quantitative 3d video microscopy of hiv transfer across t cell virological synapses anna buch...

Quantitative 3D Video Microscopy of HIV Transfer Across T Cell

Virological Synapses

Anna Buch26.10.2009

Wolfgang Hübner et al., Science, 2009

Human Immunodeficiency Virus

• familiy retroviridae

• genus lentivirus

• + ss RNA, 10 kb

• 16 distinct proteins

• enveloped virus

• conical capsid

http://www.niaid.nih.gov/factsheets/graphics/howhiv.jpg

Peterlin & Trono, (2003) Nat Rev Imm. 2:97-107

HIV Life Cycle

The virological synapse

Haller & Fackler, (2008) Biol. Chem. 10:1253-60

HIV Gag-iGFP

Hübner et al., (2007) J Virol. 22:12596-607

Gag accumulates at synaptic buttons after T cell adhesion

Figure 1

Synaptic button

Synapses appear as button-shaped disks

Figure 1, S1

CellTracker Orange CMRAHIV Gag-iGFPHIV Gag-iGFP

Movie S1

A & B: CD4+ T cells

C & D: Donor: Jurkat T cell, Target: CD4+ T cell

FRET

http://www.zeiss.de/c12567be00459794/Contents-Frame/c9bcd00b2754e25ac125727600552921

Gag homo-oligomerizes at the synapse

Figure 1

FRET: flourescence resonance transfer

Gag-iCeruleanGag-iCerulean (donor)

Gag-iVenusGag-iVenus (acceptor)

Target cells (CellTracker Orange)

Movie S2

Viral buds: concentrated at the synapse

Figure 1I, S3 A &B, S4 C

A, B, I: HIV Gag-iGFPC: HIV wt

Viral buds

Spining disk confocal microscopy

http://zeiss-campus.magnet.fsu.edu/tutorials/spinningdisk/yokogawa/index.html

Gag is recruited from a synapse-proximal region

Figure 2Movie S3

Rapid and directional transport towards the button

Figure 2Movie S4 Movie S5 Movie S6 Movie S8 Movie S9

Cell-to-cell transfer of Gag-iGFP with mean velocity of 0,12 µm/s

Figure 2Movie S10

Env is present in an internal Gag+ compartment

Figure 2

Synapses target HIV into vesicular compartments within target cells.

Productive infection of a synapsed cell

Figure 3Movie S11

Cell-associated infection is coreceptor dependent

B: HIV NL-GI (X4) → CXCR4 tropic virus AMD3100: 10 µg/ml, coreceptor antagonist

C: HIV NL-GI (R5) → CCR5 tropic virus MT4-R5: CCR5-expressing MT4 cells

Figure 4

Cell-associated infection is actin-dependent and resists neutralizing antibodies

D: HIV Gag-iGFP Cytochalasin D: 2.5 µg/ml, blocks actin polymerzation

E: HIV Gag-iGFP HIV neutralizing serum 2, negative control human serum

Figure 4

Summary• Gag puncta are directed into synaptic buttons to infect

target cells

• Gag-iGFP transport: directional and fast (0,12 µm/s)

• Synapses: button-shaped disks ( 1-3 µm)

• Gag homo-oligomerizes at the synapse

• Cell-to-cell transmission could go via endocytic routes

• Future vaccines: against cell-surface env epitopes

• Future antiviral drugs: factors required for synapse formation

Thank you for your attention!

Fluorescence resonance energy transfer analysis of HIV Gag-iCerulean

(donor) and HIV Gag-iVenus (acceptor) fluorophores upon cotransfection into Jurkat Tcells and cocultured with CD4+ T cells.

Figure S2

Transmission electron microscopy of viral buds in 75nm sections tightlypacked at the virological synapse and far from the cell-to-cell contact area.

Figure S3