Comparative analysis of HIV- - 1 1 Comparative analysis of HIV - PowerPoint PPT Presentation

Comparative analysis of HIV- - 1 1 Comparative analysis of HIV attachment and fusion efficiency attachment and fusion efficiency Manon Eckhardt University Hospital Heidelberg Department of Virology AG Müller / Kräusslich

Background – – HIV HIV entry entry Background HI V entry • • pH-independent fusion at viral determinant - Env protein the plasma membrane • gp120 interacts with CD4 • V3-loop interacts with coreceptor • cellular determinants: • receptor CD4 • coreceptors (CCR5 or CXCR4) attachment attachment fusion fusion Karlsson Hedestam et al. , Nature Reviews Microbiology 2008

Project outline outline Project multidimensional analysis of HIV-1 entry � → investigate the effect of different parameters on HIV entry: different V3-loop sequences � varying CD4 receptor density � varying density of coreceptors at the cell surface � concentration of prototype HIV entry inhibitors � computationally generate mathematical models of viral entry � (systems biology)

Experimental setup setup Experimental � hemophiliac cohort (Bonn, 1980s) � phenotyping done in Bonn based on SI/ NSI � bulk sequencing of Env-sequences (Virology Köln) � selection of 8 sequences (MPII Saarbrücken) � pure X4: 220, 286, 685 � dubious: 409, 651, 924 220 SI CTRPNNNTIKGISIGPGRAVIATRKIIGDIRQAHC 286 SI CTRPHNNIKRHRIHIGPGRSFHTTKGITGNIRQAHC � pure R5: 822, 838 409 SI CTRPGNNTRKSITRTPGRVIYATGAIIGDIRQAHC 651 SI CTRPNNNTRKSVRIGPGDIFITTDIIGNIRQAHC 685 SI CTRPNNNIMRRIHIGPGRAFYATRKIIGNIRQAHC � comparison with reference strains: 822 NSI CTRPNNNTRRSIHIAPGRAFYTTGQIIGDIRQAHC � NL4-3 838 NSI CTRPNNNTRKSIHIGPGKAFYTTGEIIGDIRQAHC 924 SI CFRPNNNTRKGIHIGPGRAFYTTGEIIGDIRRAYC � NL4-3 R5 NL4-3 CTRPNNNTRKSIRIQRGPGRAFVTIGKI GNMRQAHC � Env(-) NL4-3 R5 CTRPNNNTRKGIHI--GPGRAFYTTGEIIGDIRQAHC � insertion of V3-loop sequences in Env- expression plasmid � production of isogenic pseudovirions carrying different Envs

β - β - Lactamase Lactamase ( ( BlaM BlaM) ) assay assay (A) Generation of HCV pseudoparticles carrying Vpr.BlaM fusion protein (B) Principle of BlaM Assay 520 nm cytoplasm loaded with β -Lactamase cleavable dye CCF2 (green) 447 nm β -Lactamase Entry by fusion � Vpr.BlaM enters the cytoplasm � cleavage of CCF2 (blue product)

BlaM Assay: different read Assay: different read- - out methods out methods BlaM (A) microscopic read-out (qualitative) no virus VSVpp HCVpp L unet CD HDL 6h incubation 4,0 (B) fluorimetric read-out (96-well plate) 3,5 ratio green/blue 3,0 (bulk-quantitative) re l Bla M a c tivity no inhibitor 2,5 Bafilomycin 2,0 α -CD81 low (0,4µg/ml) 1,5 1,0 0,5 0,0 HCVpp (50ng p24) VSV-G (1ng p24) Env(-) (50ng Pseudotyp (C) FACS read-out (quantitative)

Second read read- - out out : : GFP GFP- - transduction transduction Second

Experimental setup setup II II Experimental 1. Production of isogenic pseudovirions 2. Quantitative WB to check for Env incorporation 3. Entry assay and FACS analysis 4. Simultaneous antibody-staining of receptor and/ or coreceptor(s) C8 1 6 6 _ no virus C8 1 6 6 _ HI V 0.01% 34.16% • simultanious information about entry and receptor/ coreceptor density on single cell

CD4/ CCR5- - inducible inducible cellline cellline - - Affinofile Affinofile CD4/ CCR5 Johnston et al. (Benhur Lee Lab UCLA) variation of CD4 HEK 293 cells: � CD4- � CCR5- � CXCR4+ � Tet- and Pon-inducible system to � range of endogenous expression obtain CD4 and CCR5 expression variation of CCR5 range of endogenous expression

Example for for first first - - line line data data Example

Comparison between between different different cells cells Comparison

Inhibition of HIV- - 1 1 entry entry Inhibition of HIV I nhibitors of HI V entry � fusion inhibitor (Enfuvirtide – T20) Maraviroc ( MVC) Maraviroc ( MVC) � co-receptor antagonist (Maraviroc - MVC) or AMD3 1 0 0 blocks interaction with CCR5 � Enfuvirtide ( T2 0 ) � AMD3100 (X4 antagonist) discontinued in HIV trials already 2001 � Esté & Telenti, Lancet 2007 (approved for cancer therapy) useful in tissue culture �

Flourescently labelled labelled viruses viruses to to study study attachment attachment Flourescently eGFP vif vpu Matrix (MA) ‏ M A rev pol HIV Protease tat gag HIV genome vpr nef Capsid env (CA) ‏ MA eGFP CA NC p6 Nucleocapsid (NC) ‏ p6 fluorescently labelled particles microscopy bound virus / cell and 1x10 6 cells analysis addition of binding of virus, plating on poly-L- fluorescently labelled washing, fixation Lysin coated virus LabTek-chambers HIV Env(-)

Project Scientists Project Scientists Cologne Saarbrücken Saleta Sierra Eugen Schülter Alexander Thielen Kasia Bozek Heidelberg Joachim Büch Susanna Trapp Manon Eckhardt Medical Systems Biology Initiative

Acknowledgements Acknowledgements Virology Heidelberg Barbara Müller Hans-Georg Kräusslich ZMBH Heidelberg Monika Langlotz MPI Informatics - Saarbrücken Virology Cologne - Köln Thomas Lengauer Rolf Kaiser Kasia Božek Eva Heger Alexander Thielen Saleta Sierra Aragon Joachim Büch Eugen Schülter