From the Institute of Microbiology, University of
Lausanne, CH-1011 Lausanne, Switzerland, and § Cell Biology
and Metabolism Branch, NICHD, National Institutes of Health,
Bethesda, Maryland 20892
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ABSTRACT |
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The envelope glycoprotein (Env) of human immunodeficiency virus, type 1 (HIV-1) undergoes rapid internalization after its transport to the cell surface. Env internalization is dependent upon information contained within the cytosolic domain of the protein. Here, we report that the cytosolic domain of Env binds specifically to the medium chain, µ2, of the clathrin-associated protein complex AP-2, as well as to the complete AP-2 complex. The Env cytosolic domain contains two highly conserved tyrosine-based motifs (Y712SPL and Y768HRL), both of which are capable of binding to µ2 when presented as short peptides. However, only the membrane-proximal motif Y712SPL binds to µ2 and is required for internalization in the context of the whole cytosolic domain of Env. A glycine residue (Gly711) adjacent to the Y712SPL motif is also important for binding to µ2/AP-2 and internalization. These observations suggest that the accessibility of the membrane-proximal GY712SPL to µ2/AP-2 determines its function as a signal for recruitment of HIV-1 Env into clathrin-coated pits and its ensuing internalization.
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INTRODUCTION |
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The envelope glycoprotein (Env) of human immunodeficiency virus, type 1 (HIV-1)1 plays a critical role during the viral life cycle by mediating the attachment of virions to target cells and the fusion of viral and cellular membranes (1). Incorporation of Env, therefore, is essential for the formation of infectious viral particles. The cytosolic domain of the Env transmembrane subunit gp41 is the portion of the Env protein complex that is most likely to interact with the internal structural proteins of the virus (2-5). Although the cytosolic domain of Env is absolutely required for viral dissemination in vivo it seems to be dispensable for envelope incorporation into virions and, consequently, for viral replication in vitro (6). How newly assembled virions specifically acquire Env remains therefore largely unknown.
An intriguing characteristic of the Env proteins of HIV-1 and simian
immunodeficiency virus is that they undergo rapid endocytosis after
their transport to the cell surface (7, 8). As a consequence, the
internal structural proteins of these viruses need to compete with the
internalization machinery of the cell in order to acquire Env
(9).2 Although the functional
significance of this phenomenon is not understood, it is clear that Env
behaves like other plasma membrane proteins that are rapidly
internalized from the cell surface. Rapid internalization involves
recruitment of plasma membrane proteins to clathrin-coated pits, a
process that is mediated by interaction of endocytic signals found in
the cytosolic domains of the proteins with the clathrin-associated
adaptor complex AP-2 (10-12). The AP-2 complex consists of two large
chains ( and
2), a medium chain (µ2), and a small chain
(
2). A direct interaction between µ2 and tyrosine-based
sorting signals from the cytosolic domains of several cellular integral
membrane proteins has been recently demonstrated (13-15).
To assess the diverse functions of the cytosolic domain of Env including its role in internalization from the plasma membrane, we are analyzing its interaction with cellular and viral proteins. Anti-Env antibodies allowed us to co-immunoprecipitate Env with the AP-2 complex from HIV-1-infected lymphocytes, demonstrating that these proteins associate in vivo. Using GST-Env tail fusion proteins, we then identified the µ2 chain of AP-2 as a protein that interacts with the cytosolic domain of Env. Binding of µ2 to the cytosolic domain of Env was dependent on the presence and the context of a tyrosine-based sorting motif that is crucial for Env internalization (7), but it was also influenced by a glycine residue that had not previously been identified to be important for efficient endosomal sorting. Moreover, sequence as well as context dependence of µ2 binding to the cytosolic domain of Env was mimicked by the intact AP-2 complex.
The results presented here suggest that the glutathione S-transferase (GST) gene fusion system may be useful to analyze the interaction of the cytosolic domain of Env with other cellular or viral proteins. This system may also permit further dissection of µ2 functional regions and definition of the requirements for the binding of tyrosine-based sorting signals to adaptor complexes other than AP-2.
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EXPERIMENTAL PROCEDURES |
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Recombinant Proteins-- All constructs used in the in vitro binding assays were made by ligation of polymerase chain reaction (Pwo DNA polymerase; Boehringer Mannheim)-amplified DNA fragments into pGEX-3X (Amersham Pharmacia Biotech). Primer sequences were as follows: 5'-AATCCCGGGATAGTTTTTGCTGTAC-3' and 5'-CTTAAGACCACTTGCCACCCATCTTA-3'. The polymerase chain reaction products were phosphorylated and inserted into SmaI-linearized pGEX-3X. The expression vectors 3M9 for µ2, pcwt for HIV-1 Env, and the deletion mutants of Env have been described (13, 16, 17); the latter were kindly provided by D. Gabuzda and J. Sodroski.
Site-directed mutagenesis was performed either in the GST fusion constructs or in the Env expression vectors using the Quick ChangeTM system (Stratagene). The following primers were used: R709A, 5'-GTGAATAGAGTTGCGCAGGGATATTCAC-3'; Q710A, 5'-GTGAATAGAGTTAGGGCCGGCTATTCACCATTATCG-3'; G711A, 5'-GAGTTAGGCAGGCATATTCTCCATTATCG-3'; Y712A, 5'-GAGTTAGGCAGGGAGCTTCTCCATTATCG-3'; S713A, 5'-GAGTTAGGCAGGGATATGCACCATTATCG-3'; P714A, 5'-GTTAGGCAGGGATATTCAGCATTATCGTTTC-3'; L715A, 5'-GGCAGGGATATTCGCCGGCGTCGTTTCAGACC-3'; Y768A, 5'-GTGCCTCTTCAGCGCTCACCGCTTGAGAGAC-3'; Y768C, 5'-GTGCCTCTTCAGCTGCCACCGCTTGAG-3'. Mutations were confirmed by DNA sequencing.Co-immunoprecipitation of Env and AP-2--
Chronically infected
Jurkat cells were established by transfecting the cells with
proviral DNA (HXB2 strain; Ref. 18). Cells surviving the acute phase of
virus production were propagated for 4-6 weeks in RPMI 1640 medium
supplemented with 10% fetal calf serum and antibiotics (100 units/ml
penicillin and 100 µg/ml streptomycin). Virus production was
monitored by p24 capture (Abbott) and by SDS-PAGE analysis of purified
virions. To perform the immunoblot analysis, uninfected or infected
cells were washed in PBS and then lysed in 500 µl of a Triton
X-100-based lysis buffer (50 mM Hepes, pH 7.4, 1% Triton
X-100, 10% glycerol, 100 mM NaCl, 10 mM NaF,
and 1 mM EDTA) essentially as described (19). Precleared lysates were preincubated for 1.5 h with 50 µl of Protein
A-Sepharose CL-4B beads (Amersham Pharmacia Biotech). Env was
precipitated using the anti-Env monoclonal antibody b12 (20), kindly
provided by Dennis Burton, immobilized on Protein A-Sepharose CL-4B
overnight at 4 °C. After SDS-PAGE separation, the precipitated
material was transferred onto nitrocellulose membranes. Those membranes were then probed with Sigma antibodies 100/2 (anti--adaptin) or
100/1 (anti-
-adaptin). Bound antibodies were visualized after binding of secondary antibodies by enhanced chemiluminescence (Pierce).
Expression of Fusion Proteins--
GST fusion proteins were
produced in Escherichia coli strain TB1 and purified on
glutathione-Sepharose 4B (Amersham Pharmacia Biotech). Precultures (4 ml of LB medium supplemented with 0.5 M
D-sorbitol and 2.5 mM betaine) were grown
overnight at 37 °C. They were transferred to 400 ml of LB medium
supplemented with 1 M D-sorbitol and 2.5 mM betaine and grown for 8 h at 37 °C. Cultures
were shifted to 25 °C and induced overnight with 0.1 mM
Isopropyl--D-thiogalactoside. The purification on
glutathione-Sepharose 4B beads was performed according to the
manufacturer's instructions including a heat shock step with 50 mM Tris-HCl (pH 7.4), 10 mM MgSO4,
and 2 mM ATP for 10 min at 37 °C.
Binding Assays-- [35S]Methionine-labeled µ2 was prepared using a coupled transcription-translation system (TNT®, Promega,). Precleared in vitro translated µ2 (2 µl) was incubated with 5 µg of GST fusion protein in 500 µl of binding buffer (0.05% (w/v) Triton X-100, 50 mM HEPES (pH 7.3), 0.1 mM CaCl2, 2 mM MgCl2, 100 mM KCl, 50 µM dithiothreitol, 10% (w/v) glycerol, 0.1% bovine serum albumin) for 2 h at room temperature. Loading buffer was added to washed beads, and the samples were run on SDS-PAGE. The amount of µ2 bound to the different GST fusion protein mutants was determined by measuring the radioactivity of the signals using the Instant ImagerTM system (Packard).
T cell lysate was prepared from Jurkat cells essentially as described (21). Briefly, the cells were washed three times with cytosol buffer (25 mM Hepes, pH 7.0, 125 mM CH3COOK, 2.5 mM (CH3COO)2Mg, 1.0 mM dithiothreitol, 1 mg/ml glucose), and the resulting pellet was resuspended in an equal volume of cytosol buffer containing 1 mM phenylmethylsulfonyl fluoride. The cell suspension was frozen in liquid nitrogen, thawed on ice, and drawn five times through a 21-gauge syringe. After centrifugation for 30 min at 20,000 × g, 4 °C, the supernatant was transferred to new tubes in 100-µl aliquots and stored atInternalization Assay-- Twenty-four hours after transfection with the Env expression plasmids, HeLa cells were metabolically labeled for 16 h with [35S]cysteine (50 µCi/ml). The labeled cells were chilled on ice and biotinylated for 30 min in 1 ml of PBS containing 1.5 mg/ml NHS-SS-biotin (Pierce). After three washes with PBS, 50 mM glycine, the cells were returned to 37 °C for different periods of time to allow for endocytosis to occur. To remove biotin from surface proteins that had not been internalized, cells were again chilled on ice and incubated twice for 15 min with 50 mM glutathione and for an additional 15 min with 60 mM 2-mercaptoethanesulfonic acid (MESNa). Free sulfhydryl groups were blocked by washing the cells three times with 50 mM glycine in PBS. The cells were then lysed in 1× radioimmune precipitation buffer, and viral proteins were immunoprecipitated with serum from an individual infected with HIV-1. Following immunoprecipitation, 90% of the material was boiled in 10% SDS, while the remaining 10% of the material was analyzed directly by SDS-PAGE to assess the amount of total Env in each sample. Biotinylated Env was recovered with streptavidin-agarose, followed by SDS-PAGE. The intensity of the Env signal was quantified using the Instant ImagerTM system. The proportion of internalized Env was determined by subtracting for each time point the activity of the glutathione/MESNa-resistant biotin-tagged Env at 0 °C from the activity of the glutathione/MESNa-resistant biotin-tagged Env at 37 °C and dividing the remaining activity by one-hundredth of the activity of Env from cells that had not undergone reduction.
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RESULTS |
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HIV-1 Env and AP-2 Associate in Infected Cells-- The overall goal of our work is to identify proteins interacting with the cytosolic domain of HIV-1 Env. Since Env is rapidly retrieved from the cell surface after its transport to the plasma membrane (7), we tested whether Env interacts with the clathrin-associated protein complex AP-2. As shown in Fig. 1, AP-2 was detected in anti-Env immunoprecipitates from chronically infected lymphocytes but not in anti-Env immunoprecipitates from noninfected cells. A detailed analysis of the requirements for the observed Env-AP-2 interaction cannot be performed in infected cells, because many mutations in Env will affect the efficiency of viral replication and consequently also the levels of Env expression. Env-AP-2 interactions were therefore analyzed in more detail in vitro.
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The Cytosolic Domain of HIV-1 Env Associates with µ2-- The cytosolic domain of Env contains two motifs that strongly resemble tyrosine-based endocytosis signals, one at position 712 (Y712SPL) and the other at position 768 (Y768HRL) (Fig. 3A). Both motifs are well conserved among different strains of HIV-1 (18). Recently, the medium chain (µ2) of the clathrin-associated protein complex AP-2 was found to interact with tyrosine-based signals conforming to the canonical motif YXXØ, where X is any amino acid and Ø is a an amino acid with a bulky hydrophobic side chain (13-15). Therefore, we decided to test whether the cytosolic domain of Env of HIV-1 associates with µ2. The cytosolic tail was expressed as a GST fusion protein (GST-EnvCD) in E. coli. The medium chain (µ2) of the clathrin-associated protein complex AP-2 was translated in vitro in a rabbit reticulocyte lysate. Fig. 2 shows that µ2 bound to GST-EnvCD but not to GST. For comparison, one-tenth of the in vitro translated µ2 is shown in the first lane. Typically, about 5-15% of the in vitro translated µ2 bound to GST-EnvCD.
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Binding of µ2 to the Cytosolic Domain of Env Depends on the Presence and the Context of a Tyrosine-based Motif-- To identify the binding site for µ2 in the cytosolic domain of Env, we tested the interaction of µ2 with various Env tail deletion mutants (Fig. 3B and data not shown). Deletions that did not affect the membrane-proximal tyrosine motif Y712SPL had little or no effect on the association of µ2 with the cytosolic tail. Deletion of a region containing this membrane-proximal tyrosine-based motif, however, severely reduced the binding of µ2 to the cytosolic tail. In contrast, a deletion of region containing the motif Y768HRL had less of an effect. A more detailed analysis of the requirements for µ2 binding was then performed using mutants where single amino acids were mutated to alanine (Fig. 3C). As expected from the results shown in Fig. 3B, mutation of Tyr768 had very little impact on the binding of µ2 to the cytosolic domain of Env. Similarly, substitutions of amino acids Arg709, Gln710, or Ser713, respectively, by alanine influenced binding of µ2 only marginally. In contrast, binding of µ2 was decreased by mutation of Gly711 or Pro714 and even more dramatically by mutation of Tyr712 or Leu715. Thus, residues in the membrane-proximal tyrosine-based motif but not in the membrane-distal one are critical for the binding.
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Association of the Intact AP-2 Complex with the Cytosolic Domain of
Env Shows the Same Sequence Requirements as Env-µ2 Binding--
To
test whether the same region of the cytosolic domain of Env important
for µ2 binding represents the binding motif for intact AP-2
complexes, beads coated with some of our GST-EnvCD fusion proteins were
incubated with cytosol of T cells and washed three times, and bound
material was eluted. After SDS-PAGE, immunoblotting was performed using
antibodies against either of the two large chains of the AP-2 complex
(Fig. 5). Whereas the wild-type EnvCD, as
well as the Y768C mutant, precipitated and
-adaptin, the Y712A
mutant did not. Substitution of Gly711 for alanine,
however, still allowed for the precipitation of some
- and
-adaptin. These results demonstrate that the association of not only
the isolated medium chain µ2 but also the intact AP-2 complex with
the cytosolic domain of Env is critically dependent on the integrity of
the membrane-proximal sequence Y712SPL. In addition,
Gly711 also contributes to the binding of EnvCD to AP-2.
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Binding of µ2/AP-2 to the Cytosolic Domain of Env Correlates with
Env Internalization--
The functional importance of some residues in
the cytosolic domain of Env in its sorting to intracellular
compartments was assessed using a biochemical internalization assay
(see "Experimental Procedures"). None of the deletion mutants was
tested for endocytosis, because the cytosolic domain of Env partially
overlaps with the viral Rev function, which is necessary for Env
expression in intact cells (22). Fig. 6
shows the results of our internalization studies for three of the
mutants where single amino acids had been substituted. The same
mutations in the cytosolic domain of Env that led to a decrease in
association with µ2 and the intact AP-2 complex (see Figs.
3C and 5, respectively) also reduced Env internalization. On
the other hand, mutation of Tyr768, which had little effect
on µ2/AP-2 binding, did not affect endocytosis of Env. Using a
fluorescence-activated cell sorting-based endocytosis assay, Siliciano
and colleagues (7) had already demonstrated that the Tyr712
in the membrane-proximal motif but not Tyr768 in the
membrane-distal motif was important for internalization of Env from the
cell surface. Our data thus confirm those results and also show that
glycine at position 1 with respect to the membrane-proximal
Y712SPL motif (Gly711) influences the
recruitment of Env into clathrin-coated pits. Most importantly, these
data establish a correlation between the binding of the cytosolic
domain of Env to µ2, as well as to the intact AP2 complex, in
vitro and the efficiency of Env internalization in
vivo.
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DISCUSSION |
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The intracellular domain of HIV-1 Env is absolutely required for viral dissemination, but very little is known about its functions. This prompted us to look for proteins interacting with the cytosolic domain of Env. Here we report that the medium chain µ2 of the clathrin-associated protein complex AP-2 as well as the complete AP-2 complex binds the cytosolic domain of Env fused to GST. Furthermore, Env and AP-2 form a complex in vivo, i.e. in infected lymphocytes. Binding of µ2/AP-2 to the cytosolic domain of Env correlates well with Env internalization from the cell surface. Such fusion proteins may therefore be useful not only as probes to identify other proteins interacting with the cytosolic domain of Env but also to analyze the requirements of functional interactions of the adaptor protein complexes or its subunits with the cytosolic domain of Env.
Determinants in the cytosolic domain of Env have been implicated in the directed release of HIV-1 observed in polarized epithelial cells as well as in the retrieval of Env from the cell surface (7, 8, 23, 24). In both instances, a membrane-proximal tyrosine (Tyr712) was demonstrated to be part of the signal(s) responsible for the respective sorting process. Tyrosines have been identified as critical components of sorting signals in the cytosolic domains of different cellular membrane proteins destined for various cellular compartments (10, 12). Together, these data led us to investigate whether the cytosolic domain of Env interacts with elements of the cellular sorting machinery. As a probe, we used a protein where GST was fused to the cytosolic domain of Env. It was demonstrated previously that GST fusion proteins containing tyrosine-based sorting signals can bind proteins involved in the sorting of cellular membrane proteins (13). Here, we demonstrated that the cytosolic domain of Env in the context of a GST fusion protein binds to µ2. Such an interaction between the whole cytosolic domain of Env and µ2 was not observed in the yeast two-hybrid system,3 thus demonstrating the usefulness of the in vitro binding approach in order to study these interactions.
Having established that the cytosolic domain of Env in the context of a
GST fusion protein can bind to µ2, we sought to define the
requirements for this interaction. The cytosolic domain of Env contains
two YXXØ motifs. Both motifs are highly conserved among
almost all primary viral isolates analyzed so far (18), indicating that
they belong to functionally important regions of this domain. The
results of the present study demonstrate that only the
membrane-proximal tyrosine-based motif Y712SPL, but not the
more C-terminally located motif Y768HRL, is important for
µ2 binding. The lack of a contribution of the YHRL motif to µ2
binding was not due to a lower intrinsic affinity of this motif for
µ2. Using the yeast two-hybrid system, Ohno et al. (25)
recently showed that the membrane-distal motif as such can bind to
µ2. Our peptide competition experiments shown in Fig. 4 indicated
that the YHRL motif may have even a slightly higher affinity for µ2.
The results of the in vitro binding assays presented here
thus suggest that µ2 cannot access the YHRL motif in the context of
the whole cytosolic domain. Support for this hypothesis is provided by
the analysis of Env internalization (Fig. 6). Our analysis as well as a
previous study (7) found that tyrosine at position 712 but not tyrosine
at position 768 is critical for Env internalization. Remarkably, the
correlation between binding of µ2 to the cytosolic domain of Env and
endocytosis of Env holds even for the Gly711 to Ala mutant.
Glycine at position 1 relative to the critical Y712SPL
motif has not been previously recognized to influence the internalization of membrane receptors. Mutant G711A showed an intermediate phenotype in the µ2 binding assay as well as in the endocytosis assay. Together, our data suggest that binding of µ2 to
the cytosolic domain of Env defines the specificity for the recruitment
of the protein by the AP-2 complex. Therefore, we tested whether the
cytosolic domain of Env also associates with the intact AP-2 complex
in vitro and, if so, whether the specificity of binding to
EnvCD was the same for µ2 and the intact complex. Our results showed,
first, that EnvCD-AP-2 association was likewise dependent on the
intactness of the membrane-proximal but not of the membrane-distal
tyrosine-based motif. Second, we could reproducibly precipitate the
large chains
- and
-adaptin of AP-2 with the Gly711
to alanine mutant, although its interaction with the intact AP-2 complex may be weaker than its association with µ2. Therefore, the
strength of interaction of µ2 with cognate signals in the cytosolic
domains of membrane proteins may be modulated by an interaction of µ2
with the other subunits of AP-2.
Appropriate spacing of the tyrosine signal was shown to be crucial for
the sorting of a cellular protein to lysosomes (26), presumably
mediated by the AP-1 complex. A possible explanation for the different
recognition by µ2/AP-2 of the Y712SPL motif and the
Y768HRL motif may thus be due to different placement
relative to the plasma membrane. Alternatively, folding of the
cytosolic domain of Env may be such that Y768HRL is not
accessible to µ2/AP-2. The Y768HRL motif is positioned
within one of two amphipathic regions of the cytosolic domain of Env
that have been proposed to associate with the inner face of the plasma
membrane (27, 28). It could thus be that the conformational context of
the Y768HRL motif in vivo prevents its
recognition by the AP-2 complex in cells. The situation may be
different for the region close to the membrane-spanning domain of Env.
This region is predicted to form neither -helices nor
-sheets
(29). The Y712SPL motif within that context may thus adopt
the "tight turn" structure, which was predicted to be the
conformational determinant shared by tyrosine-based internalization
signals (30). Our results do not allow us to exclude the possibility
that the membrane-distal motif contributes to the EnvCD-AP-2 binding
but that the interaction with Tyr768 is dependent on the
interaction of AP-2 with the membrane-proximal motif. However, since
the Tyr768 to Cys mutation does not affect Env
internalization (Fig. 6), we do not consider this to be a very likely
scenario.
Preliminary results suggest that the described Env fusion proteins not only bind with remarkable specificity to µ2/AP-2 but that they can also selectively interact with other cellular sorting complexes. Work is in progress to test whether the association with those complexes also correlates with the in vivo sorting of Env. Such an analysis is particularly desirable with regard to the membrane-proximal region of the Env tail, which is involved not only in Env endocytosis (Ref. 7 and this study) but also in the polarized sorting of Env (24). This situation is reminiscent of the co-linearity of endocytosis signals and basolateral sorting signals that is observed in a number of cellular proteins (31).
In summary, these studies show that the µ2 subunit of AP-2 as well as the intact AP-2 complex bind to a tyrosine-based signal within cytosolic domain of Env involved in endocytosis. A second tyrosine-based motif in the cytosolic tail of Env that is not relevant for Env internalization seems to be largely shielded from recognition by µ2/AP-2. In all, our data describe a case where components of the cellular sorting machinery discriminate between two potential binding sites based on their position in the cytosolic domain of the protein. Thus, our data demonstrate that it is not possible to predict if a putative µ2 binding sequence will function in vivo, based on in vitro studies alone, underscoring the importance of corroborating data from binding experiments with functional studies in vivo.
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ACKNOWLEDGEMENTS |
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We thank Walter Hunziker for critical reading of the manuscript and discussions, Kenneth Raj for useful technical hints, D. Gabuzda and J. Sodroski for HIV-1 Env deletion mutants, and Dennis Burton for the gift of antibody b12.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by Swiss National Science Foundation Grant 3139-040948/1 and a career award (to M. T.).
¶ To whom correspondence should be addressed: Institute of Microbiology, University of Lausanne, IMUL/CHUV 44, Rue du Bugnon, CH-1011 Lausanne, Switzerland. Tel.: 41 21 314 4099 Fax: 41 21 314 4095; E-mail: Markus.Thali{at}inst.hospvd.ch.
1 The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; MESNa, 2-mercaptoethanesulfonic acid.
2 S. Wyss and M. Thali, unpublished observation.
3 H. Ohno and J. Bonifacino, unpublished observations.
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REFERENCES |
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