Prof. Dr. Oliv­er T. Fack­ler

oliver.fackler@med.uni-heidelberg.de

Phone: ++49-(0)6221–56-1322

Fax: ++49-(0)6221–56-5003

Cell Biology and Immunology of HIV Pathogenesis

Projects

Our research address­es the cell biol­o­gy, immunol­o­gy and patho­gen­e­sis of HIV‑1 infec­tion with an empha­sis on CD4+ T lym­pho­cytes. One focus of our stud­ies is on the mol­e­c­u­lar mech­a­nisms of action by which the HIV‑1 path­o­genic­i­ty fac­tor Nef repro­grams host cell vesic­u­lar trans­port, sig­nal trans­duc­tion and motil­i­ty to opti­mize HIV‑1 spread in the host and to accel­er­ate dis­ease pro­gres­sion. Anoth­er impor­tant aspect of our work is on the host innate immune sys­tem in HIV infec­tion and on viral eva­sion mech­a­nisms. This includes dis­sect­ing how the intrin­sic immu­ni­ty fac­tor SERINC5 impairs HIV‑1 par­ti­cle infec­tiv­i­ty and how this activ­i­ty is antag­o­nized by the viral pro­tein Nef, but also stud­ies to elu­ci­date which bar­ri­ers pre­vent pro­duc­tive HIV‑1 infec­tion of rest­ing CD4+ T lym­pho­cytes. These HIV-relat­ed stud­ies involve the devel­op­ment of com­plex 3D cul­ture sys­tems for study­ing the rela­tion­ship between host cell motil­i­ty and HIV‑1 spread in tis­sue. Final­ly, we are also inter­est­ed in the cell biol­o­gy of CD4 T cell acti­va­tion and dif­fer­en­ti­a­tion. In this con­text, we par­tic­u­lar­ly focus on the new­ly iden­ti­fied role of nuclear actin fil­a­ment for­ma­tion for CD4 T cell help.

1 | Molecular mechanisms of the HIV‑1 pathogenicity factor Nef

The Nef pro­tein of HIV and SIV is crit­i­cal for high virus load and full path­o­genic­i­ty in the infect­ed host. This path­o­gen­ic poten­tial is under­scored by a trans­genic mouse mod­el in which the expres­sion of Nef alone caus­es an AIDS-like dis­ease. This activ­i­ty ren­ders Nef an attrac­tive tar­get for antivi­ral strate­gies to com­ple­ment exist­ing treat­ment. Nef does not pos­sess detectable enzy­mat­ic activ­i­ty and medi­ates all its func­tions via inter­ac­tions with host cell pro­teins. While a mul­ti­tude of activ­i­ties have been ascribed to Nef, the bio­log­i­cal prop­er­ties and pro­tein-inter­ac­tions that gov­ern its path­o­gen­ic poten­tial have still not been unam­bigu­ous­ly iden­ti­fied. Nef‘s activ­i­ties can be divid­ed into immune eva­sion func­tions and effects that direct­ly boost viral spread in the absence of host immune attack. Both these gen­er­al Nef activ­i­ties are achieved by manip­u­la­tion of host cell sig­nal trans­duc­tion and intra­cel­lu­lar trans­port process­es. Our work focuss­es on visu­al­iz­ing and dis­sect­ing the inde­pen­dent mol­e­c­u­lar mech­a­nisms employed by which Nef dis­rupts host cell vesic­u­lar trans­port, actin remod­el­ing, and motil­i­ty. This increas­ing­ly involves the use of com­plex pri­ma­ry cell cul­ture mod­els and in vivo analy­ses in mice.

Fig­ure 1. Deter­mi­nants in Nef for asso­ci­a­tion with NAKC as well as PAK2 reduce T cell polar­i­ty oscil­la­tions. Time pro­jec­tion of live-cell spin­ning-disc microscopy videos of A3.01 cells tran­sient­ly express­ing GFP or Nef.GFP. Scale bars, 5 μm. (from Lamas-Murua et al. (2018) J. Immunol., 201:2731–2743.)

2 | Innate sensing and restriction of HIV‑1

The innate immune sys­tem is increas­ing­ly rec­og­nized as a major arm of immu­ni­ty against effi­cient HIV‑1 repli­ca­tion that pos­es cell-intrin­sic bar­ri­ers to virus repli­ca­tion (restric­tion fac­tors) and elic­its antivi­ral sig­nal­ing upon recog­ni­tion of repli­ca­tion inter­me­di­ates (by sen­sors). Our cur­rent work focuss­es on two aspects of innate immune defens­es against HIV.

a) Host cell restric­tion by SERINC pro­teins

SERINC3 and 5 were recent­ly iden­ti­fied as host cell restric­tion fac­tors that potent­ly sup­press the infec­tiv­i­ty of HIV‑1 viri­ons. This antivi­ral activ­i­ty can be coun­ter­act­ed by the HIV‑1 pro­tein Nef. We are study­ing the mech­a­nisms under­ly­ing the antivi­ral action of SERINC pro­teins as well as its antag­o­nism by Nef.

Fig­ure 2. Mod­el of S5-medi­at­ed restric­tion of HIV‑1 par­ti­cle infec­tiv­i­ty and Nef antag­o­nism. In the absence of Nef, S5 is incor­po­rat­ed into prog­e­ny virus par­ti­cles and inhibits viri­on infec­tiv­i­ty. Nef pre­vents incor­po­ra­tion of S5 into bud­ding par­ti­cles and enhances viri­on infec­tiv­i­ty. Down­reg­u­la­tion from the plas­ma mem­brane and accu­mu­la­tion of the restric­tion fac­tor in endo­somes is insuf­fi­cient and dis­pens­able, respec­tive­ly, for this coun­ter­ac­tion. At least 4 mol­e­c­u­lar deter­mi­nants in Nef are required for S5 coun­ter­ac­tion (LL, ED, 12–39 and CAW). Addi­tion­al­ly, our results sug­gest that Nef antag­o­nizes viri­on asso­ci­at­ed pools of S5 via an unknown mech­a­nism. Trautz et al. (2016), J. Virol. 90:10915 – 10927.

 

 

b) HIV infection of resting CD4+ T lymphocytes

HIV‑1 infects CD4+ T lym­pho­cytes and cells of the monocytes/macrophage lin­eage. In the case of pri­ma­ry human CD4+ T lym­pho­cytes, the per­mis­siv­i­ty to HIV‑1 infec­tion ex vivo depends on the cel­lu­lar acti­va­tion state: while acti­vat­ed, pro­lif­er­at­ing CD4+ T lym­pho­cytes are high­ly sus­cep­ti­ble to infec­tion and sup­port effi­cient virus repli­ca­tion, rest­ing CD4+ T cells are large­ly non-per­mis­sive for HIV‑1 repli­ca­tion but can be infect­ed in vivo and serve as a latent viral reser­voir. Since phys­i­o­log­i­cal­ly the vast major­i­ty of all CD4+ T lym­pho­cytes are in a rest­ing state, this phe­nom­e­non pro­vides one expla­na­tion as to why the fre­quen­cy of pro­duc­tive­ly infect­ed CD4+ T lym­pho­cytes in AIDS patients is very low. In col­lab­o­ra­tion with Prof. Dr. Oliv­er T. Kep­pler (LMU Uni­ver­sität München) we iden­ti­fied the deoxynu­cle­o­side triphos­phate (dNTP) triphos­pho­hy­dro­lase SAMHD1 (ster­ile alpha motif (SAM) and histidine/aspartic acid (HD) domain-con­tain­ing pro­tein 1) as an essen­tial host cell fac­tor for this restric­tion that can be over­come by the Vpx pro­tein encod­ed by HIV‑2 and SIV and defined addi­tion­al rest­ing T cell-spe­cif­ic blocks at the lev­el of reverse tran­scrip­tion and nuclear import. Our cur­rent efforts focus on defin­ing the nature of these addi­tion­al blocks to HIV‑1 repli­ca­tion in rest­ing CD4+ T cells.

Fig­ure 3. Pro­posed mod­el for HIV restric­tions in pri­ma­ry rest­ing CD4 T cells and coun­ter­ac­tion by SIV Vpx vari­ants. (A) Both SAMHD1 (RT block 1) and an unknown fac­tor (RT block 2) are able to restrict HIV at the lev­el of reverse tran­scrip­tion. Down­stream, an unknown fac­tor lim­its nuclear import of the prein­te­gra­tion com­plex (NI block 1). (B) SIVmac239 Vpx WT tar­gets SAMHD1 for degra­da­tion to over­come RT block 1. In the pres­ence of SAMHD1 and RT block 2, SAMHD1 is the pre­ferred tar­get of SIVmac239 Vpx WT, but in the absence of SAMHD1, RT block 2 is tar­get­ed. SAMHD1 degra­da­tion-defi­cient mutants of SIVmac239 Vpx tar­get RT block 2 sim­i­lar­ly to SIVmnd‑2 and SIVr­cm Vpx through a mech­a­nism that like­ly involves pro­tea­so­mal degra­da­tion. (from Bal­dauf et al. (2017), PNAS,

3 | HIV‑1 spread in complex cell systems

Stud­ies on the mech­a­nisms gov­ern­ing HIV‑1 spread in infect­ed indi­vid­u­als are ham­pered by the fact that clas­si­cal cell cul­ture sys­tems lack tis­sue het­ero­gene­ity and orga­ni­za­tion or are large­ly refrac­to­ry to exper­i­men­tal vari­a­tion of impor­tant para­me­ters. To this end we engi­neered 3D col­la­gen matri­ces as cul­ture sys­tem to study HIV‑1 spread between CD4 T lym­pho­cytes. Inte­gra­tion of pop­u­la­tion-based and sin­gle cell math­e­mat­i­cal mod­els revealed that HIV‑1 spread in 3D is pre­dom­i­nant­ly medi­at­ed by cell-cell trans­mis­sion and that 3D envi­ron­ments pose a potent restric­tion to cell-free infec­tion. In turn, the 3D envi­ron­ment induces alter­ations to archi­tec­ture and dura­tion of cell-cell con­tacts that facil­i­tate cell-asso­ci­at­ed HIV trans­fer. In our ongo­ing stud­ies we focus on dis­sect­ing the rela­tion­ship between cell motil­i­ty and HIV spread and on increas­ing the com­plex­i­ty of this 3D ex vivo cul­ture sys­tem.

Fig­ure 4. INSPECT-3D exper­i­men­tal sys­tem and work­flow analy­sis of pop­u­la­tion analy­sis of pathogen spread in 3D col­la­gen. Schemat­ic overview of the para­me­ters that can be quan­ti­fied by INSPECT-3D. Depict­ed in the top pan­el are schemat­ic views of 2D sus­pen­sion and 3D col­la­gen cul­tures with unin­fect­ed and infect­ed cells in red and green, respec­tive­ly. Aster­ics: virus. Mid­del pan­el shows a wide field micro­graph of cell den­si­ty and arrange­ment, the low­er pan­el a still image from a movie of 3D cul­ture with unin­fect­ed and infect­ed cells. Scale bars: 40µm. From: Imle et al. (2019) Nat Com­mun, 10: 2144.

4 | Cellular mechanisms of actin dynamics / nuclear actin

The aston­ish­ing dynam­ics of the actin cytoskele­ton pro­vides the basis for fun­da­men­tal process­es in mam­malian cells such as motil­i­ty, kine­sis, vesic­u­lar trans­port and sig­nal trans­duc­tion. While mech­a­nisms and func­tion of actin dynam­ics in the cyto­plasm are rel­a­tive­ly well stud­ied, an only recent­ly emerg­ing field is the role of actin dynam­ics in the nucle­us. We recent­ly iden­ti­fied the rapid and tran­sient for­ma­tion of nuclear actin fil­a­ments as nov­el effec­tor func­tion of T cell recep­tor stim­u­la­tion in CD4 T lym­pho­cytes. Medi­at­ed by cal­ci­um tran­sients in the nucle­us and nuclear pools of the actin poly­mer­iza­tion machin­ery Arp2/3 com­plex, the pres­ence of nuclear fil­a­ments facil­i­tates expres­sion of a select­ed set of genes required for CD4 T helper func­tion. Our cur­rent efforts aim at char­ac­ter­iz­ing the mech­a­nisms gov­ern­ing these nucle­us-spe­cif­ic actin poly­mer­iza­tion event as well as at dis­sect­ing the rela­tion­ship between nuclear actin dynam­ics and gene expres­sion.

Fig­ure 5. Anal­y­sis of the nuclear F‑actin net­works in A3.01 T cells. STED microscopy of endoge­nous nuclear actin fil­a­ments with Alexa Flu­or 488 and Atto 647N Phal­loidin in A3.01 T cells with­out stim­u­la­tion (−) or after stim­u­la­tion (+) with PMA/Iono.
From Tsopoulidis et al. (2019) Sci Immunol, 4. pii:eaav1987.

Selected Publications

Com­plete pub­li­ca­tion list (PubMed)

Ananth, S., Morath, K., Trautz, B., Tibroni, N.,  Shy­taj, I.A., Ober­maier, B., Stolp, B., Lusic, M. and Fack­ler, O.T. (2020). Mul­ti­func­tion­al Roles of the N‑Terminal Region of HIV‑1SF2Nef Are Medi­at­ed by Three Inde­pen­dent Pro­tein Inter­ac­tion Sites. J. Virol., ePub ahead of print: doi.org/10.1128/JVI.01398–19.

Imle, A., Kum­berg­er, P., Schnell­bäch­er, 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., Fack­ler, O.T. (2019) Exper­i­men­tal and com­pu­ta­tion­al analy­ses reveal that envi­ron­men­tal restric­tions shape HIV‑1 spread in 3D cul­tures. Nat Com­mun, 10:2144.

Tsopoulidis, N., Kaw, S., Lake­ta, V., Kutschei­dt, S., Baar­link, C., Stolp, B., Grosse, R., Fack­ler, O.T. (2019) T cell recep­tor-trig­gered nuclear actin net­work for­ma­tion dri­ves CD4+ T cell effec­tor func­tions. Sci Immunol, 4. pii: eaav1987.

Lamas-Murua, M., Stolp, B., Kaw, S., Thoma, J., Tsopoulidis, N., Trautz, B., Ambiel, I., Reif, T., Aro­ra, S., Imle, A., Tibroni, N., Wu, J., Cui, G., Stein, J.V., Tana­ka, M., Lyck, R. and Fack­ler, O.T. (2018). HIV‑1 Nef Dis­rupts CD4+ T Lym­pho­cyte Polar­i­ty, Extrava­sa­tion and Hom­ing to Lymph Nodes via its Nef-Asso­ci­at­ed Kinase Com­plex Inter­face. J. Immunol., 201:2731–2743.

Chase, A.J., Wom­bach­er, R. and Fack­ler, O.T. (2018). Intrin­sic prop­er­ties and plas­ma mem­brane traf­fick­ing route of Src fam­i­ly kinase SH4 domains sen­si­tive to retar­get­ing by HIV‑1 Nef. J. Biol. Chem., 293:7824–7840.

Trautz, B., Wiede­mann, H., Lücht­en­borg, C., Pieri­ni, V., Kranich, J., Glass, B., Kräus­slich, H.G., Brock­er, T., Piz­za­to, M., Rug­gieri, A., Brüg­ger, B. and Fack­ler, O.T. (2017). SERINC5 restricts HIV‑1 infec­tiv­i­ty with­out alter­ing the lipid com­po­si­tion and orga­ni­za­tion of viral par­ti­cles. J. Biol. Chem., 292:13702–13713.

Bal­dauf, H.M., Stegmann, L., Schwarz, S.M., Ambiel, I., Tro­tard, M., Mar­tin, M., Burggraf, M., Lenzi, G.M., Lejk, H., Pan, X., Fregoso, O.i., Lim, E.S., Abra­ham, L., Nguyen, L., Rutsch, F., König., R., Kim., B., Emer­man, M., Fack­ler, O.T. * and Kep­pler, O.T. * (2017). Vpx over­comes a SAMHD1-inde­pen­dent block to HIV reverse tran­scrip­tion that is spe­cif­ic to rest­ing CD4 T cells. Proc. Natl. Acad. Sci. USA, 114: 2729–2734. (* cor­re­spond­ing authors).

Trautz, B., Pieri­ni, V., Wom­bach­er, R., Stolp, B., Chase, A.J., Piz­za­to, M. and Fack­ler, O.T. (2016). The Antag­o­nism of HIV‑1 Nef to SERINC5 Par­ti­cle Infec­tiv­i­ty Restric­tion Involves the Coun­ter­ac­tion of Viri­on-Asso­ci­at­ed Pools of the Restric­tion Fac­tor. J. Virol., 90:10915–10927.

Pujol, F.M., Lake­ta, V., Schmidt, F., Muken­hirn, M., Müller, B., Boulant, S., Grimm, D., Kep­pler, O.T. and Fack­ler, O.T. (2016). HIV‑1 Vpu Antag­o­nizes CD317/tetherin by Adap­tor pro­tein-1-Medi­at­ed Exclu­sion from Virus Assem­bly Sites. J. Virol., 90:6709–6723.

Galas­ki, J., Ahmad, F., Tibroni, N., Pujol, F.M., Müller, B., Schmidt, R.E., and Fack­ler, O.T. (2015). Cell Sur­face Down­reg­u­la­tion of NK Cell Lig­ands by Patient-derived HIV‑1 VPU and Nef Alle­les. J. AIDS, 72:1–10.

Tro­tard, M., Tsopoulidis, N., Tibroni, N., Willem­sen, J., Binder, M., Rug­gieri, A., and Fack­ler, O.T. (2015). Sens­ing of HIV‑1 Infec­tion in Tzm-bl Cells with Recon­sti­tut­ed Expres­sion of STING. J Virol. , 90: 2064- 2076.

Imle, A., Abra­ham, L., Tsopoulidis, N., Hoflack, B., Sak­sela, K. and Fack­ler, O.T. (2015). Asso­ci­a­tion with PAK2 Enables Func­tion­al Inter­ac­tions of Lentivi­ral Nef Pro­teins with Exo­cyst. mBio, 6: e01309-15.

Haller, C., Müller, B., Fritz, J.V., Lamas-Murua, M., Stolp, B., Pujol, F., Kep­pler, O.T. and Fack­ler, O.T. (2014). HIV‑1 Nef and Vpu are Func­tion­al­ly Redun­dant Broad-Spec­trum Mod­u­la­tors of Cell Sur­face Recep­tors Includ­ing Tetraspanins. J. Virol., 88: 14241–14257.

Fack­ler, O.T. *, Murooka, T.T., Imle, A. and Mem­pel, T.R.*(2014) Adding new dimen­sions: Towards an inte­gra­tive under­stand­ing of HIV‑1 spread. (* cor­re­spond­ing authors). Nat. Rev. Micro­bi­ol., 12:563–574.

Kutschei­dt, S., Zhu, R., Antoku, S., Lux­ton, G.G.W., Stagl­jar, I., Fack­ler, O.T. * and Gun­der­sen, G. * (2014). FHOD1 inter­ac­tion with nesprin-2G medi­ates TAN line for­ma­tion and nuclear move­ment (* cor­re­spond­ing authors). Nat. Cell Biol. 16: 708–715.

Bal­dauf, H‑M.+, Pan, X.+, Erik­son, E., Schmidt, S., Dad­dacha, W., Burggraf, M., Schenko­va, K., Ambiel, I., Wab­nitz G., Gram­berg, T., Panitz, S., Flo­ry, E., Lan­dau, N.R., Ser­tel, S., Rutsch, F., Lasitsch­ka, F., Kim, B., König, R., Fack­ler, O.T.* and Kep­pler, O.T.* (2012). The deoxynu­cle­o­side triphos­phate triphos­pho­hy­dro­lase SAMHD1 restricts HIV‑1 infec­tion in rest­ing CD4+ T cells. Nat Med, 18: 1682–1687 (* cor­re­spond­ing authors, + first authors).

Pan, X., Rudolph, J.M., Abra­ham, L., Haber­mann, A., Haller, C., Kri­jnse-Lock­er, J. and Fack­ler, O.T. (2012) HIV‑1 Nef com­pen­sates for dis­or­ga­ni­za­tion of the immuno­log­i­cal synapse by induc­ing trans-Gol­gi network–associated Lck sig­nal­ing. Blood, 119:786–797.

Stolp, B., Imle, A., Coel­ho, F.M., Hons, M., Men­diz, R.G., Lyck, R., Stein, J.V. and Fack­ler, O.T. (2012). HIV‑1 Nef Inter­feres With T Lym­pho­cyte Cir­cu­la­tion Through Con­fined Envi­ron­ments in vivo. Proc. Natl. Acad. Sci. USA, 109: 18541–18546.

Stolp, B., Raich­man-Fried, M., Abra­ham. L., Pan, X., Giese, S.I., Han­ne­mann, S., Gouli­mari, P., Raz, E., Grosse, R. and Fack­ler, O.T. (2009). HIV‑1 Nef inter­feres with host cell motil­i­ty by dereg­u­la­tion of cofil­in. Cell Host and Microbe, 6:174–186