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Quantitative 3D Video Microscopy of HIV Transfer Across T Cell
Virological Synapses
Anna Buch26.10.2009
Wolfgang Hübner et al., Science, 2009
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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
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Peterlin & Trono, (2003) Nat Rev Imm. 2:97-107
HIV Life Cycle
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The virological synapse
Haller & Fackler, (2008) Biol. Chem. 10:1253-60
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HIV Gag-iGFP
Hübner et al., (2007) J Virol. 22:12596-607
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Gag accumulates at synaptic buttons after T cell adhesion
Figure 1
Synaptic button
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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
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FRET
http://www.zeiss.de/c12567be00459794/Contents-Frame/c9bcd00b2754e25ac125727600552921
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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
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Viral buds: concentrated at the synapse
Figure 1I, S3 A &B, S4 C
A, B, I: HIV Gag-iGFPC: HIV wt
Viral buds
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Spining disk confocal microscopy
http://zeiss-campus.magnet.fsu.edu/tutorials/spinningdisk/yokogawa/index.html
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Gag is recruited from a synapse-proximal region
Figure 2Movie S3
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Rapid and directional transport towards the button
Figure 2Movie S4 Movie S5 Movie S6 Movie S8 Movie S9
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Cell-to-cell transfer of Gag-iGFP with mean velocity of 0,12 µm/s
Figure 2Movie S10
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Env is present in an internal Gag+ compartment
Figure 2
Synapses target HIV into vesicular compartments within target cells.
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Productive infection of a synapsed cell
Figure 3Movie S11
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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
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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
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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
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Thank you for your attention!
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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
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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