Dr. Mari­na Lusic

Marina.Lusic@med.uni-heidelberg.de

Phone: ++49-(0)6221–56-5007

Nuclear architecture in viral infection

Projects

We study nuclear archi­tec­ture and chro­matin orga­ni­za­tion in response to HIV‑1 infec­tion. The nucle­us is a com­plex envi­ron­ment, in which chro­matin is orga­nized to sup­port dif­fer­ent struc­tur­al and func­tion­al aspects of cell phys­i­ol­o­gy, and rep­re­sents a chal­lenge for an incom­ing viral genome, which needs to be inte­grat­ed into cel­lu­lar DNA to ensure a pro­duc­tive infec­tion. Inte­gra­tion site selec­tion has func­tion­al con­se­quences for viral tran­scrip­tion, which usu­al­ly fol­lows the inte­gra­tion event. Alter­na­tive­ly, fol­low­ing inte­gra­tion, HIV‑1 can enter the state of laten­cy due to the atten­u­at­ed or repressed viral tran­scrip­tion. Laten­cy is main­tained under cur­rent antivi­ral ther­a­pies, but once ther­a­py is inter­rupt­ed, the virus can resume its pro­duc­tive phase.

1 | HIV‑1 integration and genome organization

 a. in depth analy­sis of the genom­ic prop­er­ties of inser­tion sites in HIV‑1 tar­get cells

One of the main inter­ests of the Lusic lab is under­stand­ing the chro­matin and genom­ic fea­tures of HIV‑1 inte­gra­tion sites. Although HIV‑1 can in the­o­ry tar­get any cel­lu­lar genom­ic sequences, and despite the fact it has the well-estab­lished pref­er­ence for tran­scrip­tion­al­ly active regions of the genome, some genom­ic regions are tar­get­ed much more often than the oth­ers. By analysing dif­fer­ent inde­pen­dent stud­ies of inte­gra­tion sites both in patients, and in HIV‑1 infec­tions in vit­ro, we intro­duced a con­cept of HIV‑1 recur­rent inte­gra­tion genes (RIGs), as genes found in at least two or more inde­pen­dent stud­ies (Mari­ni et al., 2015, Lucic et al 2019). These genes most fre­quent­ly posi­tion in the out­er shells of CD4+ T cell nucle­us, 1 micron under­neath the nuclear enve­lope, where we also mapped HIV‑1 genome by 3D Immuno-DNA flu­o­res­cence in situ hybridiza­tion (FISH).
The major­i­ty of these recur­rent inte­gra­tion genes are clas­si­fied as cell-type spe­cif­ic genes with super-enhancers (SE), due to which they clus­ter in 3D nuclear space as revealed by Chro­mo­some con­for­ma­tion cap­ture, cou­pled with deep sequenc­ing (Hi‑C).

Fig­ure 1 | HIV‑1 inte­gra­tion hot-spots are prox­i­mal to super-enhancers. FOXP1 IS (red) super­im­po­si­tion on H3K27ac (orange), SE (blue), H3K36me3 (green) and BRD4 (vio­let) ChIP-Seq tracks.

b. the reor­ga­ni­za­tion of T cell nucle­us upon T cell acti­va­tion.

We are par­tic­u­lar­ly inter­est­ed in how spe­cif­ic genom­ic ele­ments, such as super-enhancers, con­trol the major tran­scrip­tion­al changes that a rest­ing T cell under­goes when acti­vat­ed. By exploit­ing tran­scrip­tome data of these two pri­ma­ry cell states, and by detect­ing radi­al posi­tion­ing of the cell-type spe­cif­ic genes with dis­tal super-enhancers by 3D Immuno-DNA FISH we could observe a 3D genome reor­ga­ni­za­tion that leads to the dock­ing of these high­ly expressed SE to the out­er shell of the nucle­us dur­ing acti­va­tion.

Fig­ure 2 | Genes prox­i­mal to super-enhancers change their nuclear posi­tion­ing upon T cell acti­va­tion. Three-dimen­sion­al immuno-DNA FISH of two RIGs, FOXP1 and STAT5B, in rest­ing and acti­vat­ed (anti-CD3/an­ti-CD28 beads, IL‑2 for 48 h) CD4+ T cells.

Fig­ure 3 | Mod­el of HIV‑1 inte­gra­tion into the 3D clus­ters of genes prox­i­mal to super-enhancers in acti­vat­ed CD4+ T cells. HIV‑1 inte­gra­tion occurs rarely in rest­ing CD4+ T cells that have large het­e­rochro­matin regions and few active genes. HIV‑1 effi­cient­ly inte­grates in acti­vat­ed CD4+ T cells, where 3D clus­ters of active genes (Recur­rent Inte­gra­tion Genes), often prox­i­mal to super-enhancers repo­si­tioned towards the out­er shells of the nucle­us, rep­re­sent inser­tion hot-spots.

C. Devel­op­ment of a method to cap­ture HIV‑1 inte­gra­tion sites and their chro­mo­so­mal con­for­ma­tion (DZIF 06.902 and DZIF 04704)

We are estab­lish­ing a tech­nique that enables the detec­tion of inte­gra­tion sites and con­tacts with neigh­bor­ing regions. The first, termed HIV‑1 Cap, has been val­i­dat­ed with sev­er­al his­tor­i­cal data sets and his­tone pro­files char­ac­ter­is­tic for HIV‑1 inser­tions gen­er­at­ed in our lab­o­ra­to­ry in pri­ma­ry CD4+ T cells (Rhein­berg­er et al., in prepa­ra­tion). We are cur­rent­ly set­ting up the 4C mod­i­fi­ca­tion of this method, HIV‑1 Gen­Cap.

Fig­ure 4 | Schemat­ic rep­re­sen­ta­tion of HIV Cap and HIV Gen­Cap method. After son­i­ca­tion (5) of the genom­ic DNA, ends are repaired, adap­tors are added and index­es are intro­duced by PCR (6). The sam­ples are hybridized with biotiny­lat­ed RNA oli­gos (7), and cap­tured with strep­ta­vidin beads (8 enriched by PCR and sequenced (9). The method can be extend­ed to com­bine inte­gra­tion site sequenc­ing with a 4C approach (left). For this pur­pose, DNA is fix­at­ed with formalde­hyde (1), digest­ed (2) and prox­im­i­ty lig­at­ed (3). After revers­ing the crosslinks, DNA is extract­ed, son­i­cat­ed and the Cap method can be per­formed

d. The role of Nucle­o­porins (Nups) in nuclear entry, inte­gra­tion site selec­tion and pos­si­bly even sub­se­quent tran­scrip­tion of the HIV‑1 genome (Fund­ed by DFG SFB 1129)

 The goal of this project is to deci­pher the role of nuclear pore com­plex­es dur­ing HIV‑1 infec­tion and sub­se­quent tran­scrip­tion (more infor­ma­tion here). We want to use cor­rel­a­tive cryo light and elec­tron microscopy (cCLEM) tech­niques in col­lab­o­ra­tion with the Beck and Kräus­slich lab­o­ra­to­ries to visu­al­ize the dock­ing of the virus to NPCs (Nuclear Pore Com­plex) and to deter­mine how exact­ly it trav­els through the pore. Once inside, we will use pro­teomics and genomics approach­es to deter­mine how NPC pro­teins guide inte­gra­tion into RIGs, and how they sub­se­quent­ly define the tran­scrip­tion­al prop­er­ties of these genes and the inte­grat­ed HIV‑1 genome in close col­lab­o­ra­tion with the Beck lab­o­ra­to­ry.

e. HIV‑1 brain reser­voir: how microglia-spe­cif­ic nuclear dynam­ics affect viral inte­gra­tion and fate (Fund­ed by DFG SPP 2202)

Microglia, brain res­i­dent cells of the macrophage lin­eage, rep­re­sent a puta­tive HIV‑1 reser­voir in the cen­tral ner­vous sys­tem. This project aims at dis­cov­er­ing HIV‑1 inte­gra­tion and laten­cy pat­terns in the nuclear envi­ron­ment of the puta­tive reser­voir in the brain — microglia. By using the HIV Gen­Cap tech­nique we will define the inte­gra­tion pro­files of in vit­ro infect­ed immor­tal­ized pri­ma­ry human microglia cells, iso­lat­ed from an adult cor­ti­cal brain surgery (pro­vid­ed by Dr Alvarez-Car­bonell). These pro­files will be com­pared to the ones from the close­ly-relat­ed periph­er­al pri­ma­ry human macrophages. More specif­i­cal­ly, we want to under­stand if lin­eage-deter­mi­nant sig­na­tures (i.e. PU.1, CTCF) and cell-spe­cif­ic genomics con­tacts influ­ence viral inte­gra­tion and its fate. These deter­mi­nants could indeed sup­port immuno­log­i­cal iner­tia of latent cells in the cen­tral ner­vous sys­tem and can be addressed to revert laten­cy. This would help to define microglia con­tri­bu­tion to HIV‑1 pro­duc­tion and per­sis­tence under anti­retro­vi­ral ther­a­py. 3D Immuno FISH (Fig­ure 5) will be used to assess the 3D local­iza­tion of HIV‑1 as well as the spa­tial dis­tri­b­u­tion of inte­gra­tion sites; they will be com­pared to the ones in CD4+ cells.

Fig­ure 5 | Microglia mod­el of HIV- 3D Immuno FISH images of inte­grat­ed HIV (green) in microglia nuclei with con­fo­cal microscopy (red lamin, blue Hoechst).

2 | Linking HIV‑1 latency and 3D genome organization

The main obsta­cle to the func­tion­al HIV cure are the latent reser­voirs of provirus estab­lished in mem­o­ry CD4+ T cells which can­not be erad­i­cat­ed with cur­rent antivi­ral treat­ments (cART). Despite some evi­dence that cART inten­si­fi­ca­tion is hav­ing pos­i­tive effects on HIV resid­ual repli­ca­tion, our under­stand­ing of HIV‑1 laten­cy and reac­ti­va­tion is still incom­plete. While explor­ing the mol­e­c­u­lar mech­a­nisms involved in the estab­lish­ment and main­te­nance of laten­cy in CD4+ T cells, we obtained sub­stan­tial evi­dence that reg­u­la­tion of oxida­tive phos­pho­ry­la­tion path­way and oxida­tive stress in the cell plays a very impor­tant role in HIV‑1 tran­scrip­tion­al con­trol and laten­cy. Our results high­light for the first time the con­nec­tion and the mol­e­c­u­lar mech­a­nism between HIV‑1 induced oxida­tive stress, viral production/latency and Promye­lo­cyt­ic Leukemia Nuclear Bod­ies (PML NBs) turnover and fur­ther strength­en the idea that PML NBs can be used both as mol­e­c­u­lar mark­ers and/or phar­ma­co­log­ic tar­gets for HIV‑1 infec­tion

 

Fig­ure 6 |  Reg­u­la­tion of antiox­i­dant respons­es, intra­cel­lu­lar iron con­tent and PML NBs abun­dance dur­ing the tran­si­tion between pro­duc­tive and latent HIV‑1 infec­tion. In unin­fect­ed cells, there is either no or very lit­tle oxida­tive stress, the num­ber of PML Nuclear bod­ies is nor­mal, and the iron lev­els are in their steady state. Upon HIV‑1 infec­tion, increased lev­els of oxida­tive stress lead to the dis­bal­ance in iron home­osta­sis and heme degra­da­tion. In pro­duc­tive infec­tion, there are high lev­els of oxida­tive stress, and high antiox­i­dant response, increased iron import fol­lowed by decreased PML NB num­bers. High antiox­i­dant response ulti­mate­ly results either in cell death or in iron home­osta­sis nor­mal­iza­tion in PML pro­tein lev­els and PML NB num­bers, when laten­cy can be estab­lished.

 

 

Selected Publications

Com­plete pub­li­ca­tion list (PubMed)

Shy­taj IL, Lucic B, For­ca­to M,  et al.  Alter­ations of redox and iron metab­o­lism accom­pa­ny devel­op­ment of HIV laten­cy. BioRX­IV doi: https://doi.org/10.1101/549014BioRXIV doi: https://doi.org/10.1101/549014

Lucic  B, Chen H‑C, Kuz­man M, Zori­ta E„ Weg­n­er J, Min­nek­er V, Wang W, Fron­zaR, Laufs S, Schmidt M, Stad­houd­ers R, Roukos V,  Vla­hovicek K, Fil­ion GJ and Lusic M. Spa­tial­ly clus­tered loci with mul­ti­ple enhancers are fre­quent tar­gets of HIV‑1 Nature Com Accept­ed for pub­li­ca­tion. IF 12,353

Muen­chau S, Deutsch R, de Cas­tro IJ, Hielsch­er T, Heber N, Niesler B, Lusic M, Stan­i­fer ML, Boulant S. (2019) Hypox­ic envi­ron­ment pro­motes bar­ri­er for­ma­tion in human intesti­na epithe­lial cells through reg­u­la­tion of miR­NA-320a expres­sion. Mol Cell Biol.  IF 3.813

Ali H, Mano M, Bra­ga L, Naseem A, Mari­ni B, Vu DM, Colle­si C, Meroni G, Lusic M, and Giac­ca, M.(2019) Cel­lu­lar TRIM33 restrains HIV‑1 infec­tion by tar­get­ing viral inte­grase for pro­tea­so­mal degra­da­tion. Nat Com­mun, 10:926. IF 12,353

Michiele­to D, Lusic M, Orlan­di­ni E, Maren­duz­zo D (2019) Phys­i­cal prin­ci­ples of retro­vi­ral inte­gra­tion. Nat Com­mun, 10:575. IF 12,353

Bejara­no DA, Peng K, Lake­ta V, Börn­er K, Jost KL, Lucic B, Glass B, Lusic M, Müller B, Kräus­slich HG. (2019)  HIV‑1 nuclear import in macrophages is reg­u­lat­ed by CPS­F6-cap­sid inter­ac­tions at the nuclear pore com­plex. Elife. IF 7,616

Lusic M. (2018) Col­or­ing Hid­den Virus­es. Elife. IF 7,616

Agos­ti­ni S, Ali H, Vard­abas­so C, Fit­ti­pal­di A, Tas­ciot­ti E, Cere­se­to A, Bugat­ti A, Rus­nati M, Lusic M, Giac­ca M (2017) Inhi­bi­tion of Non Canon­i­cal HIV‑1 Tat Secre­tion Through the Cel­lu­lar Na+,K+-ATPase Blocks HIV‑1 Infec­tion. EBio­Med­i­cine. 21:170–181.  IF 6.183

Lusic M and Robert F. Sili­ciano (2017) Nuclear land­scape of HIV‑1 infec­tion and  inte­gra­tion. Nat  Rev Microb. 15:69–82. IF 31,851

Lucic B and Lusic M. (2016) Con­nect­ing HIV‑1 inte­gra­tion and tran­scrip­tion: a step toward new treat­ments. FEBS Let­ters. 590:1927. IF 2,999

Tur­ri­ni F, Marel­li S, Kajaste-Rud­nit­s­ki A, Lusic M, Van Lint C, Das AT, Har­wig A, Berk­hout B, Vicen­zi E. (2015) HIV tran­scrip­tion­al silenc­ing caused by TRIM22 inhi­bi­tion of Sp1 bind­ing to the viral pro­mot­er Retro­vi­rol­o­gy, 12:104. IF 3,867

Mari­ni B, Kertesz-Farkas A, Lucic B, Hashim A, Lisek K, Man­ga­naro L, Pon­gor S, Luz­za­ti R, Mav­ilio F, Giac­ca M and Lusic M (2015) Nuclear archi­tec­ture dic­tates HIV‑1 inte­gra­tion site selec­tion Nature, 521: 227–31. IF 41.577

Lusic M and Giac­ca M (2014) Ground Con­trol to Major Tom: “Pre­pare for HIV Land­ing” Cell Host & Microbe, 16: 557–559.  IF 17,872

Lusic M and Giac­ca M (2014) Reg­u­la­tion of HIV‑1 laten­cy by chro­matin struc­ture and nuclear archi­tec­ture. A review arti­cle for the spe­cial issue “Func­tion­al Rel­e­vance and Dynam­ics of Nuclear Orga­ni­za­tion. J Mol BiolIF 4.894

Feli­cian G, Colle­si C, Lusic M, Mar­tinel­li V, Fer­ro MD, Zen­tilin L, Zac­chigna S, Giac­ca M (2014) Epi­ge­net­ic mod­i­fi­ca­tion at Notch respon­sive pro­mot­ers blunts effi­ca­cy of induc­ing notch path­way reac­ti­va­tion after myocar­dial infarc­tion. Circ Res, 115:636–49.  IF 15,211

Lusic M, Mari­ni B, Ali H, Lucic B, Luz­za­ti R, and Giac­ca M (2013) Prox­im­i­ty to PML Nuclear Bod­ies neg­a­tive­ly reg­u­lates HIV‑1 gene expres­sion in CD4+ T cells. Cell Host & Microbe, 13: 665–677. Research high­light in Sci­ence Vol 341 (2013) and in Cell Host & Microbe 13:625–626.  IF 17,872

Del­la Chiara G, Crot­ti A, Liboi E, Giac­ca M, Poli G and Lusic M. (2011) Neg­a­tive Reg­u­la­tion of HIV- 1 Tran­scrip­tion by a Het­erodimer­ic NF-kB1/p50 and C‑Terminally Trun­cat­ed STAT5 Com­plex. J Mol Biol, 410: 933–943. IF 4,894

Allouch A, Di Prim­io C, Alpi E, Lusic M, Aro­sio D, Giac­ca M and Cere­se­to A (2011) KAP‑1 inhibits HIV‑1 inte­gra­tion. Cell Host & Microbe, 9: 484–95. IF 17,872

Man­ga­naro L, Lusic M*, Gutier­rez MI, Cere­se­to A, Del Sal G and Giac­ca M* (2010) Con­cert­ed action of cel­lu­lar JNK and Pin‑1 restricts HIV‑1 genome inte­gra­tion to acti­vat­ed CD4+ T lym­pho­cytes. Nat Med­i­cine, 16: 329–323 (* cor­re­spond­ing authors). IF 32,621

Dieudon­né M, Maiuri P, Bian­cot­to C, Kneze­vich A, Kula A,Lusic M, and Mar­cel­lo A. (2009) Tran­scrip­tion­al com­pe­tence of the inte­grat­ed HIV‑1 provirus at the nuclear periph­ery EMBO J. 28:2231–2243. IF 9,792

Sabo’ A, Lusic M, Cere­se­to A and Giac­ca M (2008) Acety­la­tion of con­served lysines in the cat­alyt­ic core of CDK9 reg­u­lates kinase activ­i­ty and sub­nu­clear local­iza­tion. Mol Cell Biol, 28:2201–12 . IF 3,813

Vard­abas­so C, Man­ga­naro L, Lusic M, Mar­cel­lo A and Giac­ca M (2008) The his­tone chap­er­one pro­tein Nucle­o­some Assem­bly Protein‑1 (hNAP‑1) binds HIV‑1 Tat and pro­motes viral tran­scrip­tion. Retro­vi­rol­o­gy, 28:5–8. IF 3,867

Perkins KJ, Lusic M, Mitar I, Giac­ca M and Proud­foot NJ (2008) Tran­scrip­tion depen­dent gene loop­ing of the HIV‑1 provirus is dic­tat­ed by recog­ni­tion of pre-mRNA pro­cess­ing sig­nals. Mol­e­c­u­lar Cell, 29:56–68. IF 14,248