Kliniken & Institute … Institute Zentrum für… Virologie Forschung Müller

Research Group Müller

You can find our lab on the 3rd floor of the CIID (INF344).

Projects

Our group is 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 between virus and 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, the bulk data and 'snapshots' obtained using these methods 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 techniques 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 that 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.

See also: Movie on our work

Dynamics of HIV-1 particle maturation

Newly produced 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 a defined and ordered sequence by the viral protease. This proteolytic processing is accompanied by dramatic rearrangements of the virus architecture. It is well established that the processes of proteolytic and structural maturation need to be very tightly regulated to achieve formation of infectious virus. Precise characterization of this regulation is thus crucial for our understanding of HIV-1 morphogenesis.


Despite intense research on this topic, a number of important questions are still unanswered - for example these rather basic questions:

  • when, where and how is maturation initiated? 
  • what are the structural intermediates of the maturation process? 
  • what is the time course of proteolytic and structural maturation?

In oder to develop a better understanding of the complex and dynamic events occurring during HIV-1 particle assembly, polyprotein processing and structural maturation, we develop novel fluorescence-based readout systems. These are used to monitor protease activation (or activity) at individual virus assembly sites by live cell microscopy. Our analyses are complemented by biochemical and virological approaches for studying the maturation process. Structural analyses of maturation intermediates are performed in collaboration.
 

Ca. 2500 molecules of Gag polyprotein assemble at the plasma membrane to form a virus bud. Immature 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 results in dramatic rearrangement of the virus architecture. The characteristic cone-shaped capsid is a hallmark of the infectious virus. © Virology Heidelberg.

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-defined nucleoprotein complexes (designated as reverse transcriptase complex and pre-integration complex, RTC and PIC).


Uncoating of the HIV-1 capsid is functionally linked to reverse transcription and nuclear import. As a consequence, events in the post-entry phase need to be very tightly controlled in time and space. Recently it became clear that the viral capsid protein plays a central role in regulating post-entry steps. A number of host cell proteins that interact with the viral capsid have been identified over the past years; these proteins either promote or restrict viral replication.


While these basic facts are well established, the sequence and intracellular localisation of molecular events, the temporal and functional correlation between subsequent steps, and the precise 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 with high time resolution. Thereby we can quantitatively describe a dynamic sequence of events. We  therefore develop and apply improved replication competent labeled HIV variants using various genetically encoded tags, as well as novel, minimally invasive labelling strategies (genetic code expansion and click chemistry). These derivatives allow us to apply advanced microscopic methods to visualize individual events in the early stages of the HIV-1 replication cycle. 

In close collaboration with groups from different disciplines within the SFB1129 we address questions as:
•    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?
 

Post-entry events in HIV-1 replication

Group members

Barbara Müller, apl. Prof. Dr. rer. nat.
phone: +49-6221-561325
fax: +49-6221-565003
e-mail

Afrodite Trapp, secretary (Lehrkoordination)
phone: +49-6221-5632563
fax: +49-6221-567838
e-mail

Maria Anders-Össwein, Technician (MTA)
phone: +49-6221-561324
fax: +49-6221-567838
e-mail

Dr. Annica Flemming, postdoc
phone: +49-6221-561323
fax: +49-6221-567838
e-mail

Rène Sahm, PhD student
phone: +49-6221-561323
fax: +49-6221-567838
e-mail

Sandra Schifferdecker, PhD student
phone: +49-6221-561324
fax: +49-6221-567838
e-mail

Stephanie Ullrich, PhD student
phone: +49-6221-561324
fax: +49-6221-567838
e-mail

 

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)

Clarissa Reinbott, Bachelor student
Dr. Volkan Sakin, postdoc
Anna-Lena Schäfer, Bachelor student
Christina Schulte-Huxel, Bachelor student

Katja Willrodt, Master student

Selected publications

complete publication list in Pubmed

 

***co-corresponding authors
.

Müller, T.G., Sakin, V., and Müller, B. (2019). A Spotlight on Viruses - Application of Click Chemistry to Visualize Virus-Cell Interactions. Molecules 24.

Bejarano, D.A., Peng, K., Laketa, V., Börner, K., Jost, K.L., Lucic, B., Glass, B., Lusic, M., Müller, B., and Kräusslich, H.G. (2019). HIV-1 nuclear import in macrophages is regulated by CPSF6-capsid interactions at the Nuclear Pore Complex. eLife, e41800

Imle, A., Kumberger, P., Schnellbacher, N.D., Fehr, J., Carrillo-Bustamante, P., Ales, J., Schmidt, P., Ritter, C., Godinez, W.J., Müller, B.,  Rohr, K., Hamprecht, F.A., Schwarz, U.S., Graw, F., and Fackler, O.T. (2019). Experimental and computational analyses reveal that environmental restrictions shape HIV-1 spread in 3D cultures. Nature communications 10, 2144.

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, 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]

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

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.

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.