<title>Introduction</title> <p>The HIV-1 accessory protein, Nef, affects the biology of the infected cell in several
ways to achieve conditions optimal for viral replication and spread. Nef alters the
intracellular trafficking of important immune molecules, such as class I and II
major histocompatibility complex proteins (MHC-I and MHC-II), CD4, CD28, and DC-SIGN
##REF##2014052##[1]##–##REF##11285224##[5]##. Nef-dependent reduction
of surface MHC-I protects HIV-infected primary T cells from recognition and killing
by HIV-specific cytotoxic T lymphocytes (CTLs) <italic>in vitro</italic>
##REF##9450757##[6]##.
Moreover, disruption of MHC-I expression by HIV-1 and SIV Nef provides a selective
advantage under immune pressure <italic>in vivo</italic>
##REF##10608759##[7]##–##REF##15542684##[10]##. CD4 downregulation by
Nef is also essential for efficient viral spread. The rapid removal of CD4 prevents
viral superinfection ##REF##8098729##[11]##, and enables optimal viral particle production by
eliminating detrimental CD4/HIV envelope interactions in the infected cell ##REF##10375528##[12]##,##REF##10375525##[13]##.</p> <p>Mutagenesis of protein-protein interaction domains has revealed that Nef uses
genetically separable mechanisms to affect MHC-I and CD4 transport. Specifically,
disruption of MHC-I surface expression requires an N-terminal α helix, a
polyproline repeat, and an acidic domain in Nef ##REF##9582271##[14]##,##REF##9971776##[15]##,
while CD4 downregulation requires an intact dileucine motif, two diacidic motifs,
and a hydrophobic pocket in Nef ##REF##9971776##[15]##–##REF##10199403##[18]##. Amino
acids necessary for the myristoylation ##UREF##1##[19]##,##REF##11578695##[20]## and oligomerization ##REF##10799608##[21]## of Nef are
required for the disruption of both MHC-I and CD4 surface expression.</p> <p>Nef has the capacity to affect MHC-I transport at multiple subcellular locations; Nef
blocks the export of newly-synthesized MHC-I from the secretory pathway and Nef
expression results in a small increase in the rate of MHC-I internalization ##REF##12584329##[22]##. To
accomplish this, Nef directly binds to the cytoplasmic tail of MHC-I early in the
secretory pathway ##REF##15653685##[23]##–##REF##15596859##[26]##. The Nef-MHC-I
complex then actively recruits the clathrin adaptor protein complex AP-1, which
targets MHC-I from the TGN to the endo-lysosomal network where it is ultimately
degraded ##REF##15569716##[25]##. Recruitment of AP-1 primarily requires a methionine
at position 20 in the N-terminal α helical domain of Nef and a tyrosine
residue in the cytoplasmic tail of MHC-I. Additionally, the acidic and polyproline
domains of Nef have recently been shown to stabilize this interaction ##REF##18073204##[27]##,##REF##18057255##[28]##. The normal function
of AP-1 is to target proteins into the endosomal pathway and then recycle them back
to the TGN. Thus, the AP-1 interaction with the Nef/MHC-I complex explains the
targeting of MHC-I containing vesicles to the endosomal pathway and to the TGN.
However, it does not explain accelerated degradation of MHC-I, hence other cellular
factors may be involved ##REF##15569716##[25]##.</p> <p>The mechanism of Nef-induced CD4 internalization and degradation has been derived, in
part, from correlating Nef function with the requirement for domains in the
C-terminal flexible loop region of Nef that bind to cellular factors. The Nef
dileucine motif (ExxxLL<sub>165</sub>) is needed for CD4 internalization and it
binds to adaptor protein complexes AP-1, AP-2, and AP-3 ##REF##9811606##[16]##, ##REF##9564030##[29]##–##REF##8916970##[36]##. In addition, a
diacidic motif, which is also required, enhances the interaction of Nef with AP-2
##REF##18032517##[37]##. There is separate evidence that this diacidic motif
may recruit the H subunit of the vacuolar ATPase (V1H) ##REF##9620685##[38]## to promote AP-2 recruitment
##REF##12032142##[39]##.
Because the normal role of AP-2 is to link cargo to clathrin and promote
internalization, it makes sense that this molecule would be necessary and indeed,
the involvement of AP-2 has now been confirmed using RNAi knockdown in a number of
cell systems ##REF##17267500##[40]##–##REF##16103193##[42]##.</p> <p>After CD4 is internalized, it is targeted to lysosomes for degradation. There is
evidence that this step requires β-COP ##REF##10199403##[18]##, a component of COP-1
coats implicated in endosomal trafficking as well as transport through the early
secretory pathway ##REF##9412469##[43]##–##REF##9382865##[45]##. Specifically, there are
defects in the Nef-dependent transport of CD4 into acidified vesicles at the
non-permissive temperature in cells harboring a temperature sensitive ε-COP
mutant ##REF##10199403##[18]##. Nef directly interacts with β-COP ##REF##7982906##[46]##, and
a second diacidic motif in the C-terminal loop domain of Nef has been demonstrated
to mediate this interaction ##REF##10199403##[18]##,##REF##15202998##[47]##, although, this result has not been reproducible
by another group ##REF##11264386##[48]##.</p> <p>To more clearly understand the mechanism of altered MHC-I and CD4 trafficking
observed in Nef-expressing cells, we directly compared these two processes in T
cells that expressed Nef. We confirmed that Nef primarily affected MHC-I and CD4 at
different subcellular locations and we demonstrated that the cytoplasmic tails of
the respective molecules dictated which pathway was utilized. Despite the
differences in initial trafficking, we found that HLA-A2 and CD4 co-localized in a
discrete subset of vesicular structures. Upon further inspection, we determined that
these structures also contained markers of late endosomes (Rab7) and to a lesser
extent, the lysosomal marker, LAMP-1. Electron microscopy (EM) revealed that CD4 and
HLA-A2 were found within MVBs of Nef-expressing T cells. HLA-A2 (but not CD4) was
also found in tubulovesicular structures adjacent to the Golgi. In Nef expressing
cells, reduction of β-COP expression reduced the targeting of HLA-A2 from
the TGN to LAMP-1<sup>+</sup> compartments and stabilized CD4 expression
within endosomal compartments. Finally, we identified two separate domains within
Nef that were necessary for these activities and for β-COP binding. These
data support a model in which both MHC-I and CD4 are ultimately targeted to the
lysosomes in Nef expressing cells by a final common pathway.</p>
<title>Materials and Methods</title> <title>Cell lines</title> <p>CEM T cells stably expressing HA-tagged HLA-A2 (CEM HA-HLA-A2) have already been
described ##REF##15569716##[25]##. Cell lines stably expressing YFP-tagged Rab7 or
HA-HLA-A2/CD4 were made by transducing cells with murine retroviral constructs
(MSCV YFP-Rab7 or MSCV HA-A2/CD4) as previously described ##REF##12584329##[22]##, followed by
culture in selective media.</p> <title>DNA constructs</title> <p>MSCV YFP-Rab7 was constructed by cloning a filled-in a <italic>Kpn</italic>
I-<italic>Xho</italic> I fragment from pEYFP-Rab7 ##REF##14718518##[66]## into MSCV puro ##REF##7584069##[67]##.
MSCV HA-A2/CD4 was constructed using PCR mutagenesis. The first round PCR
produced two products: the first utilized 5′ primer (primer 1)
<named-content content-type="gene">5′-CGGGATCCACCATGCGGGTCACGGCG-3′</named-content> and
3′ primer (primer 2) <named-content content-type="gene">5′-CTCTGCTTGGCGCCTTCGGTGCCACATCACAGCAGCGACCAC-3′</named-content>
with MSCV HA-HLA-A2 as the template ##REF##15569716##[25]##. The second utilized
5′ primer (primer 3) <named-content content-type="gene">5′-GTGGTCGCTGCTGTGATGTGGCACCGAAGGCGCCAAGCAGAG-3′</named-content>
and 3′ primer (primer 4) <named-content content-type="gene">5′-CCTCGAGTCAAATGGGGCTACATGTCTTCTGAAATCGGTGAGGGCACTGG-3′</named-content>
using CD4 as the template. The second round utilized primers 1 and 4 from the
previous PCR reactions plus 1 µl of each purified first round PCR
reactions as template. The resulting product was digested with BamHI and XhoI
and ligated into MSCV 2.2 ##REF##7584069##[67]## digested with BglII and XhoI.</p> <p>MSCV A2/Nef has been described ##REF##15596859##[26]##. MSCV HA-A2/CD4/Nef was constructed using a
PCR mutagenesis approach. The first round PCR produced two products: the first
utilized 5′ primer (primer 1) <named-content content-type="gene">5′-CGGGATCCACCATGCGGGTCACGGCG-3′</named-content> and
3′ primer (primer 2) <named-content content-type="gene">5′-CCACTTGCCACCCATACTAGTAATGGGGCTACATGT-3′</named-content>
with MSCV HA-A2/CD4 as the template. The second utilized 5′ primer
(primer 3) <named-content content-type="gene">5′-ACATGTAGCCCCATTACTATGATGGGTGGCAAGTGG-3′</named-content>
and 3′ primer (primer 4) <named-content content-type="gene">5′- GCGAATTCTCAGCAGTTCTTGAAGTACTC-3′</named-content>
with NL4-3 Nef open reading frame as template. The second round utilized primers
1 and 4 from the previous PCR reactions plus 1 µl of each purified
first round PCR reactions as template. The resulting product was digested with
BamHI and EcoRI and ligated into MSCV IRES GFP ##REF##10514006##[68]## digested with
BglII and EcoRI.</p> <p>Nef mutants were made by using the PCR mutagenesis approach described previously
(Wonderlich et al. 2008). The mutagenesis primers were as follows:
R<sub>17/19</sub>A <named-content content-type="gene">5′-TGGCCTACTGTAGCGGAAGCAATGAGACGAGCT-3′</named-content>
and EE<sub>154–155</sub>AA <named-content content-type="gene">5′-GTTGAGCCAGATAAGGTAGCAGCGGCCAATAAAGGAGAGA-3′</named-content>.
Each primer, plus its reverse complement were utilized together with additional
5′ and 3′ primers to generate the mutated product. Wild type
NL4-3 Nef [MSCV A2/Nef IRES GFP (Roeth et al 2005)] was used
as a template for the PCR reaction, except for the double mutant,
R<sub>17/19</sub>A/EE<sub>154–155</sub>AA, in which the MSCV
R<sub>17/19</sub>A Nef IRES GFP was used as the template. Each mutated PCR
product was digested and cloned into MSCV IRES GFP ##REF##10514006##[68]## as described
previously (Wonderlich et al. 2008).</p> <p>The FG12 shRNA lentiviral vectors were constructed as previously described ##REF##12518064##[50]##.
Briefly, complementary primers were annealed together and ligated into vector
pRNAi ##REF##15653686##[69]## digested with BglII and HindIII. The sequences of
the primers were as follows (the target sequence is underlined): shNC (an siRNA
directed at GFP, with several base changes ##REF##15569716##[25]##)- sense
<named-content content-type="gene">5′-GATCCCC<underline>GCTCACACTGAAGTTAATC</underline>TTCAAGAGAGATTAACTTCAGTGTGAGCTTTTTGGAAA-3′</named-content>,
antisense <named-content content-type="gene">5′-AGCTTTTCCAAAAAGCTCACACTGAAGTTAATCTCTCTTGAAGATTAACTTCAGTGTGAGCGGG-3′</named-content>,
shβ-COP- sense <named-content content-type="gene">5′-GATCCCC<underline>TGAGAAGGATGCAAGTTGC</underline>TTCAAGAGAGCAACTTGCATCCTTCTCATTTTTGGAAA-3′</named-content>,
antisense <named-content content-type="gene">5′-AGCTTTTCCAAAAATGAGAAGGATGCAAGTTGCTCTCTTGAAGCAACTTGCATCCTTCTCAGGG-3′</named-content>;
shμ1A- (a mixture of two lentiviruses was used) (1) sense <named-content content-type="gene">5′GATCCCC<underline>TGAGGTGTTCTTGGACGTC</underline>TTCAAGAGAGACGTCCAAGAACACCTCATTTTTGGAAA-3′</named-content>,
antisense <named-content content-type="gene">5′-AGCTTTTCCAAAAATGAGGTGTTCTTGGACGTCTCTCTTGAAGACGTCCAAGAACACCTCAGGG-3′</named-content>,
(2) sense <named-content content-type="gene">5′-
GATCCCC<underline>CGACAAGGTCCTCTTTGAC</underline>TTCAAGAGAGTCAAAGAGGACCTTGTCGTTTTTGGAAA-3′</named-content>,
and antisense <named-content content-type="gene">5′-
AGCTTTTCCAAAAACGACAAGGTCCTCTTTGACTCTCTTGAAGTCAAAGAGGACCTTGTCGGGG-3′</named-content>.
The pRNAi constructs were digested with XbaI and XhoI to remove the promoter and
shRNA sequence. The resulting fragment was ligated into FG12 ##REF##12518064##[50]##,
digested with XbaI and XhoI.</p> <title>Virus preparation and transductions</title> <p>Adenovirus was prepared by the University of Michigan Gene Vector Core facility.
Adenoviral and HIV (HXB-EP ##REF##9450757##[6]##) transductions of T cells ##REF##15569716##[25]## or
373 mg astrocytoma cells ##REF##11289809##[49]## have been described previously. Murine
retroviral vector (MSCV) expressing Nef was prepared as described previously
(Roeth et al. 2005), except that in some cases the retroviral vector
supernatants were concentrated by spinning at 14000 RPM for four hours at
4°C. The viral pellet was then resuspended in media to yield a
twenty-fold concentrated stock. Lentiviruses expressing shRNA were generated
using an approach similar to that already described ##REF##12518064##[50]##. Briefly, 293 cells
were transfected with the FG12 constructs described above plus pRRE ##REF##9765382##[70]##,
pRSV-Rev ##REF##9765382##[70]## and pHCMV-G ##REF##8415602##[71]## using
Lipofectamine 2000 (Invitrogen). Supernatants from the transfected cells were
collected and used to transduce CEM T cells using a spin-transduction
protocol.</p> <title>Flow cytometry and internalization assays</title> <p>Intact cells were stained for flow cytometry analysis as previously described
##REF##12414957##[24]##. Briefly, HLA-A2 was detected with BB7.2 ##REF##7035415##[72]##
that had been purified as previously described ##REF##12584329##[22]##. Endogenous CD4 was
detected using RPA-T4 from Serotec. The secondary antibody was goat
anti-mouse-phycoerythrin (BioSource, 1∶250). For experiments using the
GFP-expressing FG12 lentivirus for shRNA expression, the GFP-positive cells were
gated to identify the subset of transduced cells (generally
&gt;90% of cells). Endocytosis assays were performed as
previously described with minor modification ##REF##12584329##[22]##. Briefly, cells
were washed once with Endocytosis Buffer [D-PBS, 10 mM HEPES, 10
µg/ml BSA (NEB)], then stained with primary antibody
(described above) for 20 minutes on ice. After washing, the cells were
resuspended in RPMI supplemented with 10% fetal bovine serum, 10 mM
HEPES buffer, 2 mM L-glutamine, penicillin and streptomycin (R10) (pre-warmed to
37°C) and replicate aliquots were removed and placed on ice for each
time point. Cells were then washed and stained with goat
anti-mouse-phycoerythrin (BioSource, 1∶250) and the samples were
analyzed using a FACScan flow cytometer (Becton Dickinson). Flow cytometry data
was processed using FlowJo v4.4.3 software (Treestar Corp.). The mean
fluorescence at time zero was set to 100%, and this value was used to
calculate the relative surface staining at each subsequent time point.</p> <title>Cell surface transport assay</title> <p>CEM cells transduced with adenoviral vectors as previously described ##REF##12584329##[22]##
were first incubated in pre-label media [RPMI –Cys
–Met (Specialty Media, Inc.)+10% dialyzed FBS
(Invitrogen)] for 15 minutes at 37°C. Pulse labeling was
performed in pre-label media with 150–200 µCi/ml Pro-mix-L
[<sup>35</sup>S] (&gt;1000 Ci/mmol; Amersham
Pharmacia) for 30 minutes at 37°C. The cells were then chased in R10
media for 15 minutes at 37°C, followed by two washes with D-PBS. To
label the protein that reached the cell surface, the cells were resuspended in
D-PBS containing 0.5 mg/ml EZ-Link sulfo-NHS-LC-Biotin (Pierce), and incubated
at 37°C for 1 hour. Surface biotinylation was quenched by washing the
cells in D-PBS+25 mM Lysine (Fisher).</p> <p>For ##FIG##0##Figure 1D##,
immunoprecipitation of proteins from cell lysates was performed as previously
described ##REF##15569716##[25]##, except that one-third of the total lysate was
used for the HLA-A2 immunoprecipitation while two-thirds of the material was
used to recover CD4. For immunoprecipitations of <sup>35</sup>S labeled
proteins, 5 µg of BB7.2 and 2.5 µg RPTA4 (BD Pharmingen)
were used for HLA-A2 and CD4 respectively. In ##FIG##0##Figure 1E## and ##FIG##2##3D##, the total cell lysate was
immunoprecipitated with anti-HA ascites (HA.11, Covance).</p> <p>For ##FIG##0##Figures 1D, 1E## and ##FIG##2##3D##, recovered proteins were
released from the beads by boiling in 100 µl of 10% SDS.
One third was analyzed directly by SDS-PAGE (Total). The remaining two thirds
was brought to a total volume of 1 ml in RIPA Buffer ##REF##15569716##[25]##, and 40 µl
of avidin-agarose (Calbiochem) was added to recover biotinylated proteins. After
2 hours at 4°C, the beads were washed three times with 1 ml RIPA buffer
and proteins were separated by SDS-PAGE (Surface).</p> <title>Immunofluorescence microscopy</title> <p>Adeno-transduced CEM cells were adhered to glass slides, fixed, permeabilized,
and stained for indirect immunofluorescence as previously described ##REF##15569716##[25]##.
Bafilomycin treatment was performed as described previously ##REF##15569716##[25]##. The
following antibodies were utilized to localize proteins via microscopy: ##FIG##1##Figure 2##, and ##SUPPL##2##Figures S1##
and ##SUPPL##5##S4##;
anti-CD4 (S3.5, Caltag Laboratories) and anti-HLA-A2 (BB7.2); ##FIG##2##Figure 3##; anti-giantin
(Covance); ##FIG##4##Figure 5##;
anti-CD4 antibody (S3.5, Caltag Laboratories), anti-LAMP-1 (H4A3, BD Pharmingen)
and anti-HLA-A2 (BB7.2). Secondary antibodies were obtained from Molecular
Probes and were used at a dilution of 1∶250: Giantin, Alexa Fluor 546
goat anti-rabbit; CD4, Alexa Fluor 546 goat anti-mouse IgG2a; LAMP-1, Alexa
Fluor 546 goat anti-mouse IgG1; BB7.2 (##FIG##1##Figures 2##, ##FIG##4##5D## and ##SUPPL##5##S4##), Alexa
Fluor 647 goat anti-mouse IgG2b; BB7.2 (##SUPPL##2##Figure S1##), Alexa Fluor 488 goat anti-mouse
IgG2b. See ##SUPPL##1##Table
S2## for a summary of antibodies used to gather data for ##SUPPL##0##Table S1##.</p> <p>For the microscopy based internalization assay in ##FIG##4##Figure 5A##, CEM T cells were allowed to adhere
to glass slides, and placed on ice. The cells were washed once with wash buffer
(D-PBS, 10 µg/ml BSA (NEB) and 2% goat serum), incubated
with anti-CD4 antibody (S3.5, Caltag Laboratories, IF, 1∶25) for 20
minutes, washed once with wash buffer, incubated with Alexa fluor 546 goat
anti-mouse IgG2a (Molecular Probes, 1∶250) for 20 minutes and washed
once with wash buffer. The zero time point was fixed with 2%
paraformaldehyde, while the remaining time points incubated at 37°C for
the indicated time. The cells were then fixed with 2%
paraformaldehyde. Images were collected using a Zeiss LSM 510 confocal
microscope and processed using Adobe Photoshop software. Three-dimensional
projections of cells were generated from Z-stacks using Zeiss LSM Image Examiner
software. Otherwise, single Z sections through the center of the cell were
displayed.</p> <title>Electron microscopy</title> <p>Electron microscopy with CEM cells transduced with adenovirus was performed by
the Harvard Medical School (HMS) Electron Microscopy Facility. Frozen samples
were sectioned at −120°C, the sections were transferred to
formvar-carbon coated copper grids and floated on PBS until the immunogold
labeling was carried out. The gold labeling was carried out at room temperature
on a piece of parafilm. All antibodies and protein A gold were diluted in
1% BSA. The diluted antibody solution was centrifuged 1 minute at
14,000 rpm prior to labeling to avoid possible aggregates. Grids were floated on
drops of 1% BSA for 10 minutes to block for unspecific labeling,
transferred to 5 µl drops of primary antibody and incubated for 30
minutes. The grids were then washed in 4 µl drops of PBS for a total
of 15 minutes, transferred to 5 µl drops of Protein-A gold for 20
minutes, washed in 4 µl drops of PBS for 15 minutes and 6 µl
drops of double distilled water. Contrasting/embedding of the labeled grids was
carried out on ice in 0.3% uranyl acetete in 2% methyl
cellulose for 10 minutes. Grids were picked up with metal loops (diameter
slightly larger than the grid) and the excess liquid was removed by streaking on
a filter paper (Whatman #1), leaving a thin coat of methyl cellulose (bluish
interference color when dry). The grids were examined in a Tecnai G<sup>2</sup>
Spirit BioTWIN transmission electron microscope and images were recorded with an
AMT 2k CCD camera.</p> <title>Western blot analyses and immunoprecipitations</title> <p>For the western blot analysis in ##FIG##2##Figures 3A##, ##FIG##3##4C##,
##FIG##6##7D##, ##FIG##8##9A##, ##SUPPL##3##S2##, and ##SUPPL##4##S3##, cells
were lysed in PBS 0.3% CHAPS, 0.1% SDS pH 8, 1 mM PMSF,
normalized for total protein and separated by SDS-PAGE. Endo H (NEB) digestion
was performed according to the manufacturer's protocol. Staining of the
western blot was performed using anti-Nef (AG11, ##REF##9883905##[73]##) and
anti-β-COP (M3A5 ##REF##3536963##[74]##), which were purified as previously described
##REF##12584329##[22]##. Additional antibodies used were HA (Covance) and
μ1 (RY/1 ##REF##7876268##[75]##). The secondary antibody for anti-Nef,
β-COP, and HA was HRP-rat anti-mouse IgG<sub>1</sub> (Zymed) and for
anti-μ1 was HRP-goat anti-rabbit (Zymed).</p> <p>For ##FIG##5##Figure 6B##, the IP-western
experiment was performed as previously published ##REF##15596859##[26]##. Briefly,
parental CEM T cells were spin-transduced with murine retroviral supernatant
expressing either empty vector, A2/Nef or A2/CD4/Nef. At 72 hours post
transduction, the cells were incubated in 20 mM NH<sub>4</sub>Cl for 4 hours.
The cells were then treated with DTBP (Pierce) for 40 minutes, quenched per the
manufacturer's protocol, and lysed in PBS with 0.3% Chaps
and 0.1% SDS. The lysate was pre-cleared and immunoprecipitated with
HLA-A2 with BB7.2 chemically crosslinked protein A/G beads (Calbiochem) ##REF##15569716##[25]##. The
immunoprecipitates were washed in TBS with 0.3% CHAPS and
0.1% SDS. A more stringent IP protocol was used in ##FIG##5##Figures 6A##, ##FIG##6##7C##, ##FIG##7##8E##, and ##SUPPL##6##S5##. For
these experiments, CEM cells were transduced with control, wild type Nef, or
mutant Nef expressing adenovirus (##FIG##5##Figure 6A## and ##FIG##6##7C##) or concentrated MSCV (##FIG##7##Figures 8E## and ##SUPPL##6##S5##). At 48 hours post-transduction, the cells
were incubated in 20 mM NH<sub>4</sub>Cl for 16 hours. The cells were not
crosslinked and were lysed in digitonin lysis buffer (1% digitonin
(Wako), 100 mM NaCl, 50 mM Tris pH 7.0, 1 mM CaCl<sub>2</sub>, and 1 mM
MgCl<sub>2</sub>). After pre-clear, the lysates were immunoprecipitated with
either BB7.2 (##FIG##5##Figures 6A## and
##SUPPL##6##S5##)
or M3A5 (##FIG##6##Figures 7C## and
##FIG##7##8E##) crosslinked to
beads. The immunoprecipitates were eluted and analyzed by western blot as
described previously ##REF##15596859##[26]##.</p> <title>Pulse-chase analysis of protein degradation</title> <p>A total of 30 million CEM T cells transduced with wild type or mutant Nef using
concentrated MSCV as described above were pulse labeled for 30 minutes with
[<sup>35</sup>S]-methionine and cysteine. Half of the
cells were collected as the zero time point and stored at −20 degrees.
The remaining cells were then chased for 12 hours in RPMI, collected and stored
at −20 degrees. Lysates were generated in lysis buffer (PBS
0.3% CHAPS, 0.1% SDS pH 8, 1 mM PMSF) and precleared over
night. They were immunoprecipitated for two hours with an anti-HLA-A2 antibody
(BB7.2) and washed once in radioimmunoprecipitation assay (RIPA) buffer (50 mM
Tris pH 8, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate,
0.1% SDS). The immunoprecipitates were then eluted by boiling in
10% SDS, reprecipitated with an antibody against HA (HA.11, Covance),
and washed two times in RIPA buffer. The final immunoprecipitates were then
separated by SDS-PAGE, the gel was dried down and analyzed using a
phosphorimager.</p>
<title>Results</title> <title>The cytoplasmic tail dictates the pathway utilized by Nef to eliminate MHC-I
and CD4 surface expression</title> <p>It is known that Nef binds to the cytoplasmic tails of both CD4 and
MHC–I, but that it affects them differently. To better understand the
similarities and differences governing these two pathways, we examined the
trafficking of CD4, HLA-A2 and a chimeric molecule in which the wild type HLA-A2
cytoplasmic tail was substituted with the CD4 cytoplasmic tail (HA-A2/CD4). A
flow cytometric analysis of steady state surface expression revealed that Nef
dramatically reduced steady state surface expression of all three molecules
(##FIG##0##Figure 1A##). Consistent
with prior studies, we found that CD4 was rapidly internalized from the cell
surface in Nef expressing T cells, whereas wild type HLA-A2 was not (##FIG##0##Figure 1B##). Substitution of
the CD4 tail for the HLA-A2 cytoplasmic tail was sufficient to confer this
phenotype (##FIG##0##Figure 1C##).
Conversely, prior studies have shown that Nef disrupts cell surface expression
of MHC-I by blocking the transport of newly synthesized MHC-I from the TGN to
the cell surface ##REF##12584329##[22]##,##REF##15653685##[23]##. As shown in ##FIG##0##Figure 1D##, Nef inhibited
HLA-A2 forward transport by approximately 75%, whereas CD4 was
unaffected at Nef levels that had a clear effect on HLA-A2 transport. Slight
effects on CD4 could be observed at higher Nef levels (##FIG##0##Figure 1D, lane 8##). The substitution of the
HLA-A2 cytoplasmic tail with the CD4 tail reduced the ability of Nef to disrupt
forward trafficking (##FIG##0##Figure
1E##). Thus, sequences in the cytoplasmic tails of CD4 and HLA-A2 determine
how Nef disrupts their trafficking.</p> <title>CD4 and a subset of HLA-A2 proteins are found in late endosomes and lysosomes
of Nef-expressing T cells</title> <p>To better understand the similarities and differences between MHC-I and CD4
trafficking in Nef-expressing cells, we compared the steady-state distribution
of these molecules in T cells using confocal microscopy (##FIG##1##Figure 2A##). We found that Nef expression
caused the bulk of MHC-I to cluster in the perinuclear region where, in
agreement with many other studies ##REF##9582271##[14]##,##REF##9586638##[30]##,##REF##11289809##[49]##, it co-localized
with markers of the TGN (data not shown). Interestingly, we also identified a
subset of HLA-A2 that co-localized with CD4 in vesicular structures (##FIG##1##Figure 2A##; arrows show example
vesicles). To further identify these structures, we simultaneously stained for
HLA-A2, CD4, and organelle markers using 3-color confocal microscopy (summarized
in ##SUPPL##0##Table
S1##). Our results indicated that CD4 was mainly found in discrete
vesicular structures, which also contained HLA-A2 (91.9% of the
CD4<sup>+</sup> vesicles co-localized with HLA-A2, ##SUPPL##0##Table S1##)
and markers of late endosomes and lysosomes. Overall, the best marker for
structures containing both HLA-A2 and CD4 was Rab7 (94%, of
CD4+ vesicles co-localized with Rab 7, ##SUPPL##0##Table S1##
and ##FIG##1##Figure 2A##, arrowheads
mark example vesicles). CD4 and HLA-A2 were also found to co-localize with
markers of lysosomes, such as LAMP-1. However, the vesicles with the most
intense LAMP-1 staining did not contain either HLA-A2 or CD4, possibly because
of degradation. Consistent with this, the co-localization of HLA-A2 and CD4 was
dramatically increased when the cells were treated with bafilomycin, which
inhibits degradation in acidic compartments (##SUPPL##2##Figure S1##).
Thus, the normal steady-state co-localization of HLA-A2 and CD4 in Nef
expressing cells was limited because degradation prevented accumulation in this
compartment.</p> <title>Colocalization of HLA-A2 and CD4 in MVBs</title> <p>To further discern these structures, we also examined them using electron
microscopy (EM). In agreement with the confocal data, our EM analysis revealed
that compared with control cells in which both HLA-A2 and CD4 were found on the
cell surface (##FIG##1##Figure 2B, panel
1##), in Nef-expressing T cells, the majority of CD4 was found in MVBs,
co-localizing with HLA-A2 (##FIG##1##Figure
2B, panel 2##). In addition, we also noted substantial HLA-A2, but not
CD4, accumulating in tubulovesicular structures adjacent to Golgi stacks (##FIG##1##Figure 2B, panel 3##). In
separate experiments these structures were also found to contain AP-1 (##FIG##1##Figure 2C##). Based on these
studies, it appears that the majority of HLA-A2 resides in tubulovesicular
structures in the region of the TGN with AP-1, whereas at any given time, a
small subset can be found in the endosomal compartment with CD4.</p> <title>Required cellular co-factors</title> <p>To further elucidate the similarities and differences between these pathways, we
examined the role of known Nef-interacting proteins implicated in intracellular
trafficking. AP-1 is a heterotetrameric adaptor protein involved in protein
sorting from the TGN and it has been previously demonstrated to interact with
MHC-I molecules in Nef expressing HIV-infected primary T cells and to direct
MHC-I into the endolysosomal pathway ##REF##15569716##[25]##. Nef is also known
to interact with β-COP ##REF##7982906##[46]##, a component of
COP-1 vesicles also involved in endosomal trafficking ##REF##9412469##[43]##–##REF##9382865##[45]##.
Indeed, expression of wild type COP 1 components is needed for targeting CD4
into acidic vesicles in Nef-expressing cells ##REF##10199403##[18]##.</p> <p>To compare and contrast the requirement for these factors in Nef-dependent CD4
and HLA-A2 trafficking, we knocked down their expression using lentiviral
vectors expressing short hairpin RNAs (shRNAs) ##REF##12518064##[50]##. All of these studies
were performed in T cells and new cell lines were generated for each experiment
to eliminate the possibility that long term growth in culture would select for
cells that had compensated for the defect. Using this system, we obtained good
knock down of the μ1 subunit of AP-1 and β-COP (##FIG##2##Figure 3A–C##). (A
small apparent effect of shβ-COP on μ1 levels observable in
##FIG##2##Figure 3A## was not
significant when adjusted for protein loading in the experiment shown here or in
replicate experiments [##FIG##2##Figure 3B##]. We also did not observe any effect of another
siRNA directed against a different target site in β-COP on μ1
expression [##SUPPL##3##Figure S2##].)</p> <title>The effect of knocking down β-COP expression on the structural
integrity of the Golgi</title> <p>Because β-COP is known to be important for intra-Golgi and ER-to-Golgi
trafficking, we asked whether the Golgi structure or MHC-I trafficking were
drastically affected by reduced β-COP expression. We found that there
was only a small reduction in the normal transport of MHC-I to the cell surface
(35% reduction, ##FIG##2##Figure
3D##). In addition, cells lacking β–COP generally
maintained overall Golgi structure as assessed by the intracellular localization
of giantin, a transmembrane protein normally residing in the
<italic>cis</italic> and <italic>medial</italic> Golgi ##REF##7691276##[51]## (##FIG##2##Figure 3E##). In contrast,
brefeldin A, an inhibitor of an ARF1 GEF necessary for β-COP activity
obliterated the normal Golgi staining (##FIG##2##Figure 3E, panel 9##). The relatively mild
phenotype of this knock-down compared to the drastic effects of brefeldin A,
suggests that brefeldin A has effects other than just disrupting COP 1 coats by
blocking ARF1 activity.</p> <p>Having established that knocking down β-COP allowed relatively normal
forward trafficking of HLA-A2, we proceeded to assess the effect of knocking
down β-COP or AP-1 in Nef-expressing cells. Consistent with previous
publications ##REF##15569716##[25]##, we found that knocking down the ubiquitously
expressed form of AP-1 (AP-1A ##REF##11157985##[52]##) largely reversed the effect of Nef on HLA-A2
(p&lt;10<sup>−4</sup>), but had a smaller and less significant
effect (<italic>p</italic>&lt;0.02) on CD4 surface expression (##FIG##3##Figure 4A and 4B##).
Surprisingly, we also observed that knocking down β-COP expression
inhibited MHC-I downmodulation by Nef and had a small but statistically
significant effect on CD4 downmodulation (p&lt;10<sup>−3</sup>;
##FIG##3##Figure 4A and 4B##). The
small effect of β-COP on CD4 surface expression indicated that
β-COP was not necessary for CD4 internalization and downmodulation from
the cell surface. However, further studies were needed to determine whether
β-COP was required to degrade the CD4 after it was internalized.</p> <title>A role for β-COP in promoting degradation of Nef cellular targets</title> <p>Prior studies had determined that expression of β-COP was necessary for
acidification of CD4-containing vesicles and thus it was hypothesized that
β-COP was needed to target vesicles containing internalized CD4 for
lysosomal degradation. Therefore, we asked whether the role of β-COP in
MHC-I trafficking was also to promote MHC-I degradation. To examine this, we
utilized an assay we had developed, which measures the loss of mature, endo
H–resistant HA-tagged HLA-A2 in Nef expressing cells by western blot
analysis. This assay system is based on previous data demonstrating
Nef-dependent degradation of the mature form of MHC-I in a manner that is
reversible by inhibitors of lysosomal degradation ##REF##15569716##[25]##. As shown in ##FIG##3##Figure 4C, under normal##,
steady state conditions, most of the HLA-A2 is resistant to endo H digestion,
indicating that it has matured through the Golgi apparatus (##FIG##3##Figure 4C, lane 2##). However, when Nef was
expressed, we observed a dramatic reduction in total MHC-I and a decrease in the
ratio of endo H resistant to sensitive protein (##FIG##3##Figure 4C compare lanes 2 and 18##, see also
##SUPPL##4##Figure
S3##). Consistent with a role for AP-1, we observed that AP-1A shRNA
largely reversed this effect of Nef (##FIG##3##Figure 4C, compare lanes 18 and 20##. See also
##FIG##3##Figure 4D## for
quantification). To detect degradation of molecules containing a CD4 tail, we
used HA-A2/CD4 (##FIG##0##Figure 1##)
and found that Nef expression accelerated the degradation of endo H resistant
forms of this molecule (##FIG##3##Figure 4C,
compare lanes 6 and 22##). However, we found that there was no effect
of reduced AP-1A expression on Nef-dependent degradation of molecules containing
the CD4 tail (##FIG##3##Figure 4C, compare
lanes 22 and 24##. See also ##FIG##3##Figure 4D## for quantification).</p> <p>When β-COP expression was reduced, we observed a small increase in the
amount of immature, endo H–sensitive protein (##FIG##3##Figure 4C, compare lanes 10 and 12##),
consistent with the 35% reduction in export of MHC-I to the cell
surface shown in ##FIG##2##Figure 3D##.
However, we also noted that reduction in β-COP expression reduced the
Nef-dependent degradation of the mature, endo H resistant form of these
molecules (##FIG##3##Figure 4C, compare lanes
26 and 28##. See also ##FIG##3##Figure 4D## for quantification) implicating β-COP in this
pathway. We were also able to confirm the model that β-COP is involved
in Nef-dependent CD4 degradation as treating cells with β-COP shRNA
reduced the degradation of the A2/CD4 chimeric molecule (##FIG##3##Figure 4C, compare lanes 30 and 32##. See also
##FIG##3##Figure 4D## for
quantification).</p> <title>β-COP is required for targeting internalized CD4 for degradation in
Nef-expressing T cells</title> <p>We next directly examined the effect of reducing β-COP expression on
Nef-dependent trafficking by confocal microscopy. For these experiments, cells
were infected with HIV or were transduced with Nef-expressing adenoviral vectors
and then the fate of internalized CD4 was assessed by confocal microscopy. Using
this assay system, we observed fairly rapid internalization of CD4 in
Nef-expressing cells, followed by loss of CD4 staining by 30 minutes (##FIG##4##Figure 5A##, compare control
cells in row 1 to Nef-expressing cells in row 3). However, in T cells expressing
β-COP shRNA, there was a three-to-four fold increase in the number of
CD4-containing vesicles, consistent with a role for β-COP in promoting
maturation of these vesicles into degradative compartments (##FIG##4##Figure 5A##, compare control treated
Nef-expressing cells in row 3 to shβ-COP–expressing cells in
row 4). Reduction of β-COP expression yielded similar results whether
Nef was introduced using HIV infection or via adenoviral vectors (##FIG##4##Figure 5B and 5C##).</p> <title>β-COP is required for targeting MHC-I to LAMP-1<sup>+</sup>
compartments in Nef-expressing T cells</title> <p>Confocal analysis of MHC-I intracellular localization revealed that expression of
β-COP shRNA in control cells increased the intracellular accumulation of
MHC-I, consistent with the slowing of export we observed in cells deficient in
β-COP (##FIG##4##Figure 5D, compare
rows 1 and 2##). Infection with Nef-expressing HIV resulted in the loss
of cell surface MHC-I and an increase in intracellular MHC-I, some of which
co-localized with LAMP-1 (##FIG##4##Figure
5D, compare rows 1 and 3##). Under these conditions, reduction of
β-COP expression reduced the degree of colocalization with LAMP-1 (##FIG##4##Figure 5D, compare rows 3 and
4##).</p> <p>To enhance our ability to observe trafficking of MHC-I into
LAMP-1<sup>+</sup> compartments, we treated the cells with bafilomycin,
which inhibits the vacuolar ATPase and thus acidification and degradation within
lysosomal compartments. As previously reported ##REF##15569716##[25]##, bafilomycin
treatment enhanced our ability to detect MHC-I in LAMP-1<sup>+</sup>
compartments in Nef-expressing T cells (##FIG##4##Figure 5D, compare rows 3 and 7##). The
expression of β-COP shRNA decreased LAMP-1 colocalization with MHC-I,
consistent with a role for β-COP in targeting MHC-I for degradation in
lysosomal compartments in Nef expressing T cells (##FIG##4##Figure 5D, compare rows 7 and 8##). Similar
results were observed whether Nef was introduced using HIV or adenoviral vectors
(##FIG##4##Figure 5E and 5F##).</p> <p>We also examined co-localization of HLA-A2 and CD4 in cells that expressed
β-COP shRNA. We observed that reduction of β-COP expression
resulted in increased staining of both proteins, and did not disrupt their
co-localization (##SUPPL##5##Figure S4##). Thus, β-COP was not necessary for targeting
these proteins into a common endosomal pathway, but rather was needed for their
subsequent targeting into a degradative pathway.</p> <title>The cytoplasmic tail of MHC-I is necessary for AP-1 binding in Nef-expressing
T cells</title> <p>To further explore the molecular mechanism for the similarities and differences
in MHC-I and CD4 trafficking in Nef-expressing T cells, we asked whether these
molecules differed as to how well they bound Nef or cellular factors. As
expected, we found that HIV Nef bound to both the HLA-A2 and the CD4 tail (##FIG##5##Figure 6A, right panel##).
However, AP-1 only co-precipitated with molecules containing the HLA-A2
cytoplasmic tail (##FIG##5##Figure 6A, right
panel##). The chimeric molecule with the CD4 cytoplasmic tail did not
bind AP-1 in Nef-expressing T cells (##FIG##5##Figure 6A, right panel##). In these
experiments, we noted that the expression level of A2/CD4 was lower than for
wild type HLA-A2, which could explain this difference. Therefore, we confirmed
these data using a fusion protein containing either HLA-A2 or A2/CD4 directly
fused to full length HIV-Nef protein. In previously published experiments it was
shown that the HLA-A2/Nef fusion protein co-precipitated AP-1 in a manner that
depended on sequences both in Nef and in the HLA-A2 cytoplasmic tail ##REF##15569716##[25]##.
Here we show again that the HLA-A2 cytoplasmic tail was necessary for this
interaction and, moreover, that the CD4 tail could not substitute for it (##FIG##5##Figure 6B, right panel##).</p> <title>Evidence that formation of a Nef–β-COP complex is an
essential step necessary for MHC-I degradation</title> <p>The Nef-β-COP interaction is well-described in the literature ##REF##7982906##[46]##
and there is evidence that β-COP interacts with a diacidic motif
(E<sub>154/155</sub>) within the Nef C-terminal loop ##REF##10199403##[18]##. However, this
region of Nef has never been implicated in MHC-I trafficking. To provide further
evidence that β-COP is needed to promote MHC-I degradation, we sought to
identify a region of Nef that is needed both for MHC-I degradation as well as
β-COP binding. We therefore examined a panel of mutations
(M<sub>20</sub>A, V<sub>10</sub>EΔ17–26 and
E<sub>62–65</sub>Q) that are specifically defective at disrupting
MHC-I trafficking ##REF##9582271##[14]##,##REF##9971776##[15]##,##REF##15596859##[26]##,##REF##12097566##[53]##. We also examined
a Nef mutant, D<sub>123</sub>G, that is defective at both CD4 and MHC-I
downmodulation ##REF##10799608##[21]##. The relative activity of these Nef mutants
in MHC-I and CD4 downmodulation is shown in ##FIG##6##Figure 7A## and quantified in ##FIG##6##Figure 7B##.</p> <p>We then examined the relative ability of each of these mutant molecules to
co-precipitate with β–COP. As shown in ##FIG##6##Figure 7C##, we found that the V<sub>10</sub>E
Δ17–26-Nef, which is defective at MHC-I downmodulation, was
also defective at binding to β-COP (compare lanes 3 and 5).
Interestingly, this deletion mutant is also defective at interacting with AP-1
##REF##15569716##[25]##. However, the β-COP binding site was
separable from the AP-1 interaction site because M<sub>20</sub>, which is
located within the deleted region, is needed for AP-1 interaction ##REF##15569716##[25]##,##REF##18073204##[27]##), but was not
necessary for β-COP binding to Nef (##FIG##6##Figure 7C, compare lanes 3 and 4##). Mutation
of the Nef dimerization motif [D<sub>123</sub>G, ##REF##10799608##[21]##], which
disrupts a number of Nef functions, including MHC-I and CD4 downmodulation, also
reduced binding to β-COP (##FIG##6##Figure 7C, compare lanes 3 and 7##). Finally, mutation of the Nef
acidic domain (E<sub>62–65</sub>Q), which disrupts binding to MHC-I
##REF##15596859##[26]##, AP-1 ##REF##18073204##[27]##,##REF##18057255##[28]##
and PACS-1 ##REF##10707087##[54]##, did not affect binding to β-COP
(##FIG##6##Figure 7, compare lanes 3 and
6##).</p> <p>As expected, we found that V<sub>10</sub>EΔ<sub>17–26</sub>
Nef, which was defective at β-COP binding, was also defective at
inducing the degradation of the endo H resistant form of HLA-A2 (##FIG##6##Figure 7D, upper panel, compare lanes
3 and 4 with lanes 5 and 6##). In contrast,
V<sub>10</sub>EΔ17–26 Nef was not defective at A2/CD4
degradation based on western blot analysis (##FIG##6##Figure 7D, lower panel, compare lanes 3 and 4 with
lanes 5 and 6##). These data suggested that there may be another
interaction domain that recruits β-COP to the Nef-CD4 complex to promote
CD4 degradation. This would be consistent with the faint band observable in the
V<sub>10</sub>EΔ<sub>17–26</sub>-Nef mutant
immunoprecipitation (##FIG##6##Figure 7C,
lane 5, longer exposure##) and prior publications demonstrating that
mutation of E<sub>154/155</sub> also affected β-COP binding ##REF##15202998##[47]##.
Thus, there may be two independent binding sites for β-COP within Nef,
each of which governs the degradation of a different cellular factor.</p> <p>To further define the β-COP binding site, and to determine whether there
were indeed two β-COP binding sites, we constructed additional Nef
mutants. We focused on the arginine residues
(R<sub>17</sub>ER<sub>19</sub>MR<sub>21</sub>R<sub>22</sub>) within the Nef
deletion Δ17–26) because previous studies had indicated that
arginine rich regions could form β-COP-binding sites ##REF##16595684##[55]##. Flow cytometric analysis of MHC-I levels on
cells expressing these mutants revealed that the R<sub>17/19</sub> pair was
necessary for maximal MHC-I downmodulation (##FIG##7##Figure 8A and 8B##). In contrast, mutation of
R<sub>21/22</sub> did not significantly affect MHC-I downmodulation
(unpublished data). An assessment of Nef-induced degradation by pulse chase
analysis of HA-HLA-A2, revealed that mutating this motif also inhibited
Nef-dependent degradation (##FIG##7##Figure
8C, compare lanes 5 and 7##, quantified in ##FIG##7##Figure 8D##). Additionally, mutation of
R<sub>17/19</sub> reduced, but did not eliminate binding of β-COP to
Nef in a manner similar to the effect of the Δ17–26 Nef
mutation (##FIG##7##Figure 8E, compare lanes
3 and 4##).</p> <p>We next examined the diacidic motif (E<sub>154/155</sub>) previously implicated
in β-COP binding. As shown in ##FIG##7##Figure 8A and 8B##, mutation of this motif did
not disrupt MHC-I downmodulation, in fact downmodulation was somewhat enhanced.
Additionally, we found that mutation of this motif did not reduce MHC-I
degradation (##FIG##7##Figure 8C, compare
lanes 5 and 11##, see also quantification in 8D). However, in agreement
with prior results, we observed a partial defect in β-COP binding with
this mutant (##FIG##7##Figure 8E, compare
lanes 3 and 6##, ##REF##10199403##[18]##,##REF##15202998##[47]##. However, this
defect was less reproducible (observed in two out of four experiments) than that
observed with disruption of R<sub>17/19</sub> (consistently observed in five out
of five experiments), suggesting that binding to R<sub>17/19</sub> can mask the
defect observed with mutation of E<sub>154/155</sub> under certain conditions.
To provide additional data supporting the possibility that both sites
contributed to β-COP binding, we constructed a double mutant,
R<sub>17/19</sub> A and E<sub>154/155</sub>A (R/E). As shown in ##FIG##7##Figure 8E, lane 5##, binding of
R/E to β-COP was further reduced relative to binding of Nef proteins
containing single mutations in each motif, strongly implicating both motifs in
β-COP binding. The phenotype of the double mutant was highly
reproducible in 5 out of 5 experiments.</p> <p>Interestingly, the R/E double mutant was not more defective than
R<sub>17/19</sub>A at downmodulating MHC-I (##FIG##7##Figures 8A and 8B##) or at promoting MHC-I
degradation (##FIG##7##Figure 8C, compare
lanes 7 and 9##, quantified in 8D), indicating that Nef did not utilize
the E<sub>154/155</sub> binding site to recruit β-COP for MHC-I
degradation. Conversely, we confirmed prior reports that the
E<sub>154/155</sub>A mutant was defective at CD4 degradation (##FIG##8##Figure 9A, compare lanes 3 and
6##) and determined moreover that there was no significant effect of
mutating R<sub>17/19</sub> on CD4 degradation, either alone or in combination
with E<sub>154/155</sub>A (##FIG##8##Figure
9A, compare lanes 3 and 4##). It is also worth noting that, in contrast
to what was observed with HLA-A2, we did not observe a clear correlation between
the relative CD4 surface expression and the relative level of total cellular CD4
(compare ##FIG##7##Figure 8B## and ##FIG##8##9B##), indicating that there was
a complex relationship between total cellular CD4 and the fraction expressed on
the cell surface.</p> <p>Because the R<sub>17/19</sub> motif is directly adjacent to M<sub>20</sub>, which
is necessary for AP-1 recruitment ##REF##15569716##[25]##,##REF##18073204##[27]##, we also examined whether these mutations, which
affect β-COP binding, also disrupted AP-1 co-precipitation. To
accomplish this, we used our standard AP-1 recruitment assay in which proteins
co-precipitating with MHC-I HLA-A2 were detected by western blot analysis. As
shown in ##SUPPL##6##Figure
S5##, mutation of R<sub>17,19</sub> (and E<sub>154/155</sub>) decreased
AP-1 binding only slightly. Thus, the defects in MHC-I downmodulation and
degradation noted with mutation of R<sub>17,19</sub> resulted primarily from
defects in β-COP binding.</p>
<title>Discussion</title> <p>Expression of HIV Nef in infected cells protects them from lysis by CTLs and this
activity of Nef is due to downmodulation of MHC-I surface expression. The Nef
protein also prevents superinfection and promotes viral spread by removing the viral
receptor, CD4 from the cell surface (for review see ##REF##16760313##[56]##). We provide evidence
that sequences in the cytoplasmic tail of these molecules are important for
determining whether Nef disrupts their trafficking from the cell surface or at the
TGN. These data, that swapping cytoplasmic domains switches the initial pathways
taken by HLA-A2 and CD4 in the presence of Nef, may seem somewhat obvious. Nef is
always the same and thus one might conclude that this information has to be
contained in the modulated protein. However, it was also possible that the
ectodomain affected Nef responsiveness by binding to other transmembrane proteins or
by altering intracellular trafficking. This was certainly a possibility for MHC-I
for which it is clear that the efficiency of peptide loading can affect trafficking
and we have found that trafficking rates affect responsiveness to Nef and AP-1
binding ##REF##15653685##[23]##.</p> <p>Prior studies have demonstrated that Nef initially binds to hypo-phosphorylated forms
of the MHC-I cytoplasmic tail early in the secretory compartment ##REF##15653685##[23]##, but
binding does not affect normal transit through the Golgi apparatus and into the TGN
##REF##15569716##[25]##.
The Nef-MHC-I complex then recruits the AP-1 heterotetrameric clathrin adaptor
protein using a binding site that is created when Nef binds the MHC-I cytoplasmic
tail. This binding site requires a methionine from the N-terminal α helix of
Nef and a tyrosine residue in the MHC-I cytoplasmic tail ##REF##15569716##[25]##. Additionally, there is
evidence that this complex is stabilized by the acidic and polyproline domains of
Nef ##REF##18073204##[27]##,##REF##18057255##[28]##. Formation of this
complex results in the re-direction of MHC-I trafficking in such a way that it is
targeted to lysosomes for degradation ##REF##15569716##[25]##. However, cellular
proteins that normally bind AP-1 are not degraded, but rather recycled to the TGN
(##FIG##8##Figure 9C##). Here we present
new evidence that Nef utilizes β-COP to promote trafficking to degradative
compartments (##FIG##8##Figure 9C##).
Knocking down expression of β-COP inhibited the degradation of MHC-I and it
did so by blocking the transport of MHC-I from intracellular vesicles to
LAMP-1<sup>+</sup> compartments. We also provide results here that
confirm β-COP is necessary for degradation of CD4 in lysosomal compartments.
Thus, we propose that AP-1 and AP-2 deliver MHC-I and CD4 respectively to endosomal
compartments where β-COP displaces AP-1 and AP-2 to target MHC-I and CD4 for
lysosomal degradation (##FIG##8##Figure
9C##).</p> <p>As described above, we found that knocking down β-COP with shRNA resulted in
stabilization of internalized CD4, however the effect on CD4 surface expression was
small, but still significant. In contrast, there was a greater effect of
β-COP knockdown on HLA-A2 surface expression. This might suggest that the
role of β-COP in the modulation of these targets was different, rather than
the same. However, this apparent paradox can be explained by our model shown in
##FIG##8##Figure 9C##. As indicated,
differences in response to β-COP knockdown can be explained by differences
in the intracellular pathways of these proteins before they interact with
β-COP. MHC-I is engaged in an AP-1-dependent endosome-to-TGN loop, and MHC-I
could “leak” out to the cell surface from the TGN in the absence
of β-COP, whereas CD4 may be unable to return to the cell surface from its
endosomal compartment. Consistent with this, we also noted a lack of correlation
between degradation and surface expression of CD4 (but not MHC-I) when Nef mutants
that were defective in β-COP binding were examined. These data indicate that
there is a complex relationship between total cellular CD4 and the fraction that is
present on the cell surface and thus intracellular pools need to be directly
examined to assess degradation rather than relying on surface expression as an
indicator of the efficiency of this process.</p> <p>It is also noteworthy that shRNA knockdown of β-COP did not fully reverse
Nef-dependent MHC-I and CD4 degradation. This may have resulted from incomplete
knockdown of β-COP. However, we also observed a similar phenotype with Nef
mutants defective at β-COP binding. Failure to fully reverse degradation may
be secondary to a default degradative pathway that exists for all proteins delivered
to endosomal pathways. Alternatively, there may be other ways Nef targets these
proteins to lysosomes, which have yet to be identified.</p> <p>Our studies indicate that there are at least three domains needed for Nef to interact
efficiently with β-COP. One of these domains (D<sub>123</sub>), is required
for dimerization of Nef and is needed to affect a variety of Nef functions ##REF##10799608##[21]##. Another
region lies within the N-terminal α helical domain of Nef that is
specifically required for disruption of MHC-I trafficking and for interactions with
AP- 1 ##REF##15569716##[25]##.
This binding site for β-COP is distinct from that used by AP-1, because
recruitment of β-COP does not require Nef's acidic domain or Nef
M<sub>20</sub>, whereas AP-1 does ##REF##15569716##[25]##,##REF##18073204##[27]##. The fact that
these Nef mutants bind β-COP, but are still defective at MHC-downmodulation
##REF##12097566##[53]##
makes sense, because these mutants are also unable to bind the MHC-I cytoplasmic
tail ##REF##15596859##[26]##.</p> <p>Additional mutants, which focused on the highly conserved stretch of arginines in the
N-terminal alpha helical domain of Nef (R<sub>17</sub>XRMRR<sub>22</sub>), revealed
that the regions involved in AP-1 and β-COP binding were very closely
apposed. However, we determined that mutation of R<sub>17/19</sub> affected
primarily β-COP binding, with only a minimal effect on AP-1 interaction.
Thus, these two Nef-interacting proteins have distinct and separable amino acid
requirements for binding.</p> <p>The identification of a β-COP binding domain within a region of Nef that is
also required for Nef to accelerate MHC-I degradation confirms the requirement for
β-COP in this pathway. In addition, the residual binding of β-COP to
these Nef mutants provided suggestive data that another binding site for
β-COP existed. Indeed, we were able to confirm prior evidence that a
diacidic motif within the C-terminal loop of Nef also promoted an interaction with
β-COP and that mutation of this motif reduced CD4 degradation ##REF##15202998##[47]##.
Finally, we demonstrated that mutation of both the RXR and the diacidic motifs
resulted in the greatest defect in β-COP binding. The double mutant did not
however result in a greater defect in either MHC-I or CD4 degradation, indicating
the role of each motif is distinct and not additive. The discovery of two distinct
β-COP binding motifs helps explain why some groups could not confirm the
role of the diacidic motif in β-COP binding ##REF##11264386##[48]## as both motifs need to
be mutated to reliably eliminate an interaction between β-COP and Nef.</p> <p>There is precedent for such redundancy. For example, there are two AP-1 binding sites
within Nef; a dileucine motif within the C-terminal flexible loop ##REF##9811606##[16]##,##REF##9811611##[31]##,##REF##10814565##[32]##,##REF##14691137##[33]## as
well as a second site that forms upon binding of Nef to the MHC-I cytoplasmic tail.
Despite the presence of two AP-1 signals, only one is active in the context of the
natural Nef-MHC-I complex ##REF##15569716##[25]##,##REF##18073204##[27]##. The dileucine
motif in the C-terminal flexible loop can become activated to affect MHC-I
transport, but only when Nef is artificially fused to the MHC-I cytoplasmic tail
##REF##18073204##[27]##. This result indicates there is no inherent inability
of this signal to affect MHC-I traffic but rather that something else, such as the
structure of the natural complex, causes the dileucine motif to be inactive ##REF##18073204##[27]##.
The dileucine motif at position 164 is located close to the diacidic motif at
position 154 that binds β-COP to promote CD4 degradation. The fact that both
of these motifs are inactive when Nef is bound to MHC-I, suggests that much of the
C-terminal flexible loop region of Nef is inaccessible under these conditions. Thus,
Nef behaves as though it assumes different structural forms in different contexts to
differentially expose distinct trafficking signals.</p> <p>We also present evidence that knock down of β-COP yielded a distinct
phenotype from BFA treatment. As described above, BFA is a chemical inhibitor of
ARF1, that is known to trigger the reversible collapse of the
<italic>cis-medial</italic> Golgi compartments to the ER ##REF##2277061##[57]##–##REF##8458343##[59]## by
inhibiting an ARF-specific guanine nucleotide-exchange protein (ARF-GEF) ##REF##1448151##[60]##,##REF##1448152##[61]##. Because ARF1 activity is necessary for
recruitment of β-COP to membranes ##REF##1631136##[62]##, it was possible
that the dramatic effects of BFA resulted from the inability for β-COP to
function normally. However, our results demonstrating that knockdown of
β-COP had no effect on overall Golgi structure indicate that the dramatic
effects of BFA are not due solely to disruption of β-COP function in the
Golgi.</p> <p>Given the important role of β-COP in the Golgi, it is surprising that
β-COP bound to Nef does not also affect transport of MHC-I through the
ER/Golgi. It is possible that our inability to detect an effect of Nef on early
transport of MHC-I ##REF##15569716##[25]## may be a result of the cell type chosen for these
studies. T cells, which are an important natural target of HIV, normally traffic
MHC-I through the early secretory pathway slowly ##REF##15653685##[23]## and thus it might be
difficult to further reduce the trafficking speed through an interaction with
β-COP. Interestingly, another group has reported a reduced ER-Golgi exit
rate for MHC-I in Nef-expressing HeLa cells ##REF##17581864##[63]##, which normally
transport MHC-I more rapidly than T cells ##REF##15653685##[23]##. We have made similar
observations in astrocytoma cells expressing higher levels of Nef than typically
needed to observe MHC-I downmodulation (Roeth and Collins, unpublished
observations). Further studies will be needed to determine whether this effect of
Nef plays a role in more physiologically relevant cell systems and whether this
effect of Nef might be dependent on β-COP expression.</p> <p>A recent report indicates that the effect of Nef on internalization of MHC-I, which
is only minimally apparent in our system, occurs via a PI3-kinase dependent pathway
##REF##18005690##[64]##.
This publication reported that CEM cells, which were used in our study, have less
PTEN (a phosphatase that inhibits PI3-kinase) than another T cell line used in their
study (H9). This deficiency might make it relatively more difficult for us to detect
an effect of chemical PI3-kinase inhibitors, but would not affect our ability to
detect a PI3-kinase-dependent trafficking pathway. In fact, one would expect the
opposite, that the PI3-kinase-dependent pathway would be more active in our system.
However, we have found that Nef has a relatively small effect on internalization of
MHC-I, and mainly affects MHC-I protein export and degradation. These data have been
corroborated in HIV-infected primary T cells ##REF##12584329##[22]##,##REF##15596859##[26]##, which were also
found to much lower levels of PTEN than H9 cells did ##REF##18005690##[64]##.</p> <p>From a teleological perspective, it makes sense that Nef would have evolved to target
early forms of MHC-I, which harbor antigens derived from the newly synthesized viral
proteins. Older forms of MHC-I already on the cell surface would be bound to normal
cellular antigens and would in fact be protective as they would inhibit killing by
natural killer cells that are stimulated to lyse cells with abnormally low MHC-I
expression. On the other hand, it makes sense that Nef, an early viral protein,
would have evolved to target surface CD4 to rapidly and efficiently remove CD4 in
order to prepare the cell for rapid release of viral particles and to render the
cell resistant to re-infection. Meanwhile, a late protein, Vpu, is expressed in
infected cells and specifically targets the newly synthesized CD4 for degradation,
preventing any additional CD4 from reaching the cell surface ##REF##1433512##[65]##.</p> <p>In sum, we have found that the HIV Nef protein commandeers the cellular trafficking
machinery efficiently by utilizing their natural activities for abnormal purposes.
The fact that these pathways may end in a final common step raises the important
possibility that inhibitors might be developed that could block multiple Nef
functions.</p>
<p>Conceived and designed the experiments: MRS ERW JFR JAL KLC. Performed the
experiments: MRS ERW JFR JAL. Analyzed the data: MRS ERW JFR JAL KLC. Wrote
the paper: MRS JFR KLC.</p> <p>To facilitate viral infection and spread, HIV-1 Nef disrupts the surface
expression of the viral receptor (CD4) and molecules capable of presenting HIV
antigens to the immune system (MHC-I). To accomplish this, Nef binds to the
cytoplasmic tails of both molecules and then, by mechanisms that are not well
understood, disrupts the trafficking of each molecule in different ways.
Specifically, Nef promotes CD4 internalization after it has been transported to
the cell surface, whereas Nef uses the clathrin adaptor, AP-1, to disrupt normal
transport of MHC-I from the TGN to the cell surface. Despite these differences
in initial intracellular trafficking, we demonstrate that MHC-I and CD4 are
ultimately found in the same Rab7<sup>+</sup> vesicles and are both
targeted for degradation via the activity of the Nef-interacting protein,
β-COP. Moreover, we demonstrate that Nef contains two separable
β-COP binding sites. One site, an arginine (RXR) motif in the N-terminal
α helical domain of Nef, is necessary for maximal MHC-I degradation. The
second site, composed of a di-acidic motif located in the C-terminal loop domain
of Nef, is needed for efficient CD4 degradation. The requirement for redundant
motifs with distinct roles supports a model in which Nef exists in multiple
conformational states that allow access to different motifs, depending upon
which cellular target is bound by Nef.</p> <title>Author Summary</title> <p>HIV is unique among viral pathogens in its capacity to cause chronic and
progressive disease in almost all infected people. To accomplish this, HIV must
evade the host immune response, especially cytotoxic T lymphocytes (CTLs), which
normally function to lyse virally infected cells. HIV encodes a factor, Nef,
which protects HIV infected cells from lysis by anti-HIV CTLs. To prevent CTL
lysis, Nef interferes with the expression of host MHC-I, which is needed for CTL
recognition of infected targets. A clear understanding of how Nef works has been
hampered by its many complex functions. In addition to MHC-I, Nef protein
disrupts the expression of multiple other cellular targets using different
mechanisms and it is unclear how one protein can accomplish all these tasks.
Here, we provide evidence that Nef acts as a highly flexible adaptor protein
that is capable of utilizing different protein binding domains depending on
which cellular target it is bound to. For example, we present evidence that Nef
binding to MHC-I creates novel motifs that result in the recruitment of AP-1 and
subsequently β-COP. This series of events results in the
mis-localization of MHC-I from the cell surface to cellular degradative
compartments, where MHC-I is destroyed.</p>
<title>Supporting Information</title>
<p>We are grateful to Dr. Linton Traub for antibody to μ1. Additionally, we are
grateful to the University of Michigan vector core for preparation of adenoviral
vectors and to the Microscopy and Image Analysis Laboratory for confocal microscope
use. We thank Dr. Joel Swanson for the pEYFP-Rab7 and we are indebted to the
laboratory of Dr. Colin Duckett for providing reagents and expertise needed for
shRNA work.</p>
<fig id="ppat-1000131-g001" position="float"><object-id pub-id-type="doi">10.1371/journal.ppat.1000131.g001</object-id><label>Figure 1</label><caption><title>The cytoplasmic tail domain of MHC-I and CD4 determines the mechanism
by which Nef affects trafficking.</title><p>(A) Reduction of surface expression of HLA-A2, CD4 and A2/CD4 as measured
by flow cytometry. CEM HA-HLA-A2 and CEM HA-A2/CD4 cells were transduced
with a control adenovirus (<italic>nef<sup>−</sup></italic>)
or adeno-Nef (<italic>nef<sup>+</sup></italic>) and stained for
surface HLA-A2 and CD4. The histograms shaded gray represent cells
treated with control adenovirus, and the solid black line indicates
cells treated with adeno-Nef. (B-C) Measurements of surface stability.
CEM HA-HLA-A2 and HA-A2/CD4 cells were treated with adenovirus as in
part A and the internalization of endogenous CD4 (B) or A2/CD4 (C) was
compared to the internalization of HLA-A2. The filled squares represent
control (<italic>nef−</italic>) cells, and the open squares
represent adeno-Nef (<italic>nef<sup>+</sup></italic>) cells.
Quantitation of part B was compiled from four independent experiments
performed in duplicate. Part C is representative of two experiments
performed in triplicate. (D–E) HLA-A2 is inefficiently
transported to the cell surface in T cells expressing Nef. CEM HA-HLA-A2
and CEM HA-A2/CD4 cells were transduced as in part A. Metabolic labeling
with continuous surface biotinylation was performed in the presence of a
cell-impermeable biotinylation reagent [NHS-biotin,
(Pierce)] to label cell surface proteins. The cells were lysed
and immunoprecipitated first with an antibody against HLA-A2 or CD4
(part D) or anti-HA (part E), then 2/3 was re-precipitated with avidin
beads to selectively precipitate the HLA-A2 on the cell surface.
Normalized surface MHC-I was calculated as follows: ((surface MHC-I
/total MHC-I×2)×100). Quantitation for parts D and E
represents the mean±standard deviation for four and three
independent experiments respectively. For part D, quantitation is
derived from data for the lower of the two Nef levels shown.</p></caption></fig> <fig id="ppat-1000131-g002" position="float"><object-id pub-id-type="doi">10.1371/journal.ppat.1000131.g002</object-id><label>Figure 2</label><caption><title>MHC-I and CD4 co-localize in a subset of vesicles in Nef-expressing T
cells.</title><p>(A) Three-way co-localization of HLA-A2, CD4 and Rab7. CEM HLA-A2 cells
stably expressing YFP-Rab7 were transduced with adeno-Nef. HLA-A2 (red),
CD4 (green), and YFP-Rab7 (blue) were simultaneously detected using
three-color confocal microscopy. The top row shows the
<italic>x</italic>–<italic>y</italic> projection of the
cell, while the bottom row displays the
<italic>x</italic>–<italic>z</italic> projection. Ten
sequential optical sections were compiled to generate a projection of
each cell about the <italic>x</italic>–<italic>z</italic>
plane. Scale bar = 5 microns. (B)
Immunogold labeling of HLA-A2 and CD4. Representative electron
micrographs of CEM HA-HLA-A2 cells treated with control adeno (column 1)
or adeno-Nef (columns 2 and 3). Thawed cryosections of cells were
labeled with anti-HA (HLA-A2) and anti-CD4 antibodies followed by 15 and
10 nm protein A-gold respectively. (C) Immunogold labeling of HA-HLA-A2
and γ-adaptin in Nef-expressing CEM T cells. Thawed cryosections
of cells were labeled with anti-HA (HLA-A2) and anti-γ-adaptin
antibodies followed by 15 and 10 nm protein A-gold, respectively.</p></caption></fig> <fig id="ppat-1000131-g003" position="float"><object-id pub-id-type="doi">10.1371/journal.ppat.1000131.g003</object-id><label>Figure 3</label><caption><title>Knockdown of β-COP does not affect HLA-A2 transport to the
cell surface or disrupt the Golgi apparatus.</title><p>(A) Analysis of protein expression in β-COP and μ1
knockdown cells. CEM HA-HLA-A2 cells were transduced with a lentivirus
expressing both GFP and a control shRNA (shNC) or an shRNA targeting
either β-COP (shβ-COP) or μ1 (shμ1). At
72 hours later, they were transduced with adeno-Nef or control
adenovirus. Three days later they were harvested, and western blot
analysis was used to assess protein levels of β-COP, μ1
and Nef. (B,C) Quantification of μ1 and β-COP expression
in shRNA treated cells. The amount of either μ1 (B) or
β-COP (C) was quantified using Adobe Photoshop software. The
average percent remaining±standard deviation for four
experiments (B) and three experiments (C) is shown. To adjust for
protein loading in part B, the nonspecific background band directly
below μ1 (shown in part A) was used to normalize protein
loading. (D) Knockdown of β-COP does not affect HLA-A2 transport
to the cell surface. CEM HA-HLA-A2 cells were transduced with lentivirus
expressing either shNC or shβ-COP as in part A. Cell surface
transport was assessed using a metabolic labeling assay with
biotinylation as described in ##FIG##0##Figure 1D##. (E) Knockdown of
β-COP does not disrupt the Golgi apparatus. CEM HA-HLA-A2 cells
were transduced with lentivirus expressing the indicated shRNA and GFP
as in part A and treated with brefeldin A (BFA) at 50 µM or
DMSO for 30 minutes. The integrity of the Golgi apparatus was assessed
by immunofluorescence staining for giantin and analyzed by confocal
microscopy. Images were taken using a Zeiss confocal microscope and
analyzed with LSM Image Browser and Adobe Photoshop software. Single
Z-sections are shown. The results shown for parts D and E are
representative of three independent experiments.</p></caption></fig> <fig id="ppat-1000131-g004" position="float"><object-id pub-id-type="doi">10.1371/journal.ppat.1000131.g004</object-id><label>Figure 4</label><caption><title>Nef requires β-COP to reduce HLA-A2 cell surface expression
and accelerate HLA-A2 degradation.</title><p>(A) β-COP and μ1 are required for Nef to reduce cell
surface expression of HLA-A2. CEM HA-HLA-A2 cells were transduced with a
lentivirus expressing GFP and a control (shNC), β-COP
(shβ-COP) or μ1 (shμ1) shRNAs and with control
adenovirus (<italic>nef<sup>−</sup></italic>) or adeno-Nef
(<italic>nef<sup>+</sup></italic>). Cell surface
expression of HLA-A2 or CD4 in the GFP-positive cells was assessed by
flow cytometry. The gray shaded histogram represents control adenovirus
(<italic>nef<sup>−</sup></italic>) treated cells and
the solid black line represents adeno-Nef
(<italic>nef<sup>+</sup></italic>) treated cells. (B)
Quantitation of HLA-A2 and CD4 downmodulation in Nef expressing cells
transduced with shRNA. The median fold downmodulation (median
fluorescence of control/median fluorescence of Nef-expressing
cells)±standard deviation derived from five (HLA-A2) and four
(CD4) independent experiments. A <italic>p</italic> value was calculated
using a two tailed t-test and significant differences were indicated
with asterisks (*<italic>p</italic>&lt;0.02,
***<italic>p</italic>&lt;10<sup>−3</sup>,
****<italic>p</italic>&lt;10<sup>−4</sup>).
(C) Knockdown of β-COP stabilizes intracellular levels of HLA-A2
and A2/CD4 in Nef expressing cells. CEM HA-HLA-A2 and CEM HA-A2/CD4 were
treated as in part A. Lysates from these cells were generated and
treated with endoglycosidase H (endo H). Protein levels of HLA-A2 and
A2/CD4 were assessed by western blot using an anti-HA antibody. Endo
H–resistant bands are marked with an R and endo
H–sensitive bands are marked with an S. The results shown are
representative of three independent experiments for HLA-A2 and two
independent experiments for A2/CD4. (D) Quantification of endo
H–resistant protein. Adobe Photoshop software was used to
quantify each band for the Nef-expressing samples. The percentage of
endo H–resistant protein in each condition was calculated as
follows: [resistant band/(resistant band+sensitive
band)]×100. The fold stabilization was then
calculated as: (% endo H–resistant in experimental
sample)/[% endo H–resistant in control
(shNC)]. The data shown is the mean of two
experiments±standard deviation.</p></caption></fig> <fig id="ppat-1000131-g005" position="float"><object-id pub-id-type="doi">10.1371/journal.ppat.1000131.g005</object-id><label>Figure 5</label><caption><title>Nef requires β-COP to target HLA-A2 and CD4 for degradation.</title><p>(A) Knockdown of β-COP stabilizes CD4<sup>+</sup>
vesicles in Nef expressing cells. CEM HA-HLA-A2 cells transduced with a
lentivirus expressing GFP and either control shRNA (shNC) or shRNA
targeting β-COP (shβ-COP) were transduced with control
adenovirus (<italic>nef<sup>−</sup></italic>) or adeno-Nef
(<italic>nef<sup>+</sup></italic>). The cells were
incubated with CD4 antibody on ice and then shifted to 37°C for
internalization for the indicated times. Images were taken with a Zeiss
confocal microscope and processed using LSM Image Browser and Adobe
Photoshop software. Single Z-sections are shown. (B) Quantitation of
CD4<sup>+</sup> vesicles is shown for 15
GFP<sup>+</sup>, <italic>nef</italic>
<sup>+</sup>
cells treated with shNC and 17 GFP<sup>+</sup>,
<italic>nef</italic>
<sup>+</sup> cells treated with
shβ-COP. The mean±standard deviation is shown. (C)
Quantitation is shown for 5 GFP<sup>+</sup>,
<italic>nef</italic>
<sup>+</sup> cells treated with shNC
and 5 GFP<sup>+</sup>,
<italic>nef</italic>
<sup>+</sup> cells treated with
shβ–COP. The mean±standard deviation is
shown. (D) CEM HA-HLA-A2 cells were transduced with a lentivirus
expressing either GFP and control (shNC) or β-COP
(shβ–COP) shRNA, infected with HIV, treated with
bafilomycin or DMSO and stained for HLA-A2 and LAMP-1 as previously
described ##REF##15569716##[25]##. Images were taken with a Zeiss
confocal microscope and processed as in part A. Single Z-sections are
shown. (E) Relative co-localization of HLA-A2 with LAMP-1 in 10
GFP<sup>+</sup>, adeno-Nef-expressing T cells treated with
shNC and 15 GFP<sup>+</sup>, adeno-Nef-expressing T cells
treated with shβ-COP. (F) Relative co-localization of HLA-A2
with LAMP-1 in 6 GFP<sup>+</sup>,
HIV-<italic>nef</italic>
<sup>+</sup> infected T cells
treated with shNC and, 6 GFP<sup>+</sup>,
HIV-<italic>nef</italic>
<sup>+</sup>–infected T
cells treated with shβ-COP. Quantitation of microscopy data was
performed independently by two blinded investigators who scored maximal
observable co-localization among all cells at an arbitrary value of 5.
Each cell was then scored relative to that. The mean±standard
deviation is shown.</p></caption></fig> <fig id="ppat-1000131-g006" position="float"><object-id pub-id-type="doi">10.1371/journal.ppat.1000131.g006</object-id><label>Figure 6</label><caption><title>Selective binding of AP-1 is dependent on the cytoplasmic tail.</title><p>(A) The HLA-A2 cytoplasmic tail is necessary for co-precipitation of
AP-1. Parental HLA-A2-negative CEM T cells (CEM) or CEM T cell lines
expressing HA-HLA-A2 or HA-A2/CD4 were transduced with adeno-Nef or a
control adenovirus. Lysates were immunoprecipitated with an antibody
directed against HLA-A2 (BB7.2) and the presence of Nef or AP-1 was
detected by western blot analysis. Results are representative of three
independent experiments. (B) The cytoplasmic tail is necessary for the
HLA-A2/Nef fusion protein to co-precipitate AP-1 in Nef expressing T
cells. CEM T cells were transduced with a murine retroviral vector
expressing no protein (vector), A2/Nef or A2/CD4/Nef fusion proteins.
These cells were immunoprecipitated with an anti-HLA-A2 antibody (BB7.2)
and western blot analysis was performed to detect co-precipitation of
AP-1. Spaces between lanes indicate where intervening lanes were cropped
out to remove irrelevant data. Results are representative of two
independent experiments.</p></caption></fig> <fig id="ppat-1000131-g007" position="float"><object-id pub-id-type="doi">10.1371/journal.ppat.1000131.g007</object-id><label>Figure 7</label><caption><title>Co-precipitation of Nef and β-COP depends on domains of Nef
that are also needed for MHC-I downmodulation.</title><p>(A) Flow cytometric analysis of Nef mutants defective at MHC-I
downmodulation. CEM T cells treated with control adenovirus
(<italic>nef<sup>−</sup></italic>), adeno-Nef
(<italic>nef<sup>+</sup></italic>) or the indicated
mutant were stained either with an anti-HLA-A2 antibody (BB7.2) or an
antibody directed at CD4. Cells were analyzed by flow cytometry as
described in <xref ref-type="sec" rid="s5">Materials and Methods</xref>.
(B) Quantitation of MHC-I and CD4 downmodulation by Nef and Nef mutants.
Fold downmodulation was determined by dividing the mean fluorescence
intensity (MFI) of control virus treated cells by the MFI of
Nef-expressing cells. The average value from three (wild-type) or two
(mutant Nef) experiments was plotted±the standard deviation.
(C) Nef D<sub>123</sub>G and V<sub>10</sub>EΔ17–26
mutants are defective at β-COP binding. CEM T cells were treated
with control adenovirus (<italic>nef<sup>−</sup></italic>),
adeno-Nef (<italic>nef<sup>+</sup></italic>), or the indicated
mutant and immunoprecipitated with a control antibody (BB7.2) or an
antibody directed against β-COP (M3A5). The presence of Nef was
detected by western blot analysis. Arrows indicate the positions of wild
type Nef and Nef V<sub>10</sub>EΔ17–26. Results are
representative of at least two independent experiments. (D)
V<sub>10</sub>EΔ17–26 Nef is defective at MHC-I, but
not CD4, degradation. CEM cells expressing HA-HLA-A2 and HA-A2/CD4 were
transduced with adeno-viral vectors encoding wild-type Nef
(Nef<sup>+</sup>), V<sub>10</sub>EΔ17–26
Nef, or a control adenoviral vector (Nef<sup>−</sup>). Two
days later, the media on half of the cells was replaced with media
containing 20 mM ammonium chloride to inhibit lysosomal degradation. The
next day, the cells were harvested, lysed, and normalized. Each sample
was split equally and one set was treated with endo H. Protein levels of
HA-HLA-A2 and HA-A2/CD4 were assessed by western blot analysis using an
anti-HA antibody. Endo H–resistant bands are marked with an R
and endo H–sensitive bands are marked with an S.</p></caption></fig> <fig id="ppat-1000131-g008" position="float"><object-id pub-id-type="doi">10.1371/journal.ppat.1000131.g008</object-id><label>Figure 8</label><caption><title>Two Nef domains recruit β-COP, but only one is used for the
degradation of HLA-A2.</title><p>(A) Flow cytometric analysis of HLA-A2 and CD4 expression in cells
expressing Nef mutants. CEM T cells stably expressing HLA-A2 were
spin-transduced with murine retroviral supernatants that express the
indicated Nef construct and a GFP cassette from an internal ribosomal
entry site. The cells were gated for GFP expression and results of
HLA-A2 (top panel) or endogenous CD4 (bottom panel) staining are shown.
R/E stands for R<sub>17,19</sub>A/E<sub>154,155</sub>A double mutant.
Open light gray curve, parental cell line; shaded dark gray curve, empty
vector; black shaded curve, wild type Nef; and open dark gray curve, Nef
mutant. (B) Quantification of down-modulation. The mean±SD
for greater than or equal to six experiments (actual number varies
depending on the mutant) is shown. (C,D) R<sub>17/19</sub> is needed for
optimal HLA-A2 degradation, whereas the E<sub>154/155</sub> is
dispensable. CEM T cells expressing HLA-A2 and Nef were generated as
described in part A. The cells were pulse labeled with
<sup>35</sup>S-labeled amino acids, chased for 0 or 12 hours in complete
medium and lysed. HLA-A2 was immunoprecipitated with the anti-HLA-A2
antibody BB7.2, separated by SDS-PAGE and quantified using a
phosphorimager. “Ig Control” indicates results from
HLA-A2-negative parental CEM cells immunoprecipitated with BB7.2
antibody. (D) Quantification of degradation. Nef activity was calculated
as follows: (the fraction of HLA-A2 remaining in control cells /the
fraction of HLA-A2 remaining in Nef expressing cells). The value
obtained for each mutant was divided by that for wild type Nef and
multiplied by 100 to calculate % wild type activity. The
mean±SD for two experiments is shown. (E) R<sub>17/19</sub>
and E<sub>154/155</sub> are required for the β-COP/Nef
interaction. CEM T cells expressing HA-A2 were transduced with a
retroviral vector expressing either wild-type Nef or the indicated Nef
mutant. The cells were immunoprecipitated with an
anti–β-COP antibody and the presence of Nef was
assessed by western blot as described in <xref ref-type="sec" rid="s5">Materials and Methods</xref>. The Ig control is
HLA-A2–negative parental CEM cells expressing wild-type Nef
immunoprecipitated with a control antibody (BB7.2) and the vector only
control is CEM cells expressing HLA-A2 transduced with the empty
retroviral vector.</p></caption></fig> <fig id="ppat-1000131-g009" position="float"><object-id pub-id-type="doi">10.1371/journal.ppat.1000131.g009</object-id><label>Figure 9</label><caption><title>Nef uses the E<sub>154/155</sub> to promote maximal CD4 degradation.</title><p>(A) Cells expressing HA-HLA-A2/CD4 were treated as in ##FIG##7##Figure 8A##, lysed and
treated with endo H as indicated. The samples were separated by SDS-PAGE
and western blotted for the HA tag on HA-A2/CD4. (B) Quantification of
degradation. Western blots were quantified using Adobe Photoshop
software. Nef activity was calculated as follows (fraction of total
protein that was endo H–resistant for wild type Nef/fraction
endo H–resistant for each mutant)×100. The
mean±SD for four experiments is shown. (C) Model for the
mechanism by which Nef affects CD4 and MHC-I trafficking. HIV Nef binds
the CD4 cytoplasmic tail at the cell surface, and recruits AP-2 and/or
the vacuolar-ATPase to facilitate internalization. CD4 is internalized
and is transported to an endosomal compartment associated with Rab7 and
β-COP. In contrast, Nef binds the MHC-I cytoplasmic tail early
in the secretory pathway, AP-1 is recruited and facilitates transport to
an intermediate endosomal compartment marked with Rab7. If AP-1 falls
off the Nef-MHC-I complex after arrival in the endosome, Nef binds
β-COP and targets MHC-I (and CD4) to lysosomes for degradation.
If AP-1 remains bound, it promotes recycling of the Nef-MHC-I complex to
the TGN. LY = lysosome,
LE/MVB = late endosome/multi-vesicular
body.</p></caption></fig>
<supplementary-material content-type="local-data" id="ppat.1000131.s001"><label>Table S1</label><caption><p>Analysis of CD4<sup>+</sup> structures in Nef-expressing T cells.
CEM HLA-A2 cells were transduced with adeno-Nef and analyzed by three-color
confocal microscopy as described in <xref ref-type="sec" rid="s5">Materials
and Methods</xref>. Discrete CD4<sup>+</sup> structures were
identified and scored for co-localization with HLA-A2 or the indicated
organelle marker protein. Data from at least two independent experiments
were combined for each protein analyzed.</p><p>(0.02 MB DOC)</p></caption></supplementary-material> <supplementary-material content-type="local-data" id="ppat.1000131.s002"><label>Table S2</label><caption><p>Combinations of antibodies used for immunofluorescence staining for
experiments summarized in ##SUPPL##0##Table S1##.</p><p>(0.04 MB DOC)</p></caption></supplementary-material> <supplementary-material content-type="local-data" id="ppat.1000131.s003"><label>Figure S1</label><caption><p>Bafilomycin treatment increases MHC-I and CD4 co-localization in
Nef-expressing cells. CEM HA-HLA-A2 cells were transduced with a control
adenovirus (nef<sup>−</sup>) or adeno-Nef
(nef<sup>+</sup>) as described in <xref ref-type="sec" rid="s5">Materials and Methods</xref>. At 72 hours later, the cells were treated
with bafilomycin or solvent control (DMSO) and stained with antibodies
directed against HLA-A2 and CD4 as described in <xref ref-type="sec" rid="s5">Materials and Methods</xref>. Images were taken with a Zeiss
confocal microscope and processed with LSM Image Browser and Adobe Photoshop
software. Single Z-sections are shown.</p><p>(1.44 MB TIF)</p></caption></supplementary-material> <supplementary-material content-type="local-data" id="ppat.1000131.s004"><label>Figure S2</label><caption><p>A second siRNA directed at β-COP disrupts Nef-dependent MHC-I
trafficking. (A) Western blot analysis of protein expression in 373 mg
astrocytoma cells transfected with the indicated siRNA. Previously published
protocols ##REF##15569716##[25]## were used to transfect 373 mg astrocytoma
cells with control siRNA (siGFP ##REF##15569716##[25]##) an siRNA
targeting β-COP (siβ-COP, sense <named-content content-type="gene">5′-GGAGAUGUAAAGUCAAAGA-3′</named-content>, antisense
<named-content content-type="gene">5′-UCUUUGACUUUACAUCUCC-3′</named-content>, Ambion)
or an siRNA targeting the AP-1 μ subunit (si μ 1 ##REF##15569716##[25]##). The data is representative of three
experiments. (B) β-COP and μ 1 are required for Nef to
efficiently reduce cell surface expression of HLA-A2. HLA-A2 cell surface
expression on astrocytoma cells from (A) was assessed by flow cytometry as
described in <xref ref-type="sec" rid="s5">Materials and Methods</xref>. The
fold downmodulation of HLA-A2 (mean fluorescence intensity of control/mean
fluorescence intensity of Nef-expressing cells) for each condition is shown
in the upper left corner. (C) Quantitation of HLA-A2 fold downmodulation in
Nef expressing cells treated with siRNA. The mean fold
downmodulation±standard deviation from three experiments is
shown.</p><p>(0.24 MB TIF)</p></caption></supplementary-material> <supplementary-material content-type="local-data" id="ppat.1000131.s005"><label>Figure S3</label><caption><p>(A) Characterization of HA-HLA-A2 protein forms using western blot analysis.
CEM T cells expressing HA-HLA-A2 were lysed and treated with either Endo H
or neuraminidase. The samples were then analyzed via Western blot. (B) CEM T
cells expressing HA-HLA-A2 and Nef or a control adenoviral vector were
lysed, normalized for total protein, digested with endo H, and probed for
HA-HLA-A2 by Western blotting with an anti-HA antibody.</p><p>(0.24 MB TIF)</p></caption></supplementary-material> <supplementary-material content-type="local-data" id="ppat.1000131.s006"><label>Figure S4</label><caption><p>Shβ-COP does not disrupt co-localization of CD4 and HLA-A2, but does
increase the amount of stainable protein within the cell. HLA-A2 CEM cells
were transduced with a lentivirus expressing control (shNC) or β-COP
(shβ-COP) shRNA. After 3 days, the cells were transduced with
adeno-Nef. After three additional days, the cells were stained with
antibodies directed against HLA-A2 and CD4 as in ##SUPPL##2##Figure
S1##. Images were taken with an Olympus FV-500 confocal microscope and
processed with Adobe Photoshop software. Single Z-sections are shown.</p><p>(0.54 MB TIF)</p></caption></supplementary-material> <supplementary-material content-type="local-data" id="ppat.1000131.s007"><label>Figure S5</label><caption><p>Mutation of R17/19 and E154/155 only slightly diminishes the amount of Nef
and AP-1 coprecipitating with HLA-A2. CEM cells expressing HA-HLA-A2 were
transduced with a retroviral vector expressing either wild type Nef or the
indicated Nef mutant. The cells were immunoprecipitated with an anti-HLA-A2
antibody (BB7.2), and the presence of Nef was assessed by Western blot as
described in <xref ref-type="sec" rid="s5">Materials and Methods</xref>.
“Control” indicates lysates from parental CEM T cells
that lack HLA-A2, but that express wild-type Nef. “Vector
only” indicates CEM T cells expressing HA-A2 transduced with empty
retroviral vector.</p><p>(0.29 MB TIF)</p></caption></supplementary-material>
<fn-group><fn fn-type="COI-statement"><p>The authors have declared that no competing interests exist.</p></fn><fn fn-type="financial-disclosure"><p>This research was supported by NIH grant RO1 AI46998. JR, EW, and JL were also
supported by the University of Michigan Cellular and Molecular Biology Training
Program. MS was supported by the University of Michigan Research Training in
Experimental Immunology Training Grant and the Herman and Dorothy Miller
Award.</p></fn></fn-group>
<graphic xlink:href="ppat.1000131.g001"/> <graphic xlink:href="ppat.1000131.g002"/> <graphic xlink:href="ppat.1000131.g003"/> <graphic xlink:href="ppat.1000131.g004"/> <graphic xlink:href="ppat.1000131.g005"/> <graphic xlink:href="ppat.1000131.g006"/> <graphic xlink:href="ppat.1000131.g007"/> <graphic xlink:href="ppat.1000131.g008"/> <graphic xlink:href="ppat.1000131.g009"/>
<media xlink:href="ppat.1000131.s001.doc"><caption><p>Click here for additional data file.</p></caption></media> <media xlink:href="ppat.1000131.s002.doc"><caption><p>Click here for additional data file.</p></caption></media> <media xlink:href="ppat.1000131.s003.tif"><caption><p>Click here for additional data file.</p></caption></media> <media xlink:href="ppat.1000131.s004.tif"><caption><p>Click here for additional data file.</p></caption></media> <media xlink:href="ppat.1000131.s005.tif"><caption><p>Click here for additional data file.</p></caption></media> <media xlink:href="ppat.1000131.s006.tif"><caption><p>Click here for additional data file.</p></caption></media> <media xlink:href="ppat.1000131.s007.tif"><caption><p>Click here for additional data file.</p></caption></media>
[{"label": ["2"], "element-citation": ["\n"], "surname": ["Schwartz", "Marechal", "Le Gall", "Lemonnier", "Heard"], "given-names": ["O", "V", "S", "F", "J"], "year": ["1996"], "article-title": ["Endocytosis of major histocompatibility complex class I molecules\nis induced by the HIV-1 Nef protein."], "source": ["Nature Medicine"], "volume": ["2"], "fpage": ["338"], "lpage": ["342"]}, {"label": ["19"], "element-citation": ["\n"], "surname": ["Aiken", "Konner", "Landau", "Lenburg", "Trono"], "given-names": ["C", "J", "N", "M", "D"], "year": ["1994"], "article-title": ["Nef induces CD4 endocytosis: requirement for a critical dileucine\nmotif in the membrane-proximal CD4 cytoplasmic domain."], "volume": ["76"], "fpage": ["853"], "lpage": ["864"]}]
PLoS Pathog. 2008 Aug 22; 4(8):e1000131