of November 1, 2016.
This information is current as
Immunological Synapse
Signaling Nanoarchitecture at the
Membrane Territories Controlling the 
HIV-1 Nef Impairs the Formation of Calcium
Helena Soares
Joana G. Silva, Nuno P. Martins, Ricardo Henriques and
ol.1601132
http://www.jimmunol.org/content/early/2016/10/19/jimmun
 published online 19 October 2016
J Immunol 
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The Journal of Immunology
HIV-1 Nef Impairs the Formation of Calcium Membrane
Territories Controlling the Signaling Nanoarchitecture at the
Immunological Synapse
Joana G. Silva,* Nuno P. Martins,† Ricardo Henriques,‡,x and Helena Soares*
The ability of HIV-1 to replicate and to establish long-term reservoirs is strongly influenced by T cell activation. Through the use of
membrane-tethered, genetically encoded calcium (Ca2+) indicators, we were able to detect for the first time, to our knowledge, the
formation of Ca2+ territories and determine their role in coordinating the functional signaling nanostructure of the synaptic
membrane. Consequently, we report a previously unknown immune subversion mechanism involving HIV-1 exploitation, through
its Nef accessory protein, of the interconnectivity among three evolutionarily conserved cellular processes: vesicle traffic, signaling
compartmentalization, and the second messenger Ca2+. We found that HIV-1 Nef specifically associates with the traffic regulators
MAL and Rab11b compelling the vesicular accumulation of Lck. Through its association with MAL and Rab11b, Nef co-opts Lck
switchlike function driving the formation Ca2+ membrane territories, which, in turn, control the fusion of LAT-transporting
Rab27 and Rab37 vesicles and the formation of LAT nanoclusters at the immunological synapse. Consequently, HIV-1 Nef
disengages TCR triggering from the generation of p-LAT and p-SLP nanoclusters driving TCR signal amplification and diver-
sification. Altogether our results indicate that HIV-1 exploits the interconnectivity among vesicle traffic, Ca2+ membrane terri-
tories, and signaling nanoclusters to modulate T cell signaling and function.
The Journal of Immunology, 2016, 197: 000–000.
T
he ability of HIV-1 to replicate in T cells is dependent on
the T cell activation state (1, 2). The accessory HIV-1
protein Nef enhances viral replication by modulating
multiple signaling pathways through a plethora of interactions with
cellular proteins (3). According to the literature, Nef may interact
with as many as 60 cellular factors and affect the function of .180
proteins (3). Notwithstanding, there is a pressing need to progress
from this encyclopedic listing of Nef interactions into a more
conceptual approach and determine how Nef intersects the regula-
tory mechanisms controlling the activation and/or differentiation
state of the infected cell.
T cell activation is coordinated at a specialized interface that
forms upon contact with an APC, known as immunological syn-
apse. TCR triggering induces the recruitment and activation of the
kinases Lck and ZAP70, which, in turn, phosphorylates LAT.
p-LAT recruitment of SLP76 allows for the formation of a sig-
naling supramolecular scaffold, nucleating various signaling
complexes involved in remodeling the T cell cytoskeleton (4),
T cell development (5), and activation (6). Thus, the assembly of
LAT-SLP76 signaling complexes, also called the LAT signal-
osome, is critical for T cell activation. The vesicular traffic has
emerged as a central player in the assembly of the signaling
machinery at the immunological synapse (7).
The TCR as well as the membrane-associated Lck and LAT
signaling molecules exploit the vesicular traffic to concentrate at
the immunological synapse (7). Recent works have started to
address the molecular mechanisms that regulate the exocytic tar-
geting of different vesicular compartments at the immunological
synapse (8–10).
Along these lines, we have determined that the regulated release
of LAT vesicles to the immunological synapse determines the
functional nanostructure of the synaptic membrane and the coor-
dination of the immune response (7, 8). We showed that Lck acts
as the signal switch and Ca2+ acts as the mediator of a vesicle
fusion feedback loop that builds a functional immunological
synapse capable of driving T cell activation and cytokine pro-
duction (8). Thus, deciphering the spatial organization of signaling
proteins is not only key to understanding the mechanisms that
underlie immune cell activation, but also to identifying common
design principals that are likely to be co-opted by HIV-1.
In this work, we aimed to decipher molecular mechanisms
underpinning the control of T cell activation to identify the
common design principals that are likely to be co-opted by HIV
infection. We uncovered a previously unknown immune subversion
mechanism involving HIV-1 exploitation, through its Nef accessory
protein, of the interconnectivity among three evolutionarily-conserved
cellular processes: vesicle traffic, signaling compartmentalization, and
*Immunobiology and Pathogenesis Group, Chronic Diseases Research Center,
NOVA Medical School, NOVA University of Lisbon, 1150-082 Lisbon, Portugal;
†Unit of Imaging and Cytometry, Gulbenkian Institute for Science, 2780-156 Oeiras,
Portugal;
‡Quantitative Imaging and Nanobiophysics Group, Medical Research
Council Laboratory for Molecular Cell Biology, University College London, London,
United Kingdom; and xDepartment of Cell and Developmental Biology, University
College London, London WC1E 6BT, United Kingdom
ORCIDs: 0000-0003-2318-3893 (J.G.S.); 0000-0002-8104-0957 (N.P.M.); 0000-
0002-2043-5234 (R.H.); 0000-0002-5881-3843 (H.S.).
Received for publication June 28, 2016. Accepted for publication September 16,
2016.
This work was supported by the Fundac¸a˜o para a Cieˆncia e Tecnologia under the FCT
Investigator Programme (H.S.), iNOVA4Health (Grant UID/Multi/04462/2013 to H.S.),
the Biotechnology and Biological Sciences Research Council (Grant BB/M022374/1 to
R.H.), and the Medical Research Council (Grant MR/K015826/1 to R.H.).
H.S. conceived the project; H.S. and R.H. designed experiments; J.G.S., N.P.M., R.H.,
and H.S. performed and analyzed experiments; and H.S. wrote the paper and all of the
authors commented on it.
Address correspondence and reprint requests to Helena Soares, Immunobiology and
Pathogenesis Group, Chronic Diseases Research Center, NOVA Medical School,
Faculdade de Cieˆncias Me´dicas, NOVA University of Lisbon, 1150-082 Lisbon,
Portugal. E-mail address: helena.soares@nms.unl.pt
Abbreviations used in this article: ATCC, American Type Culture Collection; FRET,
fluorescence resonance energy transfer; FynMA, Fyn MA domain; HA, hemaggluti-
nin; LckMA, Lck membrane anchoring domain; LATTM, LAT MA domain; MA,
membrane anchoring; TIRF, total internal reflection; TM, transmembrane; TPS,
thapsigargin.
Copyright � 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601132
 
Published October 19, 2016, doi:10.4049/jimmunol.1601132
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the second messenger Ca2+. We found that Nef selectively interacts
with Lck vesicle traffic regulators Rab11b and MAL restraining
Lck from the immunological synapse. We pioneered the use of
membrane-tethered, genetically encoded Ca2+ indicators (11) to
detect and characterize the formation of Ca2+ territories at the TCR-
stimulated membrane. Interestingly, we determined that Nef impairs
the formation of Ca2+ membrane territories at the immunological
synapse even though it has no effect on cytosolic Ca2+ fluxes (12).
Finally, by restraining Lck away from the immunological synapse,
Nef co-opts the switch regulatory function of synaptic Lck in pro-
moting the generation of Ca2+ membrane territories controlling
LAT vesicle fusion and the signaling nanoarchitecture of the im-
munological synapse.
Materials and Methods
Vectors and transfection
Lck membrane anchoring (MA) domain (LckMA)-LAT-GFP and LAT MA
domain (LATTM)-Lck-GFP were constructed by PCR-fusion of the Lck
kinase aa 1–18 and LAT aa 28–262 and of the LAT aa 1–27 and Lck aa 19–
501, respectively. pN1-Fyn-GCaMP3 was obtained by replacing the
membrane targeting sequence of Lck with the one of Fyn, through Gibson
assembly, on pN1-Lck-GCaMP3 construct obtained from Adgene through
Gibson Assembly. The construct of Nef allele NL4.3 and its mutants fused
to GFP or mCherry were gifts from F. Niedergang (Institut Cochin, Paris,
France). The Gts-Nef (revGlc-NefDSH4) and Fyn MA domain (FynMA)-
Lck-GFP (FynN18-Lck.GFP) constructs were provided by O. T. Fackler
(University Hospital Heidelberg, Heidelberg, Germany); the latter was
used to clone FynMA-Lck-mCherry. The MAL-GFP construct was a gift
from M. A. Alonso (Universidad Autonoma de Madrid, Madrid, Spain);
the Rab11b-GFP and Rab27-GFP constructs were a gift from D. Barral
(Chronic Diseases Research Center, Lisbon, Portugal), and the Rab37-GFP
construct was a gift from B. Goud (Institut Couchin, Paris, France). DNA
constructs were inserted into Jurkat and primary CD4 T cells using the
Invitrogen Neon Transfection system. Transiently transfected cells were
analyzed 24 h after transfection. Primary CD4 T cells were transfected
once with small interfering RNA oligonucleotide pools (Invitrogen) tar-
geting Lck. Silenced cells were analyzed 72 h after transfection.
Virus production and infection
To generate viral stocks, we transfected 293T cells with the HIV-1 NL4.3 and
HIV-1 NL4.3 DNef constructs. Jurkat cells were infected with HIV-1 NL4.3
and HIV-1 NL4.3 DNef virus for 3 d before imaging as described before (13).
Cells, reagents, and immunofluorescence
Jurkat cell clones E6.1 and Lck2/2 Jurkat JCAM1.6 cells were grown in
complete RPMI 1640 medium containing 10% (v/v) FBS, nonessential
amino acids, and L-glutamine. Human peripheral blood cells were stimu-
lated with PHA (1 mg/ml) and grown for 7 d in RPMI 1640 medium with
10% FCS and 20 IU/ml IL-2. Glass coverslips coated with poly-lysine
overnight at 4˚C and, when stated, further incubated with stimulatory
(anti-CD3 MEM-92 [EXBIO] and CD28 [Genentech]) or nonstimulatory
(anti-CD45 mAb GAP 8.3; American Type Culture Collection [ATCC])
Abs at a concentration of 10 mg/ml overnight at 4˚C, as previously de-
scribed (8). In some cases, thapsigargin (5 mM; TPS) was added during the
stimulation period. For imaging of Fyn-CaGMP3, transfected cells were
imaged on RPMI 1640 medium at 37˚C. For all other conditions, cells
were then fixed with 4% paraformaldehyde for 30 min at room tempera-
ture, incubated with blocking buffer (PBS BSA 1%) with or without 0.05%
saponin, and stained with primary Abs against Lck (3A5; Santa Cruz),
LAT and p-LAT (Cell Signaling), p-SLP (J141; BD Biosciences), CD45
(GAP 8.3; ATCC), hemagglutinin (HA; ab9110 and ab18181; Abcam), and
Gag (National Institutes of Health AIDS reagent program) for 1 h, washed,
and incubated for 45 min with Alexa 568– and Alexa 647–conjugated
secondary Abs (Life Technologies).
Imaging, image processing, and quantification
Confocal images were obtained using a Zeiss LSM 710 confocal micro-
scope (Carl Zeiss) over a 633 objective. 3D image deconvolution was
performed using Huygens Essential (version 3.0; Scientific Volume Im-
aging), and 2D images were generated from a maximum intensity pro-
jection over a 3D volume cut of 1-mm depth centered either on the
intracellular compartment when visible or on the cell center. For the
quantification of Lck and LAT subcellular distribution, the plasma mem-
brane of CD4 T cells was surface labeled with anti-CD45 mAbs. Plasma
membrane segmentation was implemented through the generation of bi-
nary mask delimitating the internal and external outlines of the plasma
membrane using the Fiji image analysis software.
Fluorescence resonance energy transfer imaging
A first set of fluorescence resonance energy transfer (FRET)-donor (GFP-
tagged proteins) and FRET-acceptor (mCherry-tagged proteins) confocal
images were acquired at low excitation intensity. Acceptor photobleaching
with a 561-nm laser was then carried out through strong illumination of a
region of interest corresponding to the intracellular compartment of each
cell. mCherry showed 90% decrease in signal within the defined region of
interest. A second set of images was acquired at low excitation intensity
after acceptor photobleaching. Both prebleaching and postbleaching
datasets were corrected for drift and analyzed with the AccPbFRET plugin
for ImageJ software (GraphPad).
dSTORM Imaging
Glass coverslips were washed two to three times in optical grade acetone,
soaked overnight, and sonicated in 0.1 M KOH for 20 min. Coverslips were
then thoroughly rinsed in deionized water and dried. The glass coverslips
were coated overnight at room temperature with 0.001% poly-L-lysine
(Sigma) diluted in PBS. Dried coverslips were subsequently incubated
with stimulatory (anti-CD3 MEM-92 [EXBIO] and CD28 [Genentech]) or
nonstimulatory (anti-CD45 mAb GAP 8.3 [ATCC]) Abs at a concentration
of 10 mg/ml overnight at 4˚C. Cells were resuspended in imaging buffer,
and 500,000 cells were dropped onto the coverslips and incubated for
5 min at 37˚C. In some cases, TPS (5 mM) was added during the incu-
bation period. Cells were then fixed with 4% paraformaldehyde for 30 min
at room temperature. To maximize the number of detected molecules, care
was taken to minimize photobleaching: cells were embedded in oxygen
scavenger buffer, imaged on the same day of labeling, and kept in the dark
until imaged. dSTORM images were then acquired with a Nikon Ti mi-
croscope coupled with a Hamamatsu Flash Orca 4.0 sCMOS camera and a
1003 Plan Apo l 1.45NA oil immersion objective at a 50-Hz rate.
Wavelength lasers lines of 561 nm (Coherent Genesis MX 561-500 STM)
and 638 nm (Vortran Stradus 642-110) were used to excite Alexa Fluor 568
and Alexa Fluor 647, respectively, using emission filters 595/50 and 655LP
from Chroma Technology to further reduce and limit cross-talk. For a
better signal-to-noise ratio, a pseudo–total internal reflection (TIRF) illu-
mination (oblique illumination) was used. Acquisition was done through
mManager freeware (14), and acquired data were then processed and
reconstructed through Fiji (15) using the superresolution plugin Thun-
derSTORM (16). Once events were detected, a data table was exported
with x–y coordinates of each molecule, photon count, and spatial preci-
sion, which could later be converted into an image. These datasets were
then processed to check for possible cluster formation.
For spatial point pattern of molecular detection, Ripley’s K-function was
then calculated through a Fiji macro of commands as follows:
Z Z
for i �j
KðrÞ ¼ A +
n
i¼1
+
n
j¼1
�dij
n2
�
where dij ¼ 1 if dij , r; otherwise 0
where A is the area of the analyzed region (which depends on the radius), r is
the radius of the analyzed area, n is the number of detected particles, and d is the
distance between points i and j. This counts the number of molecules inside the
circle area around each detected particle and normalizes it to the average
molecular density of all areas measured to access possible cluster formation.
Flow cytometry
Jurkat cells were stained with FITC-labeled mAbs against CD4 (RPA-T4;
BD Biosciences) and with unconjugated mAbs against HA (ab9110;
Abcam) followed by incubation with Alexa 647–labeled anti-rabbit Ab
(Life Technologies). Cell-associated fluorescence was collected on a
FACSCalibur and analyzed using the FlowJo software (BD Biosciences).
For Ca2+ flow measurements, Jurkat cells (5.106/ml) resuspended in phenol
red–free RPMI 1640 were loaded with 1 mM of Indo-1 (Molecular Probes)
at 37˚C in the dark for 45 min. Cells were washed and resuspended in the
same medium. Ca2+ measurements were performed on a MoFlo flow
cytometer (Beckman Coulter). Cells were acquired for 90 s to establish the
baseline followed by ionomycin addition (10 mg/ml). A ratiometric anal-
ysis was performed using the FlowJo software (BD Biosciences).
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Results
The N terminus of HIV-1 Nef promotes the selective
accumulation of Lck, but not LAT, in the vesicular
compartment
In recent years, the vesicle traffic of signaling molecules has emerged
as a central regulator of T cell activation (7–10). In particular, we
have determined that the formation of nanoterritories where sig-
naling is coordinated to promote T cell activation is regulated by a
vesicular traffic amplification loop involving the sequential vesicle
delivery of Lck and LAT at the immunological synapse (8). Even
though the main determinant of HIV-1 pathogenesis is the modu-
lation of TCR signaling by the viral protein Nef, it remains poorly
understood how Nef exploits the interconnectivity between vesic-
ular traffic and synaptic signaling underpinning T cell activation.
To address this open question, we started by determining the
effect of Nef on Lck and LAT subcellular distribution (7). As
previously reported, Nef retains Lck in an enlarged perinuclear
vesicular compartment (7, 13, 17) (Fig. 1A, 1B), both in Jurkat
and in primary CD4 T cells (Fig. 1A, 1B). Unexpectedly, Nef does
not alter the subcellular distribution of LAT, despite their adjacent
localization in the tightly filled T cell cytoplasm (Fig. 1A, 1B).
To gain mechanistic insights on how Nef specifically alters Lck
subcellular distribution, we tailored the expression of a large set of
GFP-tagged Lck and LAT chimeric proteins (Fig. 1C) and assessed
their subcellular distribution (Fig. 1D, 1E). Previous works have
reported that the biological activity of Nef is controlled by the spec-
ificity of its membrane targeting (18), but also that the LckMA, which
encodes Lck traffic properties, is necessary and sufficient to undergo
Nef-mediated retention (18). First, we confirmed that rerouting Nef
to the Golgi compartment prevents Lck vesicular accumulation
(Fig. 1A–C, Gts-Nef), underscoring the relevance of specific mem-
brane targeting to Nef biological activity. Next, to clarify whether the
LckMA is necessary and sufficient to undergo Nef-mediated retention,
we used an Lck construct encoding solely the MA domain consisting
of its first 18 aa and devoid of additional protein interaction surfaces
fused to GFP (LckMA), and expressed it in Lck-deficient Lck2/2
JCAM1.6 cells. Cotransfection of Nef-mCherry and LckMA-GFP in
Lck2/2 JCAM1.6 cells caused the vesicular accumulation of the
LckMA-GFP to the similar extent to the one verified with endogenous
Lck (Fig. 1A, 1B, 1D, 1E), confirming that the LckMA is sufficient to
undergo Nef-mediated vesicular accumulation (19).
Lck and the closely related Fyn kinase display distinct MA do-
mains and consequently are segregated to distinct vesicular com-
partments (20). Because Nef does not alter the vesicular distribution
of the closely related Fyn kinase (17), we then expressed in Jurkat
T cells a chimeric protein with all the Lck functional motifs except
for the MA domain, which has been replaced by the one of Fyn
(Fig. 1C, FynMA-Lck). Despite the presence of all the Lck inter-
action motifs in the FynMA-Lck chimeric protein, Nef did not cause
the vesicular accumulation of FynMA-Lck (Fig. 1D, 1E).
We next interchanged Lck and LAT trafficking properties by
constructing chimeric proteins in which the LckMAwas replaced by
the LATTM (Fig. 1C, LATTM-Lck) and the LATTM was replaced
by the LckMA (Fig. 1C, LckMA-LAT). Conferring LAT traffic
properties to Lck abrogated its retention by Nef and reciprocally,
endowing LAT with Lck traffic properties led to LAT vesicular
accumulation in Nef-expressing Jurkat cells (Fig. 1D, 1E).
These results indicate that the N-terminal domain of Lck is required
for its selective vesicular accumulation mediated by Nef.
HIV-1 Nef selectively associates with Lck traffic regulators
The specification of the vesicular identity and traffic route rely on
the Rab family GTPases (21). Lck and LAT reside and traffic in
distinct vesicular compartments, with little to no overlap in their
Rab specification (8). Whereas Lck localizes to Rab11b+ and MAL+
vesicles (8, 22, 23), LAT resides in vesicles marked with Rab27 and
Rab37 (8). We transiently coexpressed each Rab-GFP protein with
Nef-mCherry, in Jurkat and in primary T cells, and assessed their
colocalization in the vesicular compartment. To exclude the possi-
bility that Nef colocalization with MAL and Rab11b could be
caused by Nef interaction with Lck (24, 25), we first investigated
whether Nef colocalized preferentially with Lck traffic regulators, in
Lck2/2 JCAM1.6 and primary T cells (Fig. 2A). In both Jurkat and
primary T cells, Nef highly colocalized with Lck traffic regulators
Rab11b and MAL (∼80%; Fig. 2A, 2B) and only partially colo-
calized with Rab27 and Rab37 (∼20%; Fig. 2A, 2B), even when
Lck (LATTM-Lck) was targeted to LAT transporting Rab27 and
Rab37 vesicles (∼8%; Fig. 2A, 2B). Conversely, rerouting Nef to
the Golgi (Gts-Nef) abolished its colocalization with Rab11b and
MAL (∼5%; Fig. 2A, 2B). Altogether, these results indicate that
Lck vesicular accumulation requires Nef association with its traffic
regulators Rab11b and MAL, and that this association is established
independently of Lck.
To analyze whether Nef does in fact associate with Lck traffic
regulators, we performed FRET analysis through acceptor photo-
bleaching, which detects molecular proximities ,10 nm (26).
Nef associated with both Rab11b and MAL even in the absence
of Lck (∼20% FRET efficiency; Fig. 3A, 3B, Lck2/2 JCAM1.6).
However, Nef did not associate with LAT traffic regulators
Rab27 and Rab37 (∼3% FRET efficiency; Fig. 3A, 3B, Jurkat
cells). Consistent with our previous findings, rerouting Nef to the
Golgi abolished Nef association with Lck traffic regulators
Rab11b and MAL (∼2% FRET efficiency; Fig. 3A, 3B, Jurkat
cells Gts-Nef).
These results show that HIV-1 Nef associates in situ Rab11b
and MAL, and that this association is supported by Nef traffic
properties.
Nef co-opts the switchlike regulatory function of synaptic Lck
in promoting the generation of LAT signaling nanoclusters
T cell activation relies on the generation of an amplification sig-
nalosome formed by direct association of p-LAT– and p-SLP76–
Gads complexes (27). Given that Lck synaptic clustering acts as
the signal switch required for the formation of LAT signaling
territories (8), we investigated whether the vesicular retention
would impair the functional architecture of LAT signaling in Nef-
expressing cells. We triggered T cell activation by incubating the
T cells for 5 or 12 min on coverslips coated with Abs to CD3 and
CD28 followed by fixation. We visualized Nef effects on Lck and
LAT vesicle release at the immunological synapse by 3D confocal
microscopy (Fig. 4, top panels), and we determined Nef alter-
ations to the functional architecture of the synaptic membrane
using dual-color dSTORM combined with TIRF (Fig. 4, bottom
panels) (28).
As expected, Nef expression or HIV-1 infection prevented the
Lck vesicular compartment delivery at the immunological syn-
apse (Fig. 4A, 4B, 4D, top panels). However, regardless of the
fact that Nef does not interact with LAT vesicular traffic (Figs. 1,
2), LAT vesicle delivery is also impaired in Nef-expressing cells
(Fig. 4A, 4B, 4D, top panels). Regarding the signaling archi-
tecture of activated T cells, we observed that both Nef and HIV-1
infection reduced the number and size of signaling active p-LAT
nanoclusters at the immunological synapse (Fig. 4B, 4D, 4G,
4H), as well as their capacity to recruit and form signaling
complexes with p-SLP76 (Fig. 4B, 4D, 4I, 4J). All of these de-
fects were rescued when T cells were infected with a Nef-deleted
virus (Fig. 4E–J, HIV-DNef), but not by stimulating HIV-1–infected
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cells for longer (Fig. 4F–J, 129 HIV). This indicates that the sig-
naling deficit in HIV-1–infected cells is unlikely to be a manifes-
tation of delayed kinetics.
These data suggest that, by retaining Lck in a vesicular compartment,
Nef impairs the vesicle fusion positive feedback loop that drives the
formation of p-LAT nanoclusters at the immunological synapse. To
FIGURE 1.
Nef promotes the vesicular retention of Lck, but not of LAT. (A) Deconvoluted confocal images of the effect of Nef and Gts-Nef (green) on the
subcellular distribution of endogenous Lck and LAT (magenta) in Jurkat T cells (top panels) and in primary CD4 T cells (bottom panels). (B) Population analysis
quantifying the 3D fluorescence intensity in the vesicular compartment relative to total fluorescence of Lck and LAT in Jurkat cells (top panel) and in primary CD4
T cells processed as in (A) for n = 10 cells per experimental condition. (C) Schematic representation of the chimeric proteins used. To alter Nef trafficking properties,
we replaced its MA domain by a Golgi targeting sequence (18). To alter Lck trafficking properties, we replaced its MA domain (LckMA) with either FynMA (FynMA-
Lck) or LATTM (LATTM-Lck). To alter LAT trafficking properties, we replaced its TM domain by the LckMA (LckMA-LAT). (D) Deconvoluted confocal images of
the effect of Nef (green) on the subcellular distribution of the Lck anchoring domain in Lck-deficient JCAM1.6 cells (top row) and on Lck and LAT chimeric proteins
(bottom rows) in Jurkat T cells. (E) Population analysis quantifying the effect of Nef on the subcellular distribution of endogenous and chimeric Lck and LAT
proteins, measured by 3D fluorescence intensity in the vesicular compartment relative to total fluorescence, in Jurkat T cells for n $ 9 cells per experimental
condition. (A and D) Arrowheads highlight accumulation of the signaling molecules in the vesicular compartment. Scale bars, 5 mm. Each symbol represents a cell.
Data are representative (A and D) or are the sum of (B and E) three independent experiments. **p = 0.0021, ***p , 0.0001 (Mann–Whitney test). n.s., not significant.
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formally test this hypothesis, we recovered Lck clustering at the im-
munological synapse of Nef-expressing cells by coexpressing the
chimeric FynMA-Lck protein whose traffic is not affected by Nef (Fig.
1C, 1D). FynMA-Lck expression completely recovered LAT vesicle
delivery (Fig. 4C, top panels), the number and size of p-LAT nano-
clusters, as well as their ability to recruit p-SLP76 (Fig. 4C, 4G–J).
FIGURE 2.
Nef selectively colocalizes with Lck traffic regulators Rab11b and MAL. (A) Deconvoluted confocal images of Jurkat and primary T cells
illustrating the degree of Nef colocalization (in white) with (top panel) the Lck traffic regulators Rab11b/MAL in Lck-deficient JCAM1.6 and in primary
CD4 T cells, and (middle panels) with the LAT traffic regulators Rab27/Rab37 in cells coexpressing or not LATTM-Lck. Bottom panels show the degree
of Gts-Nef colocalization with the Lck traffic regulators Rab11b/MAL. Right images show inset amplification. Scale bar, 5 mm. (B) Population analysis of
the volume of colocalization in cells treated as in (A) for n = 10 per experimental condition. ***p , 0.0001 (Mann–Whitney test). n.s., nonsignificant.
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Our results collectively demonstrate that, by retaining Lck in a
vesicular compartment, Nef impairs the vesicle fusion positive
feedback loop that drives the formation of p-LAT nanoclusters and
the subsequent recruitment of p-SLP76.
HIV-1 Nef impairs the formation of the Ca2+ membrane
territories
Given that Lck clustering might participate in organizing TCR
signaling (8, 29) possibly via local Ca2+ fluxes (8, 30), we inves-
tigated the impact of HIV-1 Nef on the formation of Ca2+ domains
after TCR engagement. We reasoned that if membrane-localized
Ca2+ are indeed determinant for the signaling architecture of the
immunological synapse, the formation of Ca2+ territories should be
detectable in the TCR-stimulated membrane. To pursue our hy-
pothesis, we constructed a membrane-tethered, genetically encoded
Ca2+-sensor Fyn-GCaMP3, whose membrane localization could not
be altered by HIV-1 Nef (11). Our experimental setup enabled us to
directly detect the formation of Ca2+ territories at the synaptic
membrane, by measuring the increase in Fyn-GCaMP3 fluorescence
intensity. TCR engagement, by incubating the T cells on coverslips
coated with Abs to CD3 and CD28, promoted the formation of Ca2+
territories at the membrane of control and HIV-DNef–infected cells
T cells, but not of Nef-expressing or HIV-infected cells (Fig. 5A,
5B). Interestingly, coexpressing FynMA-Lck and Nef reverted the
formation of Ca2+ territories at the stimulatory membrane, indi-
cating that Lck signaling at the immunological synapse is required
for the formation of Ca2+ territories. Finally, Nef-expressing cells
exhibited no intrinsic functional defect in the Ca2+ channels, be-
cause treatment with Ca2+ flux–inducing drugs TPS and ionomycin
restored the formation of Ca2+ territories at the synaptic membrane
of Nef-expressing cells and also induced similar cytosolic levels of
Ca2+ influx, respectively (Fig. 5A–C).
FIGURE 3.
Nef selectively associates with the regulators of Lck vesicular traffic Rab11b and MAL. (A) FRET imaging of Lck-deficient JCAM1.6 or
Jurkat vesicular compartments by acceptor photobleaching. Top two rows, Imaging before photobleaching of the mCherry-tagged FRET acceptors (pre-
bleach; acceptor) and of the GFP-tagged FRET donors (prebleach; donor). Third and fourth rows, Imaging postphotobleaching of the mCherry-tagged
FRET acceptors (postbleach; acceptor) and of the GFP FRET donors (postbleach; donor). Bottom row displays calculated [(Donorfinal 2 Donorinitial)/
Donorfinal] FRET efficiency maps. Bleached areas are indicated by white circles. Scale bar, 5 mm. (B) Population analysis of FRET efficiencies for n = 10
per experimental condition. Each symbol represents a cell. Data are representative (A) or are the sum (B) two independent experiments. ***p # 0.0001
(Mann–Whitney U test).
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These data indicate that HIV-1 impairs the formation of Ca2+
territories at the synaptic membrane by removing Lck from the
immunological synapse.
HIV-1 Nef impairs the formation of calcium territories
orchestrating the signaling nanoarchitecture at the synaptic
membrane
Finally, to assess the direct impact of Nef on the orchestration of
TCR signaling nanoarchitecture, we determined the effect of im-
paired Ca2+ territories on LAT vesicle docking and/or fusion at the
synaptic membrane. LAT vesicles are decorated by the exocytic
Rab27 and Rab37, by the v-SNARE Ti-VAMP, and by the Ca2+
sensor synaptotagmin-7, which mediate docking and/or fusion of
vesicles with target membranes (8, 9, 21, 31). To distinguish
between vesicle docking and vesicle fusion, we used an engi-
neered LAT molecule with an extracellular HA tag that allows for
surface labeling of LAT (LAT-HA) (9). Population comparison of
HA labeling of control and Nef cotransfected T cells by flow
cytometry and by confocal microscopy showed that both control and
Nef cotransfected T cells expressed equivalent levels of LAT-HA
(Fig. 6A) with similar subcellular distribution at the plasma
membrane and within the vesicular compartment (Fig. 6B, 6C).
Activation on coverslips coated with anti-CD3 and anti-CD28 Abs
of control and HIV-DNef–infected T cells induced the fusion of
the vesicular pool of LAT-HA at the synaptic membrane and the
formation of LAT-HA nanoclusters measured by surface labeling
FIGURE 4.
Nef co-opts the switchlike function of synaptic Lck in promoting TCR signal amplification. (A–F) Confocal orthogonal view (top panels) and
dSTORM-TIRF images (bottom panels) of control (A), Nef-expressing (B), Nef and FynMA-Lck coexpressing (C), HIV (D and F), or HIV-DNef–infected (E)
Jurkat cells fixed while interacting with glass coated with Abs against CD3 and CD28 (CD3/28) for 5 (A–E) or 12 min (F) and immunostained for Lck and/
or LAT (top panels) or for p-LAT (green) and p-SLP76 (magenta) (bottom panels). (G–J) Population analysis quantifying the effect of Nef on (G) the
number of clusters of p-LAT per square micrometer, (H) the mean p-LAT cluster area per cell, (I) the number of clusters of p-SLP76 per square micrometer
and (J) the mean p-SLP76 cluster area per cell, measured by superresolution microscopy in cells treated as in (A)–(F) for n $ 9 cells per experimental
condition. Each symbol represents a cell. Data are representative (A–E) or are the sum of (G–J) two independent experiments. Scale bars, 5 mm. ***p ,
0.0001 (Mann–Whitney test).
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(Fig. 6D, 6H), which were indistinguishable in number, size, and
density from endogenous p-LAT nanoclusters (Figs. 4, 6D, 6H–J).
These results indicate that LAT vesicle fusion is required for the
orchestration of a LAT signaling nanoarchitecture. In Nef-
expressing or in HIV-infected cells, there was a stark decrease
in the number, size, and density of surface LAT-HA, reflecting Nef
impairment of LAT vesicle fusion at the synaptic membrane
(Fig. 6E, 6G, 6I, 6J). Importantly, restoration of Ca2+ territories by
TPS treatment in anti-CD3– and anti-CD28–stimulated, Nef-
expressing cells completely recovered LAT-HA nanoclustering
in number, size, and density (Fig. 6F, 6I, 6J). These data collec-
tively indicate that HIV-1 exploits the exquisite interconnectivity
between Lck signaling and the formation of Ca2+ territories to
impair the orchestration of a functional immunological synapse.
Discussion
Cross-talk among vesicle traffic, Ca2+ gradients, and TCR sig-
naling coordinates T cell activation (7, 32). In this article, we
report a previously unknown immune subversion mechanism in-
volving HIV-1 Nef exploitation of the interconnectivity among
vesicle traffic, Ca2+ membrane territories, and TCR signaling.
This inhibitory mechanism required Nef to co-opt Lck switchlike
function driving the formation of Ca2+ membrane territories
controlling the vesicular fusion amplification loop impelling LAT
nanoclustering at the immunological synapse.
Nef has been characterized as a potent, multifunctional modulator
of T cell activation (3). Recent works have shown that the vesicular
traffic has a central role in regulating the assembly of signaling
complexes at the immunological synapse for T cell activation (7, 32,
33). We propose that Nef has evolved to co-opt the traffic check-
points regulating the functional nanoarchitecture of the immunolog-
ical synapse rather than by inhibiting a series of signaling molecules
involved in sequential steps of TCR signaling cascade (34). We have
found that Nef interacts with Lck traffic regulators Rab11b and
MAL, but not with LAT traffic regulators Rab27 and Rab37 (8,
22, 23). These interactions resulted in the selective retention of
Lck in an enlarged vesicular compartment, in Nef-expressing
cells. Interchanging Lck and LAT trafficking properties impaired
Lck retention and reciprocally led to LAT vesicular accumulation
in Nef-expressing cells. We used several different experimental
approaches to determine whether Nef interacts directly with MAL;
however, the results of these experiments were inconclusive. One
possible explanation for these inconsistences is that TM proteins
containing multiple hydrophobic domains, as is the case of MAL,
often possess different tertiary structures and binding affinities
when in solution relative to those occurring within a lipid bilayer.
Such changes might confound solution-based analysis such as
coimmunoprecipitation and far-Western detection, or even render
physical interaction between Nef and MAL impossible in these
conditions. In contrast, FRET analysis performed under physio-
logic conditions with MAL in its native conformation within a
lipid bilayer yielded that Nef and MAL are found at interacting
distances. In fact, we detected Nef association with both Rab11b
and MAL in Lck-deficient cells, precluding the possibility that
Nef-Rab11b/MAL in situ interaction could be the result of a tri-
partite association between Lck-MAL/Rab11b-Nef. We found that
rerouting of Nef trafficking from the Rab11+ MAL+ recycling
compartment to the close by Golgi vesicles completely abolished
Nef activity. Our results suggest that Nef-mediated Lck vesicular
retention is the result of the specific Nef-membrane association to
the Rab11b and MAL trafficking compartment. Consistent with
our results, a recent study has found that Nef biological activity,
including Lck vesicular accumulation, is supported by the speci-
ficity of its MA domain (18). Curiously, an early work on Nef
pathogenicity had found that the MA domain of Nef was required
for Nef-mediated retention of Lck through the formation of a
protein complex (25). In addition, we and others showed that Lck
vesicular accumulation does not depend on its SH3-mediated in-
teraction with Nef, requiring only the LckMA (19). It is thus
possible that by promoting Lck vesicular accumulation, Nef as-
sociation with Rab11b and MAL facilitates Nef modulation of
Lck kinase activity through interaction with its SH3 domain.
FIGURE 5.
Nef impairs the formation of Ca2+
territories at the synaptic membrane. (A) The
membrane-tethered and genetically encoded Ca2+
indicator (Fyn-GCaMP3) was used to image
the formation of Ca2+ territories at the synaptic
membrane of control, Nef-expressing (Nef), Nef
and FynMA-Lck coexpressing (Nef; FynMA-Lck),
and HIV- or HIV-DNef–infected Jurkat cells
while interacting with glass coated with Abs
against CD3 and CD28 (aCD3/28) or with anti-
CD45 (aCD45; Ctr) for 3 min before imaging.
Scale bar, 5 mm. (B) Population analysis of the
formation of Ca2+ membrane domains at the
immunological synapse in Jurkat cells processed
as in (A) (arbitrary units). (C) Cytosolic Ca2+ flux
measurements (showing the ratio of indo-blue to
indo-violet) in control (blue) and Nef-expressing
(red) Jurkat cells stimulated with ionomycin
(arrow). Each symbol represents a cell. Data are
representative (A and C) or are the sum of (B)
three independent experiments. ***p # 0.0001
(Mann–Whitney U test). n.s., not significant.
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Even though Nef motifs responsible for promoting the inter-
nalization and vesicular accumulation of the transferrin receptor
(35), CD80/CD86, and MHC class I (36) differ, all of these
molecules share the striking commonality of trafficking through a
Rab11b compartment. It is tempting to propose that once inter-
nalized, the transferrin receptor, CD80/CD86, and MHC class I
are prevented to recycle back to the plasma membrane as the
consequence of Nef interaction with their common traffic regu-
lator, Rab11b. Thus, Nef interaction with Rab11b reported in this
study might allow for a unified view for the molecular mecha-
nisms underpinning the vesicular retention of distinct molecules
across distinct cell types (lymphocytes, macrophages, and den-
dritic cells) and might provide valuable insight into the role of
Rab11b in HIV-1 budding (37).
The spatial organization of signaling proteins determines the
T cell activation outcome (7–10, 32, 38, 39). Thus, to understand
Nef modulation of T cell activation, it is imperative to move from
simply identifying the individual TCR signaling molecules tar-
geted by Nef to determining the molecular mechanism under-
pinning Nef co-opt of the spatial organization of signaling
components. Superresolution microscopy has made important
contributions toward revealing the molecular mechanisms
FIGURE 6.
Nef impairs the formation of Ca2+ membrane territories controlling the signaling nanoarchitecture at the immunological synapse. (A)
Cytofluorimetry of the expression of LAT-HA in control (blue shaded histogram) and Nef-expressing Jurkat cells (red line) and labeled with mAb to HA.
(B) Deconvoluted confocal images illustrating the absence of Nef effect on the subcellular distribution of LAT-HA. (C) Population analysis quantifying the
absence of Nef effect on the subcellular distribution of LAT-HA, measured by 3D fluorescence intensity in the vesicular compartment relative to total
fluorescence. (D–H) Orthogonal view (top panels) and dSTORM-TIRF images (bottom panels) of control (Ctr) (D), Nef-transfected (Nef) (E), Nef-
transfected and TPS-treated (Nef TPS) (F), HIV-1–infected (HIV) (G), or HIV-1 DNef-infected (HIV-DNef) (H) Jurkat cells expressing LAT-HA, fixed
while interacting with glass coated with Abs against CD3 and CD28 for 5 min and immunostained for Lck and HA (top panels) or surface LAT-HA
nanoclusters (bottom panels). (I and J) Population analysis quantifying the effect of Nef on (I) the number of clusters of LAT-HA at the synaptic membrane
per square micrometer and (J) the mean area of LAT-HA cluster at the synaptic membrane for n = 10 cells per experimental condition, measured by
superresolution microscopy in cells treated as in (D)–(H) (bottom panels). Each symbol represents a cell. Data are representative (A, B, D–H) or are the sum
of (C, I, and J) three independent experiments. Scale bars, 5 mm. ***p # 0.0001 (Mann–Whitney U test). n.s., not significant.
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controlling the signaling nanoarchitecture of the immunological
synapse (8–10, 38, 39). By combining dual-color dSTORM with
TIRF microscopy, we found that by interfering with the MAL-
mediated route of Lck transport, Nef disengages TCR triggering
from LAT vesicle fusion and alters LAT signaling nanoarchitecture
at the synaptic membrane. Accordingly, Nef expression decreased
the number, density, and size of p-LAT nanoclusters. The formation
of elongated TCR signaling nanoclusters has been positively corre-
lated with the efficiency of signal transmission (8, 38, 40). Inter-
estingly, we noted a shift in the synaptic signaling pattern of
Nef-expressing cells from the elongated nanoclusters, observed in
control cells, toward circular p-LAT, which correlated with a de-
creased ability of p-LAT to recruit p-SLP76 and the consequent
impaired assembly of the LAT signalosome. Finally, bypassing
Lck vesicular retention was sufficient to rescue p-LAT nano-
clustering at the immunological synapse of Nef-expressing cells.
This further reinforced that Nef impairment of LAT synaptic de-
livery is not the consequence of a direct interaction, but rather the
consequence of Nef co-opting the switchlike regulatory function
of synaptic Lck in promoting the vesicle fusion and the conse-
quent formation of p-LAT nanoclusters.
Lck clustering has been implicated in the organization of TCR
signaling (8, 29) possibly via local Ca2+ fluxes (8, 30). Thus, by
retaining Lck, Nef must impair the reorganization of the synaptic
membrane that allows for the fusion of LAT vesicles. To map the
impact of Nef on the formation of Ca2+ domains at the synaptic
membrane, we pioneered the use of genetically encoded, membrane-
tethered Ca2+ indicators (11), which detects highly localized Ca2+
signals that soluble probes fail to detect, to map the impact of Nef on
the formation of Ca2+ domains at the synaptic membrane. Nef im-
pairs the formation of Ca2+ membrane territories at the synaptic
membrane even though it has no effect on cytosolic Ca2+ fluxes (12).
Once again, bypassing Lck vesicular retention was sufficient to
rescue the formation of Ca2+ membrane territories at the TCR-
stimulated membrane, in Nef-expressing cells. These results illus-
trate the exquisite control that Nef exerts on the subcellular distribution
of Ca2+ levels. Even though intracellular Ca2+ controls a remarkable
breadth of cellular processes, including secretion, motility, and dif-
ferentiation, local Ca2+ domains are essential to regulate specific
signaling events (41–43). Thus, by impairing the formation of Ca2+
territories at the stimulated membrane, Nef can block the feedback
loop amplifying TCR signaling and lower T cell sensitivity to Ag
without affecting other Ca2+-dependent functions that might be re-
quired for HIV-1 propagation. Impairment of Ca2+ membrane
territories is possibly involved in Nef inhibition of other cellular
processes, such as migration (44, 45) and phagocytosis (46),
which might need to be reinterpreted accordingly.
In conclusion, this work shows that Nef has evolved to co-opt the
traffic checkpoints regulating the functional nanoarchitecture of
the immunological synapse. Obviously, alterations to the regulatory
mechanisms controlling T cell signaling thresholds and signal ampli-
fication could underpin the establishment of long-term reservoirs. Thus,
the provision of such a mechanism is necessary to identify molecular
targets for the development of more efficient antiviral therapies.
Acknowledgments
We thank Nuno Moreno for contributing to setting up the superresolution mi-
croscope, Cla´udia Campos and Vasco Barreto for help in cloning, and Cla´udia
Bispo for help in intracellular calcium assay. We thank the National Institutes
of Health AIDS Reagent Program for providing the HIV-1 NL4.3 and HIV-1
NL4.3 DNef constructs, the Ab against Gag, and the human IL-2.
Disclosures
The authors have no financial conflicts of interest.
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