apl. Prof. Dr. Bar­bara Müller

Barbara.Mueller(at)med.uni-heidelberg.de
Phone: +49 6221 56–1325
Fax: +49 6221 56–5003

Dynamic events in HIV‑1 replication

Projects

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.

Com­plete Pub­li­ca­tion List (PubMed)
ORCID-iD: 0000–0001-5726–5585

*** 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.
  • 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.
  • 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.