1. Quantitative 3D Video Microscopy of HIV Transfer Across T Cell Virological Synapses Anna Buch 26.10.2009 Wolfgang Hübner et al., Science, 2009

2. 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

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

4. The virological synapse Haller & Fackler, (2008) Biol. Chem. 10:1253-60

5. HIV Gag-iGFP Hübner et al., (2007) J Virol. 22:12596-607

6. Gag accumulates at synaptic buttons after T cell adhesion Figure 1 Synaptic button

7. Synapses appear as button-shaped disks Figure 1, S1 CellTracker Orange CMRA HIV Gag-iGFP Movie S1 A & B: CD4 + T cells C & D: Donor: Jurkat T cell, Target: CD4 + T cell

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

9. Gag homo-oligomerizes at the synapse Figure 1 FRET: flourescence resonance transfer Gag-iCerulean Gag-iCerulean (donor) Gag-iVenus Gag-iVenus (acceptor) Target cells (CellTracker Orange) Movie S2

10. Viral buds: concentrated at the synapse Figure 1I, S3 A &B, S4 C A, B, I: HIV Gag-iGFP C: HIV wt Viral buds

11. Spining disk confocal microscopy http://zeiss-campus.magnet.fsu.edu/tutorials/spinningdisk/yokogawa/index.html

12. Gag is recruited from a synapse-proximal region Figure 2 Movie S3

13. Rapid and directional transport towards the button Figure 2 Movie S4 Movie S5Movie S6Movie S8 Movie S9

14. Cell-to-cell transfer of Gag-iGFP with mean velocity of 0,12 µm/s Figure 2 Movie S10

15. Env is present in an internal Gag + compartment Figure 2 Synapses target HIV into vesicular compartments within target cells.

16. Productive infection of a synapsed cell Figure 3 Movie S11

17. Cell-associated infection is coreceptor dependent B: HIV NL-GI (X4) → CXCR4 tropic virusAMD3100: 10 µg/ml, coreceptor antagonist C: HIV NL-GI (R5) → CCR5 tropic virusMT4-R5: CCR5-expressing MT4 cells Figure 4

18. 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

19. 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

20. Thank you for your attention!

21. 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

22. Transmission electron microscopy of viral buds in 75nm sections tightly packed at the virological synapse and far from the cell-to-cell contact area. Figure S3