müller B, Hell SW,  Kräusslich HG (2016). Stimulated Emission Depletion Nanoscopy Reveals Time-Course of Human Immunodeficiency Virus Proteolytic Maturation . ACS Nano, 10: 8215-22
FEBS Letters Special Issue (2016) Integrative analysis of pathogen replication and spread. http://febs.onlinelibrary.wiley.com/hub/issue/10.1111/feb2.2016.590.issue-13/
Konvalinka, J., Kräusslich, H.G., and Müller, B. (2015). Retroviral proteases and their roles in virion maturation. Virology 479-480:403-13
Schimer J, Pavova M, Anders M, Pachl P, Sacha P, Cigler P, Weber J, Majer P, Rezacova P, Kräusslich HG, Müller B***, Konvlinka J***, (2015). Triggering HIV polyprotein processing inside virions by rapid photodegradation of a tight-binding photodestructable protease Inhibitor. Nature Communications 6:6461
Peng K, Muranyi W., Glass B, Laketa V, Yant SR, Tsai L, Cihlar T, Müller B, Kräusslich, HG. (2014) Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid. eLife 2014 10.7554/eLife.04114
Mattei, S., Anders, M., Konvalinka, J., Kräusslich, H.G., Briggs, J.A.G. and Müller, B. (2014) Induced maturation of human immunodeficiency virus.  J Virol. 88:13722-31
Schur, F.K.M., Hagen, W.J.H., Rumlová, M., Ruml, T., Müller, B., Kräusslich, H.G., and Briggs, J.A.G. (2014) The structure of the immature HIV-1 capsid in intact . virus particles at 8.8 Å resolution. Nature. 2014 Nov 2. doi: 10.1038/nature13838
Rahman, S.A., Koch, P., Weichsel, J., Godinez, W.J., Schwarz, U., Rohr, K., Lamb, D.C., Kräusslich, H.G., and Müller, B. (2014). Investigating the role of F-actin in human immunodeficiency virus assembly by live-cell microscopy. J Virol. 88:7904-14
Müller, B., Anders, M., and Reinstein, J. (2014) In vitro analysis of HIV-1 particle dissociation: Gag proteolytic processing influences dissociation kinetics. PLoS ONE 9:e99504
Müller B, Kräusslich HG (2014). HIV-1 Maturation. Springer Encyclopedia of AIDS DOI 10.1007/978-1-4614-9610-6_59-1
Müller, B., and Krijnse-Locker, J. (2014). Imaging of HIV assembly and release. In: Methods in molecular biology, Springer Verlag, 1087, 167-184
Müller B and Heilemann M (2013). Shedding new light on viruses: super-resolution microscopy for studying human immunodeficiency virus. Trends in Microbiology 21:522-33
Könnyü B, Sadiq SK, Turányi T, Hírmondó R, Müller B, Kräusslich HG, Coveney PV, Müller V. 2013. Gag-Pol Processing during HIV-1 Virion Maturation: A Systems Biology Approach. PLoS Comput Biol. 9(6):e1003103.
Muranyi, W., S. Malkusch, B. Müller, M. Heilemann, and H. G. Kräusslich.2013. Super-resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites dependent on the Envelope C-Terminal Tail.
PLoS Pathog, 9:e1003198
de Marco A, Heuser AM, Glass B, Krausslich HG, MÜller B*, Briggs JA*. 2012. The role of the SP2 domain and its proteolytic cleavage in HIV-1 structural maturation and infectivity. J Virol 86:13708

Chojnacki J, Müller B. 2013. Investigation of HIV-1 Assembly and Release Using Modern Fluorescence Imaging Techniques. Traffic 14(1):15-24
Chojnacki J, Staudt T, Glass B, Bingen P, Engelhardt J, Anders M, Schneider J, B. M, Hell S, Kräusslich HG. 2012. Maturation Dependent HIV-1 Surface Protein Redistribution Revealed by Fluorescence Nanoscopy. Science 2012, 338:524-528.
Bozek K, Eckhardt M, Sierra S, Anders M, Kaiser R,  Kräusslich HG, Müller B*, Lengauer T*. 2012. An expanded model for HIV-1 cell entry phenotype based on multi-parameter single-cell data. Retrovirology,
Eckhardt M, Anders M, Muranyi W, Heilemann M, Krijnse-Locker J, Müller B. A SNAP-tagged derivative of HIV-1--a versatile tool to study virus-cell interactions. PLoS One. 2011;6(7):e22007. Epub 2011 Jul 22.
Baumgärtel V, Ivanchenko S, Dupont A, Sergeev M, Wiseman PW, Kräusslich HG, Bräuchle C, Müller B*, Lamb DC* (2011)  Dynamics of HIV budding site interactions with an ESCRT component visualized in live cells. Nat Cell Biol,13: 469-474

Jochmanns D, Anders M, Keuleers I, Smeulders L, Kraeusslich HG, Kraus G, Mueller B. (2010) Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease. Retrovirology 7(1):89. [Epub ahead of print]
Hermle J, Anders M, Heuser AM, Müller B. (2010) A simple fluorescence based assay for quantification of human immunodeficiency virus particle release. BMC Biotechnol. 10(1):32.
Ivanchenko S,  Godinez WJ, Lampe M, Kräusslich HG, Eils R, Rohr K, Bräuchle C, Müller B*, and DC Lamb (2009) Dynamics of HIV-1 Assembly and Release. PLoS Pathogens 5:e1000652
Müller B*, Anders M, Akiyama H, Welsch S, Glass B, Nikovics K, Clavel F, Tervo HM, Keppler OT and H.G. Kräusslich. (2009) Human immunodeficiency virus Gag processing intermediates trans-dominantly interfere with HIV-1 infectivity. JBC 284:29692-703

Koch P, Lampe M, Godinez W, Müller B, Rohr K, Kräusslich HG and M. Lehmann. (2009) Visualizing pseudotyped HIV-1 particles in real time by live cell microscopy, Retrovirology, 6(1):84.

Carlson LA, Briggs JA, Glass B, Riches JD, Simon MN, Johnson MC, Müller B, Grünewald K, Kräusslich HG. 2008. Three-dimensional analysis of budding sites and released virus suggests a revised model for HIV-1 morphogenesis. Cell Host Microbe. 4:592-9

Lampe M, Briggs JA, Endress T, Glass B, Riegelsberger S, Kraeusslich HG, Lamb DC, Braeuchle C, Mueller B. (2007) Double-labelled HIV-1 particles for study of virus-cell interaction. Virology 360, 92-104. Epub 2006 Nov 9.

Mueller B, Daecke J, Fackler OT, Dittmar MT, Zentgraf H, Kraeusslich HG (2004) Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative. J Virol 78, 10803-10813.

von Schwedler UK, Stuchell M, Mueller B, Ward DM, Chung HY, Morita E, Wang HE, Davis T, He GP, Cimbora DM, Scott A, Kraeusslich HG, Kaplan J, Morham SG, Sundquist WI (2003) The protein network of HIV budding. Cell 114:701-13.

Mueller B, Patschinsky T, Kraeusslich HG (2002) The late-domain-containing protein p6 is the predominant phosphoprotein of human immunodeficiency virus type 1 particles. J Virol 76:1015-1024.

Mueller B, Tessmer U, Schubert U, Kraeusslich HG (2000) Human immunodeficiency virus type 1 Vpr protein is incorporated into the virion in significantly smaller amounts than gag and is phosphorylated in infected cells. J Virol 74:9727-9731.

Gross I, Hohenberg H, Wilk T, Wiegers K, Grattinger M, Mueller B, Fuller S, Kraeusslich HG (2000) A conformational switch controlling HIV-1 morphogenesis. EMBO J 19:103-113.