apl. Prof. Dr. Bar­bara Müller

Barbara.Mueller@med.uni-heidelberg.de

Phone: ++49-(0)6221–56-1325

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.

Selected Publications

Com­plete pub­li­ca­tion list (PubMed)

OrcID: 0000–0001-5726–5585

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.

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. Pro­ceed­ings of the Nation­al Acad­e­my of Sci­ences of the Unit­ed States of Amer­i­ca 115, E9401-E9410.

Sakin, V., Hanne, J., Dun­der, J., Anders-Öss­wein, M., Lake­ta, V., Nikic, I., Kräus­slich, H.G., Lemke, E.A., and Müller, B. (2017). A Ver­sa­tile Tool for Live-Cell Imag­ing and Super-Res­o­lu­tion Nanoscopy Stud­ies of HIV‑1 Env Dis­tri­b­u­tion and Mobil­i­ty. Cell chem­i­cal biol­o­gy 24, 635–645 e635. IF 5,592

Schimer, J., Pavo­va, M., Anders, M., Pachl, P., Sacha, P., Cigler, P., Weber, J., Majer, P., Reza­co­va, P., Kräus­slich, H.G., Müller, B.* and Kon­va­lin­ka, J.* (2015). Trig­ger­ing HIV polypro­tein pro­cess­ing by light using rapid pho­todegra­da­tion of a tight-bind­ing pro­tease inhibitor. Nature com­mu­ni­ca­tions 6, 6461.

Mat­tei, S., Flem­ming, A., Anders-Öss­wein, M., Kräus­slich, H.G., Brig­gs, J.A.*, and Müller, B.* (2015). RNA and nucle­o­cap­sid are dis­pens­able for mature HIV‑1 cap­sid core assem­bly. Jour­nal of virol­o­gy 89, 9739–9747.

Schur, F.K., Hagen, W.J., Rumlo­va, M., Ruml, T., Müller, B., Kräus­slich, H.G., and Brig­gs, J.A. (2015). Struc­ture of the imma­ture HIV‑1 cap­sid in intact virus par­ti­cles at 8.8 A res­o­lu­tion. Nature 517, 505–508.

Mat­tei, S., Anders, M., Kon­va­lin­ka, J., Kräus­slich, H.G., Brig­gs, J.A., and Müller, B. (2014). Induced mat­u­ra­tion of human immun­od­e­fi­cien­cy virus. Jour­nal of virol­o­gy 88, 13722–13731.

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. Jour­nal of virol­o­gy 88, 7904–7914. IF 4,368

Cho­j­nac­ki, J., Staudt, T., Glass, B., Bin­gen, P., Engel­hardt, J., Anders, M., Schnei­der, J., Müller, B., Hell, S.W., and Kräus­slich, H.G. (2012). Mat­u­ra­tion-depen­dent HIV‑1 sur­face pro­tein redis­tri­b­u­tion revealed by flu­o­res­cence nanoscopy. Sci­ence 338, 524–528.

de Mar­co, A., Heuser, A.M., Glass, B., Kräus­slich, H.G., Müller, B.*, and Brig­gs, J.A.* (2012). Role of the SP2 domain and its pro­te­olyt­ic cleav­age in HIV‑1 struc­tur­al mat­u­ra­tion and infec­tiv­i­ty. Jour­nal of virol­o­gy 86, 13708–13716.

Eck­hardt, M., Anders, M., Muranyi, W., Heile­mann, M., Kri­jnse-Lock­er, J., and Müller, B. (2011). A SNAP-tagged deriv­a­tive of HIV‑1–a ver­sa­tile tool to study virus-cell inter­ac­tions. PloS one 6, e22007.

Baumgär­tel, V., Ivanchenko, S., Dupont, A., Sergeev, M., Wise­man, P.W., Kräus­slich, H.G., Bräuch­le, C., Müller, B.*, and Lamb, D.C.* (2011). Live-cell visu­al­iza­tion of dynam­ics of HIV bud­ding site inter­ac­tions with an ESCRT com­po­nent. Nature cell biol­o­gy 13, 469–474. IF 19,064

Jochmans, D., Anders, M., Keuleers, I., Smeul­ders, L., Kräus­slich, H.G., Kraus, G., and Müller, B. (2010). Selec­tive killing of human immun­od­e­fi­cien­cy virus infect­ed cells by non-nucle­o­side reverse tran­scrip­tase inhibitor-induced acti­va­tion of HIV pro­tease. Retro­vi­rol­o­gy 7, 89.

Ivanchenko, S., Godinez, W.J., Lampe, M., Kräus­slich, H.G., Eils, R., Rohr, K., Bräuch­le, C., Müller, B.*, and Lamb, D.C.* (2009). Dynam­ics of HIV‑1 assem­bly and release. PLoS pathogens 5, e1000652.

Lampe, M., Brig­gs, J.A., Endress, T., Glass, B., Riegels­berg­er, S., Kräus­slich, H.G., Lamb, D.C., Bräuch­le, C., and Müller, B. (2007). Dou­ble-labelled HIV‑1 par­ti­cles for study of virus-cell inter­ac­tion. Virol­o­gy 360, 92–104.

von Schwedler, U.K., Stuchell, M., Müller, B., Ward, D.M., Chung, H.Y., Mori­ta, E., Wang, H.E., Davis, T., He, G.P., Cimb­o­ra, D.M., Scott A., Kräus­slich, H.G., Kaplan J., Morham, S.G., Sundquist W.I., (2003). The pro­tein net­work of HIV bud­ding. Cell 114, 701–713.