Quantitative 3D Video Microscopy of HIV Transfer across T Cell Virological Synapses

Author(s): Wolfgang Hübner, Gregory P. McNerney, Ping Chen, Benjamin M. Dale,

Ronald E. Gordon, Frank Y. S. Chuang, Xiao-Dong Li, David M. Asmuth, Thomas Huser
and Benjamin K. Chen
Source: Science , Mar. 27, 2009, New Series, Vol. 323, No. 5922 (Mar. 27, 2009), pp. 1743-
1747
Published by: American Association for the Advancement of Science

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 REPORTS H
7. B. Heinrich, The Hot-Blooded Insects: Strategies and 24. Wild type, 15.7 ? 0.4 (mean ? SEM); atu mutant,
upon the transgenic expression of DmDG (Fig.
Mechanisms of Thermoregulation (Harvard Univ. Press, 32.3 ? 1.8; and DmDG-transgenic atu mutant
21). Among mitochondrial dehydrogenases in in Cambridge, AAA, 1993). (atu/atu; actin5C-GAL4/UAS-DmDG), 25.2 ? 1.6 (n = 3).
sects, PDH is the only enzyme that is reported to 8. AA. ]. Greener, R. G. Roberts, FEBS Lett. 482, 13 (2000). 25. Wild type, 124.9 ? 1.9 (mean ? SEM; n = 202); atu
show Ca2+-dependent activation (18), and there 9. W. AA. Deng et al., Development 130, 173 (2003). mutant, 148.5 ? 2.3 (n = 194); and DmDG-transgenic
10. AA. S. Patel, L. G. Korotchkina, Biochem. Soc. Trans. 34, atu mutant (atu/atu; actin5C-GAL4/UAS-DmDG),
fore, it is possible that the reduced expression
217 (2006). 115.5 ? 2.9 (n = 119).
of DmDG causes the sustained increase in the
11. S. K. Kim, E. J. Rulifson, Nature 431, 316 (2004). 26. Wild type, 139.0 ? 4.7 (mean ? SEM; n = 98); atu
[Ca2+]j, which, in turn, induces the activation of 12. A. A. Steiner, L. G. Branco, Annu. Rev. Physiol. 64, 263 mutant, 151.8 ? 7.4 (n = 88); and DmDG-transgenic atu
PDH, resulting in increased mitochondrial oxi (2002). mutant (atu/atu; actin5C-GAL4/UAS-DmDG), 101.9 + 6.3
dative metabolism. Based on these observations, 13. S. AAahajan, N. Tuteja, Arch. Biochem. Biophys. 444, 139 (n = 79).
(2005). 27. We thank Y. Nakano for statistical analysis; H. Suzuki
we propose that a reduction in the DmDG con
14. ]. E. Kammenga et aL, PLoS Genet. 3, e34 (2007). and J. Ikenouchi for assistance with electron microscopy;
tent initiates a chain of sequential reactions, i.e., 15. ]. G. McCormack, A. P. Halestrap, R. AA. Denton, R. Niwa, M. O'Connor, A. P. Gould, and M. Kamakura
increased membrane fluidity, activation of Ca2+ Physiol. Rev. 70, 391 (1990). for providing GAL4 flies; N. Juni for helpful discussions;
influx, elevated mitochondrial metabolism, and 16. P. Gailly, Biochim. Biophys. Acta 1600, 38 (2002). A. Yamaguchi, Y. Yamaguchi, and M. Nishikawa for
17. R. Barresi, K. P. Campbell,;. Cell Sci. 119, 199 (2006). technical assistance; and R. Matsuda, H. Takeshima,
eventually, altered thermoregulatory behavior (fig.
18. ]. G. McCormack, R. AA. Denton, Biochem. J. 196, 619 Y. Nagai, and M. Ui for valuable advice and
S9). It remains an enigma, however, how the meta (1981). encouragement. This work was supported in part by
bolic changes in a cell are translated into neural 19. L Liu, O. Yermolaieva, W. A. Johnson, F. AA. Abboud, Special Coordination Funds for Promoting Science and
code that induces behavioral change at the level of AA. ]. Welsh, Nat Neurosci. 6, 267 (2003). Technology from MEXT (Ministry of Education, Culture,
20. Wild type, 7.3 ? 1.8% (mean ? SEAA); atu mutant, 67.3 ? Sports, Science and Technology), Japan, and by a
the whole animal (see also supporting online text).
3.7%; and DmDG transgenic atu mutant (atu/atu; research grant from The Novartis Foundation (Japan) for
actin5C-GAL4/UAS-DmDG), 7.3 ? 1.8%. Error bars the Promotion of Science. D.Y. was supported by
References and Notes represent SEAA (n - 3). Specially Promoted Research grant 1802012 from MEXT
1. T. ]. Crowley, G. R. North, Science 240, 996 (1988). 21. Control actin5C-GAL4 line (actin5C-GAL4/+), 15.7 ? and The Tohoku Neuroscience Global Centers of
2. P. ]. Mayhew, G. B. Jenkins, T. G. Benton, Proc. Biol. Sci. 4.2% (mean ? SEM); "DmDG-knockdown" line Excellence program.
275, 47 (2008). (actin5C-GAL4/UAS-dsRNA), 59.0 ? 3.6%; and control
3. T. H. Bullock, Biol. Rev. Camb. Philos. Soc. 30, 311 (1955). UAS-dsRNA line (+/UAS-dsRNA), 25.4 ? 7.7%. Error Supporting Online Material
[Link]/cgi/content/full/323/5922/1740/DCl
4. M. ]. Angilletta Jr., P. H. Niewiarowski, C A. Navas, bars represent SEM (n = 7).
Materials and Methods
J. Therm. Biol. 27, 249 (2002). 22. Wild type, 1.6 ? 0.1 (mean ? SEM); atu mutant,
SOM Text
5. I. A. Johnston, A. F. Bennett, Eds., Animals and 3.0 ? 0.1; and DmDG-transgenic atu mutant
(atu/atu; actin5C-GAL4/UAS-DmDG), 2.2 ? 0.2. Error Figs. SI to Sll
Temperature: Phenotypic and Evolutionary Adaptation
Tables SI
(Cambridge Univ. Press, Cambridge, 1996). bars represent SEM (n = 6).
References
6. C. V. Gisolfi, M. Francisco, The Hot Brain: Survival, 23. Wild type, 20.9 ? 1.5 (mean ? SEM; n = 9); atu mutant,
Temperature and the Human Body (MIT Press, 27.0 ? 1.8 (n = 8); and DmDG-transgenic atu mutant 9 September 2008; accepted 27 January 2009
Cambridge, MA, 2000). (atu/atu; actin5C-GAL4/UAS-DmDG), 20.2 ? 1.2 (n = 8). 10.1126/science.ll65712

dynamic actin and tubulin (9), cell signaling (10),

Quantitative 3D Video Microscopy

and lipid raft recmitment (77). T cell virological
synapses transfer virus with high efficiency (12),

of HIV Transfer Across T Cell yet how this route fundamentally differs from
cell-free infection remains unclear.
To examine the spatial and temporal organi

Virological Synapses zation of synapse formation, we used an infectious,

fluorescent HIV clone, carrying a Gag-internal,
interdomain insertion of the green fluorescent
Wolfgang Hiibner,1 Gregory P. McNerney,3 Ping Chen,1 Benjamin M. Dale,1
Ronald E. Gordon,2 Frank Y. S. Chuang,3 Xiao-Dong Li,4 David M. Asmuth,4 protein (GFP), called HIV Gag-iGFP (13). This
Thomas Huser,3'4 Benjamin K. Chen1* virus faithfully reveals Gag localization, allowing
infected cells and viral particles to be tracked with
high sensitivity (12). Time-lapse fluorescence mi
The spread of HIV between immune cells is greatly enhanced by cell-cell adhesions called
croscopy of virological synapse formation showed
virological synapses, although the underlying mechanisms have been unclear. With use of an
infectious, fluorescent clone of HIV, we tracked the movement of Gag in live CD4 T cells and
that 24% of HIV Gag-iGFP-expressing Jurkat
cells formed stable adhesions to primary CD4 T
captured the direct translocation of HIV across the virological synapse. Quantitative, high-speed
cells within 4 hours (Fig. 1 and table SI A). After
three-dimensional (3D) video microscopy revealed the rapid formation of micrometer-sized
adhesion, 80% formed focal Gag accumulations
"buttons" containing oligomerized viral Gag protein. Electron microscopy showed that these
at the contact site with an average 82-min interval
buttons were packed with budding viral crescents. Viral transfer events were observed to form
(Fig. 1, A and B). In contrast, an Env-deficient
virus-laden internal compartments within target cells. Continuous time-lapse monitoring showed
clone was unable to induce cell-cell conjugates or
preferential infection through synapses. Thus, HIV dissemination may be enhanced by virological
synapse-mediated cell adhesion coupled to viral endocytosis. Gag accumulation (table SIB), illustrating that
adhesion precedes Gag redistribution.
Human immunodeficiency virus (HIV) in synapses between virus-carrying dendritic cells
division of Infectious Diseases, Department of Medicine, Im
fection leads to depletion of CD4 T cells and CD4 T cells is highly efficient (4, 5). For hu munology Institute, Mount Sinai School of Medicine, New York,
throughout the lymphoid system. Both man T cell lymphotropic virus type I, viral synapses NY 10029, USA. department of Pathology, Mount Sinai
cell-free and cell-associated infection routes con between T cells are essential for dissemination School of Medicine, New York, NY 10029, USA. 3NSF Center for
Biophotonics Science and Technology, University of California
tribute to viral dissemination in vivo (7). In vitro, (6). For HIV, infected and uninfected CD4 T cells
infection with cell-associated HIV can be Davis (UCD), Sacramento, CA 95817, USA. 4Department of In
form virological synapses that organize viral recep ternal Medicine, University of California Davis Medical Center,
thousands fold more efficient than infection with tors CD4, CXCR4, and Env (7). These infectious Sacramento, CA 95817, USA.
cell-free virus (2), and inhibition of cell-cell con contacts are regulated by cell adhesion through *To whom correspondence should be addressed. E-mail:
tacts severely limits replication (3). Infection through integrins and intercellular adhesion molecules (8), [Link]@[Link]

[Link] SCIENCE VOL 323 27 MARCH 2009 1743

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REPORTS
In fixed samples, high-resolution confocal im released into the target cell, and then migrated exceeds individual clathrin- or caveolin-associated
aging revealed prominent Gag accumulations at distally with a mean velocity of 0.12 um/s (Fig. structures, which are uniformly small (100 to
the synapse (Fig. 1C). In three-dimensional (3D) 2E and movie S8). Notably, puncta 1.5 um in 200 nm) (17). By using quantitative confocal mi
reconstructions, these appeared as button-shaped diameter were observed (fig. S6A), and on oc croscopy, we found that the accumulation and
discs, 1 to 3 pm in diameter (Fig. ID and movie casion an entire synaptic button was transferred maintenance of Gag puncta in target cells was
SI). Synaptic buttons were also observed in HTV (movie S9). Large vesicular structures were also remarkably stable over time (fig. S6, B to D, and
Gag-iGFP-oxpressing primary CD4 T cells cocul observed to fractionate into smaller vesicles while movie S10).
tured with homologous primary CD4 cells (fig. moving toward the distal pole ofthe cell (movies The GFP signal in flow-sorted HIV+ CD4 tar
SI). We assessed viral assembly at the synapse S4 and S8). The size of these translocated puncta get cells was uniformly punctate, without evidence
by measuring Gag oligomerization with fluores
cence resonance energy transfer (FRET) (13-15)
between Cerulean and Venus variants of HTV
Gag-iGFP, which form a donor-acceptor FRET
pair (16). Excitation of the Cerulean donor in co
transfected Jurkat cells generated a robust Venus
shifted FRET signal at synaptic buttons that is
indicative of Gag homo-oligomerization (Fig. IE).
____________________________ _-_-_-_-_-_---_-__-_^

Photobleaching the Venus acceptor at a synapse

lead to increased donor emission, providing ad
ditional evidence for FRET (Fig. 1, F to H, and
fig. S2). Three-dimensional reconstruction of FRET
images revealed concentrated Gag oligomeriza
tion at synapses (movie S2).
With transmission electron microscopy, we
observed that 100-nm budding viral crescents at B 1}_
the virological synapse protruded from the donor

iin: ESSSIEl^S^' _-......

cell with bud tips directly abutting the target cell
membrane (Fig. II). Viral buds were also ob
served far from the synapse, although at lower
densities (fig. S3). Native, non-GFP-expressing
HTV induced similar budding crescents, ruling
out that GFP induced these accumulations (fig.
S4). In thick 150-nm sections, near-complete 0 30 60 90 120 150 180 210 240
viral buds and a virus-containing invagination time (min)
-?*? no contact ? conjugate no butt
in the synapsed target cell were observed (fig. S4,
A and B).
To capture the dynamics of Gag trafficking,
reorganization, and viral transfer with higher tem
poral and spatial resolution, we recorded high
speed, spinning disc confocal fluorescence images.
Forty-three putative synaptic events encompassing
1187 min revealed dynamic Gag movements dur
ing virological synapse formation (table S2). New
synaptic button formation (n = 4) was captured
where patches of membrane-associated Gag moved
toward the cell adhesion site within minutes
(Fig. 2A and movie S3). At existing buttons, a /Y\\ _^_^_^_^H_
ring-shaped zone of Gag depletion often sur
rounded the synaptic button (Fig. 2B), indicative
f____H______r
?_.--^____H|j|^_V
200 / Nx_ _____________ _____________!
of a synapse-proximal region from which Gag wavelength (nm) _H_I_________________H____ __________________H_I
was recruited.
HTV Gag-iGFP-labeled structures (n = 8) close Fig. 1. Gag accumulates at synaptic buttons after T cell adhesion. (A) Time-lapse fluorescence i
of synapse formation between an HIV Gag-iGFP-expressing Jurkat cell and a CD4 T cell. G
to existing buttons moved rapidly and direction
(top) and GFP/phase contrast overlay (bottom). Cells (a) before stable contact, (b) in stable
ally into the button (Fig. 2C, fig. S5, and movies
(outlined), and (c and d) showing synaptic buttons (arrowheads). (B) Timing of synapse fo
S4 to S6). The structures moved into the synapse
following 24 HIV+ ]urkat cells; each line represents an interactive cell. (C) Confocal fluorescen
with average velocities of 0.10 to 0.25 pm/s and
of an HIV Gag-iGFP-expressing ]urkat T cell (green) synapsed with three primary CD4 T c
peaks up to 0.8 pm/s (Fig. 2C and fig. S5). Other
labeled with CellTracker Orange CMRA (Invitrogen, Carlsbad, CA)]. Positioning of perpendicul
small, mobile Gag puncta emerged from and marked at edges. (D) Reconstructed 3D view of (C). (E to H) FRET analysis of Gag-iCerulean (don
then moved back into the synaptic button (Fig. Gag-iVenus (acceptor) fluorophores at the synaptic button. (E) Three-color overlay donor Cerule
2D and movie S7). The fast, directional move 405-nm excitation), FRET channel (green, 405-nm excitation), and target cells [red, 543-nm ex
ment of Gag was seen predominantly from near stained with CellTracker Orange CAATAAR (Invitrogen)]. (F) Emission spectra at synaptic button
by puncta. and postacceptor photobleaching. (G and H) Normalized FRET (NFRET) signal (13) before and after
During cell-to-cell viral transfer (n = 10, table photobleaching in boxed area. (I) Transmission electron micrographs of the synaptic junction bet
S2), fluorescent Gag signal protruded from but Gag-iGFP-expressing donor, D, and target, T, cells. Low (top) and high (bottom) magnification
tons, penetrated the attached target cell, was sections.

1744 27 MARCH 2009 VOL 323 SCIENCE [Link]

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 REPORTS
of syncitia, and confocal imaging suggested that example one, the infected cell synapsed with the Under these contact-dependent infection con
puncta were not surface-associated (fig. S7). Anti target cell for 18 hours, the cells separated, and at ditions, productive infection by cell-associated
Env staining of the Gag-iGFP puncta required cell 32 hours a diffuse, bright GFP signal indicated HIV NL-GI was inhibited by CXCR4-antagonist,
permeabilization, indicating that Env was present productive infection (Fig. 3, A C, and movie SI 1). AMD3100 (Fig. 4B). Furthermore, productive in
in an internal Gag+ compartment (Fig. 2, F and Bystander target cells remained negative. Over fection by cell-associated R5-tropic virus HIV
G). Transmission electron microscopy of the target 67 hours, 112 conjugates tracked resulted in seven NL-GI (JRFL) was dependent on expression of
cells revealed multivesicular structures, which were productively infected MT4 target cells (table S3). the chemokine receptor, CCR5 (Fig. 4C). The re
not seen in control, unexposed cells, that contained In five cases, synapses were observed, and in sults suggest that infection through T cell synapses
viruslike densities inside 1- to 2-pm conrpanments four cases virus transfer was recorded (Fig. 3, A does not bypass the coreceptor requirement.
(Fig. 2H). We conclude that synapses target HIV to C, and movie SI2). Under culture conditions Synapse-mediated viral transfer is potently
into vesicular compartments within recipient cells. that limited new cell-cell interactions, produc inhibited by actin inhibitors such as cytochalasin
To track the fate of cells after synapse forma tively infected cells arose preferentially after ob D (9, 12). We find that cytochalasin D had little
tion, we performed continuous, long-duration im served virological synapse events. effect on cell-free HIV infection yet effectively
aging. Jurkat donor cells were cotransfected with Because synapse-mediated viral transfer is inhibited productive infection by cell-associated
HIV Gag-iGFP and HTV NL-GI, an fflV molec coreceptor-independent (12,19), we tested whether HTV (Fig. 4D). Additionally, a well-characterized
ular clone that expresses GFP in place of the viral infection through T cell synapses requires core patient antisera, which can potently block cell
early gene nef (18). This approach can visualize ceptor expression. Infection of MT4 cells by cell free infection but not transfer of virus through
viral transfer (as puncta), as well as productive associated HTV was inhibited when cells were virological synapses (72), did not efficiently
infection (as diffuse GFP) in the target cell. In separated by a 0.4-pm transwell barrier (Fig. 4A). block infection of the homologous cell-associated

a |^^n __H_H________N_______H_______H B __________!

__________!y,,~Tx_______L _______ l_______pi_________M '* ^________fl
1_i__^__i^_i | _____Hkl______E ___H____k _________ __________i
? _____________r^______i __^__^__^__^__^__^h__^__^__^__^__t_____I______________?______l______l^_____________l _____________________!

Ca __________Pp^lV-_ffl_________P^___R___3__^ ? 5.5,-10.8 j Mi
^^^y^B^V J^H^V __________l_____r^__________i __________________ 0^45 I

^. __H_______________H_Bfe_________??_____l ___________ if 'Vn. , ? ,1

/~*z^^^^^H^^^^^H^^^^^H^E|^^H __E__^Hp33 ^^i. fl^l ="' II
____-~~_____________l_________^ _-___i-_--_--________ 0 30 60 90
D a ^Hra^^HBBHnH^m bHH c 4*j - |o." - d J?l
|^^P^^_^_i_H__H^^^H_HI_^^^_^H__^_V^^^_______l ____________ ^____l st3-5 j / V \ o'

_________________________________________t________________________Er^___ _________________________________________ 1 5 ______-_____?____-_--_---^--__------_----i 0 OnrTTTTTTTTTTT I I I I I

fl_____________l_N__-__-__^ HM-___fl__i____l
E a ^|_^|_^H_^___________________1______________H C ______________________ J] XI- d"?] avgvel.:0.12|im/s
^^^^^^ ^^^^^^ ^ ^^^^ ^^^^^^B ___H__|__^__H o'e 5 >v^/ 0.6e
____________________i___________________H_____________________l___________________H a.^v/ ?-7?

________________________________H___i-^_____________________________ __H9___________ _^4-5 a

V___________________H___________________H___________________H___________________H _____________________ o _ 4

Z___P^________P______H_H___P____ __fPi^__NH / I20' I

^^H|^HPIM|^^|^^HP^^^W^^^^__________|_________B^^HI^^ *_____________ ___________RP^_____I________III__________I 2 iui. 1 i 1 11 i ii a n 0*^P^^^^4ka^r~T~^i~r^rMr-i~nr~r~^
^^HjHp ^i^__|__^__|i_iii^^ _____l_HHOPH_! 0 30 60 90 120 o <y* <& c? o* c? o* time
c?(s)o*
velocity (pm/s)
p _ccEnv_HIV Gag-iGFP_Env/Gag/Celltracker .r.v^:r.^^^x^^m _fl__M__BH__
^^^^^^^^^^^^^^^^I^^^^^^^^^^^^^^^^I^^^^Bfl^^^^^^^^^l _ I Jwil \^'i________l!_________________!

_^^_^_^_^_^_^_^_^_^_^_l_^_^_^_^_^_^_^_^_^_^_^_i_^_^_^_^_H-________________l _ ot-__--.vh_' -> a\ ' '^j____________B_______!_H_S__^_|

H________________l_______________^ 0 ;f\ ',_________________ ^________H_______I
G__H__^__^__^__HH__H__H__^__^__^__H__H__^__^__^__^__H >,255 ?n~^_f___________________l_____________H_l
* __^__^__^__^__^__^__^__H__^__^__^__^__^__^__^__^____^__^__^__^__^__^__^____| ;A:ili_f_|_|_H_^_^_^_H____________________l
^^^^^^^^^^^I^^^^^^^^^^^^^^^^^^^^^^hi s :"!'S_______________________I__________B^______I

^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^M 12 ________________________ ^____-___-__-______

Fig. 2. Dynamic recruitment of Gag puncta to the synapse and viral to the synapse center and relative velocity are graphed over time, (d) His
transfer
into a target cell compartment revealed with rapid spinning-disc togram distribution of the tracked objects velocities. (F and G) Immunostain
3D confocal
fluorescence microscopy. (A) Formation of a buttonlike accumulation ingofof Gag
Gag at puncta requires membrane permeabilization. (F) Nonpermeabilized,
anti-Env immunostain (red) does not stain the Gag-iGFP+ puncta (green) within
the site of adhesion, z projection at time = 0 (left), selected 3D reconstructions
of contact site (arrows) over time (right four images). (B) Athe zone of target
CD4 Gag cell (CellTracker Blue CMF2HC, Invitrogen), whereas surface
depletion, 2 to 3 pm wide, surrounds the synaptic button (dottedEnv-staining
yellow line). at synapse is observed. Three-color intensity profile along the
(0 Patches of synapse-proximal Gag merge into the synapse. (D)12-pm line (right). (G) Permeabilization of fixed cells reveals anti-Env
A Gag-iGFP
puncta moves out of and into the synapse. (E) During a transferimmunostain
event, Gag (red) at the GFP puncta (green) within the CD4 target cell
puncta emerge from the synapse, separate, and then move to the (blue).
distal(H) Transmission electron micrograph of vesicles containing corelike
pole.
In (0 to (E), (a) selected frames highlight movement of Gag-iGFP puncta
structures in a CD4 cell engaged in synapse with an HIV-infected Jurkat cell.
Low (left)
(yellow), (b) Object path is overlayed on the initial image, (c) Object and high (right) magnification of 70-nm sections.
distance

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REPORTS
vims (Fig. 4E). Thus, inhibitor studies clearly The live imaging of HTV cell-to-cell transfer from which HTV is directly transferred into adja
distinguish the mechanisms of cell-free from reveals that dynamic Gag movements in infected cent target cells. Although endocytic entry of cell
those of cell-associated infection. cells organize Gag puncta into synaptic buttons free HTV contributes only modestly to productive

syn- y^ ____________R!fl____________E!H

Donor ^^^^^^^H^^Hj^^^H^^Jj^^^^H^^^^^^^HHtti^^^^H^^^^^^^H
to _______________ _______________ _________________!____

transfer to ^^^^^H^^^^^H^^^^^H^^^^^H^^^^^H^^^^^H
Images show a i^^^^^^^Hi^^^^^^^Hi^^^^^^^Hi^^^^^^^Hi^^^^^^^Hi^^^^^^^H
where the (number ___________8___I_________________I______________H
separates from donor hours ______________H______________H______________H_
expresses H^^^^^Ki^^^^^^^Bi^^^^^^lSi^^^l^^^Hi^^^^^^^Hi^^^^^^^H
Top row shows _______________ _______________ __
images; bottom, GFP/phase overlays. ___|___mH______K_ll 91-1
(B) Four examples of synapsed MT4 tar- B ^^^^^^^Q^^^^^^^Q C ij 1
cells that subsequently expressed ______C______E__j_________________j ? 7 r ?J ^
HIV (numbers 2 to 5). (0 Fluorescence ___ _____________!_________________! ? 6 / t&*
intensity of the target to _______________ _________________! / /_f ^
Numbers 6 and 7 are control bystander ^^^^^^^H^^^^^^^H / mJur /.^ /
on bottom. ^^^^^^^n^^^^^^^ra ly^r p
cells. Duration of cell contact indicated ___________BB_________BBB 1 3 / ^A4^Jr ^ '

_________________|_________________| 0' ^rii^pr, vr^Vi-7bj^iaaM^da^a<M3a^iilhrg tmniii "j'A ntfjjjTT?o??_in?<niin?

_______________ _____ ________ 05 10 15 20 25 30 35 40 45 50 55 60 65 7
_______________________ ______ B-i ;i ^^^

Fig. 4. Cell-associated infection HIV NL-GI (X4) HIV NL-GI (R5)

is coreceptor-dependent and A j-.- B j-r-1 C j-1
actin-dependent and can resist 9.3% ; control 10.9% MT4-R5 6.7%
a neutralizing antiserum. (A) HIV ? >;^, ' .;'
NL-GI-expressing Jurkat cells 3 __i__||^___^? __!_'- '' :V fiftff- .": -; '..
were cocultured with CellTracker 3 "S_I_fP"P^ ^^$^0 JsBm &&_fe
Blue CMF2HC-labeled MT4 cells ? ^??^W*:: ' ] ^^ *' ? * i ^^* *""'"
in the absence or the presence [ p^|%-}'
of a 0.4-pm transwell barrier be- ? ^^,,[,,,, ,",,,,^:, /,";?',, J j,,.,-., ,]?.,.,.?,, J U--i .L-., ,.
tween cells. Productive infection j_ ,-,-. i-.- <-1-1
(GFP expression) in CellTracker- | ; 0.4% : AMD 3100 2.2% ! MT4 0.3%
labeled target cells was measured = g ^ :$&..'.
by flow cytometry at 48 hours. (B) | s \.^&? - ^ : -^___ : ____>
Coreceptor
(10 pg/ml) antagonist AAAD3100
inhibits infection ofg ?8 t. "^
HpT' t. ""-^SSP^
||g| p-' "- JRK:
^p" * :
target cells by cell-associated **: '".
X4-tropic virus, HIV NL-GI, at '___j_
48 hours. Productive infection in I , GFp I , Gpp
gated target cells indicated by
GFP expression and is plotted Cytochalasin D Donor serum Patient serum
against forward scatter width ^ j-1-1 j-j-1 ^ ]-1-1 j-1
(FSC-W). (O Cell-associated 5.5% : 2.6% 7.2% : 4.5%
R5-tropic virus infects CCR5- ? ?
expressing MT4 cells but not 3 .: -^ 3 __M|;V^^*_iSt

Cytochalasin D (2.5 |JM) inhibits o w,., , I,,..., . I w.., .1. .I ? W

cell-associated infection (top) but 5 *-.-1 i-.-1 s j- -1 |-.-1
fails to block infection with cell- jg : 10.7% 10
free virus (bottom). (E) A neu- ? _l . o> _| .
tralizing antiserum that blocks ? = a ;__! ?S % liifi^vir_;__^ ''J(_i^v' "'
effective at blocking homologous o t ^SFv?r^P ? "'^;V/" o t ^' v ^^
cell-associated infection (top). Re- '
suits representative of at least_
three independent experiments. I i Qpp I , Qpp

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 REPORTS *
3. M. Sourisseau, N. Sol-Foulon, F. Porrot, F. Blanchet, 23. M. Marsh, A. Helenius, Cell 124, 729 (2006).
infection (20-22), our results suggest that the cell
0. Schwartz,]. Virol. 81, 1000 (2007). 24. D. J. Wyma et aL, ]. Virol. 78, 3429 (2004).
to-cell transmission could favor endocytic routes. 4. P. U. Cameron et aL, Science 257, 383 (1992). 25. S. Wyss et al., ]. Virol. 79, 12231 (2005).
Thus when spreading via synapses, it is possi 5. D. McDonald etal., Science 300,1295 (2003); published 26. R. N. Germain, M. J. Miller, M. L Dustin, M. C Nussenzweig,
ble that HIV resembles a majority of viruses online 1 May 2003 (10.1126/science.l084238). Nat. Rev. Immunol. 6, 497 (2006).

that enter preferentially through endocytosis (e.g., 6. T. Igakura et aL, Science 299, 1713 (2003); published 27. We thank R. H. Cheng, V. Simon, M. Klotman,
online 13 February 2003 (10.1126/science.l080115). R. Iyengar, and A. Del Portillo for critiques and
influenza, adenoviruses, picomaviruses, alpha
7. C Jolly, K. Kashefi, M. Hollinshead, Q. ]. Sattentau, discussions; R. Huq for microscopy support; S. Izadmehr
viruses) (23). Given this scenario, the tight cou 1. Exp. Med. 199, 283 (2004). for image analysis; M. Grisotto and V. Sahi for cell
pling of Env fusogenicity with particle maturation 8. C. Jolly, I. Mitar, 0- J- Sattentau, ]. Virol. 81, 13916 sorting; H. Bell for electron microscopy support; and
(24, 25) may activate viral fusion vWthin a target (2007). S. Lira for imaging support. Work was supported
9. C. Jolly, I. Mitar, Q. ]. Sattentau, 7. Virol. 81, 5547 by NIH grant AI074420-02, Burroughs Wellcome
cell compartment that is cloistered from neutraliz
(2007). Fund Investigator Award, and Hirschl Weill-Caulier Career
ing antibodies (12). Alternatively, the prominent 10. N. Sol-Foulon etal., EMBO). 26, 516 (2007). Scientist Award to B.K.C. Imaging was supported
endocytic process that accompanies synapse for 11. C. Jolly, Q. J. Sattentau, ]. Virol. 79, 12088 (2005). by Mount Sinai School of Medicine-Microscopy Shared
mation may create viral reservoirs in intracellu 12. P. Chen, W. Hubner, M. A. Spinelli, B. K. Chen,;. Virol. Resource Facility grants NIH-NCI 5R24 CA095823-04,
81, 12582 (2007). NSF-DBI-9724504, and NIH-S10RR09145-01;
lar compartments.
13. W. Hubner et al., J. Virol. 81, 12596 (2007). by the NSF Center for Biophotonics Science and
Future vaccine strategies may be focused 14. A. Derdowski, L. Ding, P. Spearman,;. Virol. 78, 1230 Technology (cooperative agreement PHY012099);
against unique cell-surface Env epitopes that (2004). a UCD Health System Research Award to T.H.; and
block cell-associated infection, and future anti 15. D. R. Larson, Y. M. Ma, V. M. Vogt, W. W. Webb, the UCD Clinical and Translational Science Center grant
J. Cell Biol. 162, 1233 (2003). NIH-NCRR ULRR024146 (T.H. and D.M.A.).
viral drugs may target factors required for synapse
16. M. A. Rizzo, G. H. Springer, B. Granada, D. W. Piston,
formation. Ultimately the dynamics of virological Supporting Online Material
Nat. Biotechnol. 22, 445 (2004).
synapse formation must be understood within 17. M. Lakadamyali, M. J. Rust, X. Zhuang, Cell 124, 997
[Link]/cgi/content/full/323/5922/1743/DCl
Materials and Methods
lymphoid tissues, where high density and lympho (2006).
Figs. SI to S7
cyte mobility (26) are likely to promote synaptic 18. G. B. Cohen et al., Immunity 10, 661 (1999). Tables SI to S3
19. J. Blanco et al., ]. Biol. Chem. 279, 51305 (2004).
viral spread. Movies SI to S12
20. O. T. Fackler, B. M. Peterlin, Curr. Biol. 10, 1005 (2000).
References and Notes 21. V. Marechal et al., ]. Virol. 75, 11166 (2001).
1. A. T. Haase, Nat Rev. Immunol. 5, 783 (2005). 22. E. Schaeffer, V. B. Soros, W. C Greene, 7. Virol. 78, 1375 22 October 2008; accepted 30 January 2009
2. D. S. Dimitrov et al., J. Virol. 67, 2182 (1993). (2004). 10.1126/science.ll67525

pressed, SB transposase catalyzed the transposi

A Transposon-Based Genetic Screen in tion of T2/Onc, a mutagenic SB transposon (Fig.

1A) (7). T2/Onc contains a murine stem-cell virus

Mice Identifies Genes Altered in long terminal repeat and splice donor site (MSCV
LTR-SD), which can deregulate the expression

Colorectal Cancer of a nearby proto-oncogene. T2/Onc also carries

splice acceptor sites in both DNA strands and a
bidirectional polyadenylate signal, which can in
Timothy K. Starr,1* Raha Allaei,1 Kevin A. T. Silverstein,2 Rodney A. Staggs,2 activate the expression of a tumor suppressor gene.
Aaron L. Sarver,2 Tracy L. Bergemann,3 Mihir Gupta,4 M. Gerard O'Sullivan,5 Because SB transposition is biased toward re
llze Matise,5 Adam ]. Dupuy,6 Lara S. Collier/ Scott Powers,8 Ann L. Oberg,9 integration of the transposon into the same chro
Yan W. Asmann,9 Stephen N. Thibodeau,9 Lino Tessarollo,10 Neal G. Copeland,11 mosome as the donor transposon (a phenomenon
Nancy A. Jenkins,11 Robert T. Cormier,12 David A. Largaespada1* referred to as "local hopping"), we used two
T2/Onc transgenic lines that each carried approx
Human colorectal cancers (CRCs) display a large number of genetic and epigenetic alterations, imately 25 copies of the T2/Onc transposon in a
some of which are causally involved in tumorigenesis (drivers) and others that have little functional concatamer on different donor chromosomes (chrs
impact (passengers). To help distinguish between these two classes of alterations, we used a 1 and 15) (7).
transposon-based genetic screen in mice to identify candidate genes for CRC. Mice harboring A histochemical analysis of the triple trans
mutagenic Sleeping Beauty (SB) transposons were crossed with mice expressing SB transposase in genic mice (Rosa26-LsL-SBll, T2/Onc, and Villin
gastrointestinal tract epithelium. Most of the offspring developed intestinal lesions, including Cre) showed that SB transposase was strongly
intraepithelial neoplasia, adenomas, and adenocarcinomas. Analysis of over 16,000 transposon expressed in epithelial cells of the gut and pan
insertions identified 77 candidate CRC genes, 60 of which are mutated and/or dysregulated in creas but undetectable in other tissues (fig. S2).
human CRC and thus are most likely to drive tumorigenesis. These genes include APC, PTEN, and We created a cohort of 28 triple transgenic mice
SMAD4. The screen also identified 17 candidate genes that had not previously been implicated in and 72 double transgenic control mice carrying
CRC, including POLI, PTPRK, and RSP02. all possible dual combinations of the three trans
genes. Mice in this first cohort were monitored
daily for 18 months. We generated a second co
Recent genomic studies have revealed that To help identify potential driver genes in CRC, hort of 50 triple transgenic mice that were main
human colorectal cancers (CRCs) undergo we developed a forward genetic screen in mice tained in a separate facility for 12 months and
numerous genetic and epigenetic altera by using a Sleeping Beauty (SB) system to gen also monitored daily.
tions (1-4). These alterations probably derive from erate insertional mutations. To confine transposi Triple transgenic mice died at a faster rate
a mixture of "drivers" that play a causal role in tion to the gastrointestinal tract, SB11 transposase than double transgenic controls, beginning around
tumor formation and progression and "passen cDNA, preceded by a LoxP-flanked stop cassette, one year of age (Fig. IB). Examination of the
gers" that have little or no effect on tumor growth. was knocked into the Rosa26 locus (fig. SI) (5). gastrointestinal tract of moribund animals revealed
The design of targeted therapeutics for CRCs is These mice were then crossed with Villin-Cre trans discrete raised lesions ranging from 2 mm to as
dependent on the ability to distinguish drivers genic mice to activate SB transposase in epithelial large as 5 mm in diameter in the small and large
from passengers. cells of the gastrointestinal tract (6). Once ex intestine. In the first cohort, 100% (12 out of 12)

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