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

Comparative analysis of HIV Comparative analysis of HIV-

• 1

1 attachment and fusion efficiency attachment and fusion efficiency

Manon Eckhardt University Hospital Heidelberg Department of Virology AG Müller / Kräusslich

Background Background – – HIV HIV entry entry

HI V entry

• pH-independent fusion at

the plasma membrane

• cellular determinants:
• receptor CD4
• coreceptors (CCR5 or CXCR4)

fusion fusion attachment attachment

Karlsson Hedestam et al., Nature Reviews Microbiology 2008

• viral determinant - Env protein
• gp120 interacts with CD4
• V3-loop interacts with coreceptor

• 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)

Project Project outline

• utline

Experimental Experimental setup setup

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) insertion of V3-loop sequences in Env-

expression plasmid

production of isogenic pseudovirions

carrying different Envs

pure X4:

220, 286, 685

dubious:

409, 651, 924

pure R5:

822, 838

comparison with reference strains:

NL4-3 NL4-3 R5 Env(-)

220 SI CTRPNNNTIKGISIGPGRAVIATRKIIGDIRQAHC 286 SI CTRPHNNIKRHRIHIGPGRSFHTTKGITGNIRQAHC 409 SI CTRPGNNTRKSITRTPGRVIYATGAIIGDIRQAHC 651 SI CTRPNNNTRKSVRIGPGDIFITTDIIGNIRQAHC 685 SI CTRPNNNIMRRIHIGPGRAFYATRKIIGNIRQAHC 822 NSI CTRPNNNTRRSIHIAPGRAFYTTGQIIGDIRQAHC 838 NSI CTRPNNNTRKSIHIGPGKAFYTTGEIIGDIRQAHC 924 SI CFRPNNNTRKGIHIGPGRAFYTTGEIIGDIRRAYC NL4-3 CTRPNNNTRKSIRIQRGPGRAFVTIGKI GNMRQAHC NL4-3 R5 CTRPNNNTRKGIHI--GPGRAFYTTGEIIGDIRQAHC

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 447 nm cytoplasm loaded with β-Lactamase cleavable dye CCF2 (green) Entry by fusion Vpr.BlaM enters the cytoplasm cleavage of CCF2 (blue product)

β-Lactamase

L unet CD

6h incubation

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 HCVpp (50ng p24) Env(-) (50ng Pseudotyp re l Bla M a c tivity

HDL

VSV-G (1ng p24) no inhibitor Bafilomycin α-CD81 low (0,4µg/ml)

ratio green/blue

(B) fluorimetric read-out (96-well plate) (bulk-quantitative) (A) microscopic read-out (qualitative) (C) FACS read-out (quantitative)

no virus VSVpp HCVpp

BlaM BlaM Assay: different read Assay: different read-

• out methods
• ut methods

Second Second read read-

• out
• ut :

: GFP GFP-

• transduction

transduction

Experimental Experimental setup setup II II

• 3. Entry assay and FACS analysis

34.16%

C8 1 6 6 _ HI V

• 1. Production of isogenic pseudovirions
• 2. Quantitative WB to check for Env

incorporation

0.01%

C8 1 6 6 _ no virus

• 4. Simultaneous antibody-staining of

receptor and/ or coreceptor(s)

• simultanious information about entry and receptor/ coreceptor density on single cell

CD4/ CCR5 CD4/ CCR5-

• inducible

inducible cellline cellline -

• Affinofile

Affinofile

• HEK 293 cells:
• CD4-
• CCR5-
• CXCR4+
• Tet- and Pon-inducible system to
• btain CD4 and CCR5 expression

Johnston et al. (Benhur Lee Lab UCLA)

range of endogenous expression range of endogenous expression

variation

• f CCR5

variation

• f CD4

Example Example for for first first -

• line

line data data

Comparison Comparison between between different different cells cells

I nhibitors of HI V entry

fusion inhibitor (Enfuvirtide – T20) co-receptor antagonist (Maraviroc - MVC)

• blocks interaction with CCR5

AMD3100 (X4 antagonist)

• discontinued in HIV trials already 2001

(approved for cancer therapy)

• useful in tissue culture

Inhibition of HIV Inhibition of HIV-

• 1

1 entry entry

Esté & Telenti, Lancet 2007

Enfuvirtide ( T2 0 ) Maraviroc ( MVC)

• r AMD3 1 0 0

Maraviroc ( MVC)

Flourescently Flourescently labelled labelled viruses viruses to to study study attachment attachment

Capsid (CA)‏

M A

Matrix (MA)‏ Nucleocapsid (NC)‏

p6

HIV Protease

eGFP gag

pol env nef tat rev vpu vpr vif

HIV genome

MA eGFP CA NC p6

plating on poly-L- Lysin coated LabTek-chambers microscopy and analysis binding of virus, washing, fixation 1x106 cells addition of fluorescently labelled virus

fluorescently labelled particles

bound virus / cell HIV Env(-)

Project Scientists Project Scientists

Alexander Thielen Kasia Bozek Joachim Büch Eugen Schülter

Saarbrücken Cologne

Saleta Sierra Manon Eckhardt

Heidelberg

Medical Systems Biology Initiative

Susanna Trapp

Acknowledgements Acknowledgements

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