Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Correspondence
Mark Harris
mharris{at}bmb.leeds.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Studies in vitro have shown Nef to be a functionally pleiotropic protein, influencing viral infectivity and cellular signalling pathways, as well as modulating the surface expression of a number of cellular proteins (for reviews see Fackler & Baur, 2002; Arora et al., 2002
). The molecular mechanisms by which Nef exerts these effects are complex and only partly resolved. Nef has no known enzymatic functions but is thought to effect its many and diverse influences, over both HIV-1 replication and the cellular environment, through an extensive range of interacting partners.
One well-documented and highly conserved functional role of Nef is the ability to down-modulate CD4 from the cell surface (Garcia & Miller, 1991). CD4, a type I integral membrane protein, is required for T-cell activation and also serves as the primary receptor for HIV and SIV. It has been suggested that this function of Nef could promote virus release, impair CD4+ helper T-cell function, alter T-cell receptor signalling and prevent superinfection (Arora et al., 2002
). Furthermore, down-modulation of CD4 correlates closely with enhanced virus replication in both peripheral blood mononuclear cells (PBMC) and in human lymphoid tissue ex vivo (Glushakova et al., 2001
; Stoddart et al., 2003
). Nef does not alter CD4 expression levels or transport through the exocytic pathway but acts to accelerate its internalization from the cell surface. Myristoylation of Nef is needed for membrane localization and is essential for down-modulation of CD4; in addition, the cytoplasmic tail of CD4 is both necessary and sufficient for this Nef-mediated effect (Anderson et al., 1994
).
Endocytosis of CD4 has been most extensively studied in non-lymphoid cells stably expressing human CD4 in the absence of p56lck (Pelchen-Matthews et al., 1993). These studies have shown that protein kinase C (PKC)-mediated phosphorylation of serine residues within the cytoplasmic tail of CD4 [induced by phorbol 12-myristate 13-acetate (PMA) activation of PKC], greatly accelerates the rate of endocytosis. A di-leucine motif (Leu-413/414) within the cytoplasmic tail is essential for endocytosis, but only functions when the serine residues are phosphorylated (Pitcher et al., 1999
). It has been proposed that adaptor complexes, which couple CD4 to clathrin lattices thus initiating clathrin-dependent endocytosis, bind to the di-leucine motif, and this binding is partly dependent on the CD4 phosphorylation state.
In the presence of Nef there is an enhanced localization of CD4 to clathrin-coated-pits (Foti et al., 1997) and a colocalization of CD4 with members of the AP-2 adaptor complex. Nef interacts with the µ-chain of AP-2 (Piguet et al., 1998
), which has led to the suggestion that Nef acts as an adaptor protein, binding both to components of the cellular endocytic machinery and, either directly or via an as yet unknown co-factor, to CD4. There are currently no data demonstrating such an interaction in mammalian cells and it has been hypothesized that the NefCD4 interaction is either weak, or exists only transiently and is disrupted upon CD4 internalization. The existence of this complex is supported, however, by studies in non-mammalian systems: the first evidence for a NefCD4 complex came from studies in insect cells co-infected with recombinant baculoviruses expressing Nef (as an N-terminal fusion with GST) and CD4 the interaction required both Nef myristoylation and the cytoplasmic tail of CD4 (Harris & Neil, 1994
). In addition the interaction has been described in both the yeast two-hybrid system (Rossi et al., 1996
), and in vitro assays (NMR, fluorescence spectroscopy) using purified recombinant Nef and peptides derived from the cytoplasmic tail of CD4 (Grzesiek et al., 1996
; Preusser et al., 2001
). Following on from studies that showed that the CD4 di-leucine motif was required for Nef-mediated down-modulation, the yeast two-hybrid and in vitro studies demonstrated that this motif was also required for Nef binding. It has been proposed that a di-leucine motif in a C-terminal loop of Nef acts as an endocytosis signal recruiting a NefCD4 complex to clathrin-coated pits (Aiken et al., 1994
).
In this study we have reassessed the role of the CD4 di-leucine motif for both Nef binding and Nef-mediated down-modulation. In order to attempt to address some of the inconsistencies in the published literature, which may result from the use of fragments of CD4 in a non-physiological context, we have analysed the role of the di-leucine motif in the context of full-length, native CD4. Using both the previously described insect cell-based in vivo binding assay, and an ELISA assay, we show that the CD4 di-leucine motif is dispensable for Nef binding. In contrast, using HeLa cells stably expressing a CD4 mutant lacking the di-leucine motif, we show that this motif is indeed essential for CD4 down-modulation, mediated either by Nef or PMA treatment. Finally, by exploiting the inability of CD4(LL-AA) to undergo internalization, we present the first experimental evidence for a NefCD4 complex in intact mammalian cells.
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METHODS |
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Cell culture.
Spodoptera frugiperda (Sf9) insect cells were maintained in TC-100 medium (Gibco-Invitrogen) supplemented with 10 % foetal calf serum (FCS), 100 IU penicillin ml-1 and 100 µg streptomycin ml-1. HeLa cells were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10 % FCS, 2 mM glutamine, 100 IU penicillin ml-1 and 100 µg streptomycin ml-1. HeLa cell lines expressing either wild-type human CD4 or a truncation mutant lacking the majority of the cytoplasmic domain (CD4stop399) were obtained from Mark Marsh (University College, London, UK) (Pitcher et al., 1999). HeLa cells expressing CD4(LL-AA) were generated by co-transfecting pSG5CD4(LL-AA) with an expression vector conferring resistance to neomycin, pSG2-Neo, in a ratio of 10 : 1. Resistant colonies were selected in media supplemented with 1 mg G418 ml-1 and screened by flow cytometry, described below.
In vivo interaction assays.
Sf9 cells (5x106) were seeded into a 25 cm2 flask and subsequently infected with recombinant baculoviruses at 5 p.f.u. per cell. Cells were harvested at 48 h post-infection and lysed in 250 µl of Glasgow Lysis Buffer (GLB: 10 mM PIPES/NaOH pH 7·2, 120 mM KCl, 30 mM NaCl, 5 mM MgCl2, 1 % Triton X-100 and 10 % glycerol) supplemented with protease inhibitors (aprotinin, 2 µg ml-1; leupeptin, 1 µg ml-1; pepstatin A, 1 µg ml-1; AEBSF, 0·2 mM). For HeLa experiments, cells were transfected with the appropriate pSG5 constructs by lipofection (Lipofectamine, Invitrogen) and harvested into GLB at 48 h post-transfection. For the binding assay 50 µl of the GLB lysates was incubated with 10 µl (packed volume) of glutathioneagarose beads for 2 h at 4 °C. Beads were washed once with GLB/0·5M KCl and a further three times with GLB. Bound proteins were analysed by 12 % SDS-PAGE followed by immunoblotting with either sheep polyclonal sera raised to Nef or CD4. Blots were visualized with ECL reagents (Amersham).
Flow cytometry.
Infected Sf9 cells (106) were harvested at 24 h post-infection, washed once with PBS, once again with PBS/1 % FCS and then incubated with a 1 : 40 dilution of a phycoerythrin (PE)-conjugated monoclonal anti-CD4 (CalTag) in PBS/1 % FCS. After a 30 min incubation at 4 °C cells were washed three times with PBS/1 % FCS and analysed on a Becton Dickinson FACSCalibur with Cell Quest software. HeLa cell lines expressing full-length CD4 (HeLaCD4), or CD4 mutants HeLaCD4stop399 and HeLaCD4(LL-AA) were transfected by lipofection with bicistronic vectors expressing either wild-type Nef (strain NL4.3), or a mutant containing multiple in-frame stop codons, and GFP (pCG-NL4.3-IRES-GFP or pCG-NL4.3STOP-IRES-GFP) (Carl et al., 2001). As controls (black lines) the three cell lines were transfected with pEGFP-N1 (Clontech); the negative control (dotted lines) represents control HeLa cells transfected with pEGFP-N1. Cells were harvested 48 h post-transfection and stained as outlined above. The levels of cell-surface CD4 were assayed on the GFP-positive cell populations. PMA treatment (10 ng ml-1) was carried out 15 min before harvest.
ELISA protein interaction assay.
C-terminally His-tagged Nef (strain BH10) was expressed and purified by standard techniques. Serial dilutions of purified Nef-His6 were incubated in 96-well plates (Greiner Bio-one PS-microplate) in 50 µl of PBS/0·1 % Tween (PBT) overnight at 4 °C. Plates were washed in PBT and blocked in PBT containing 5 % non-fat dried milk (PBTM) for 2 h at room temperature. Plates were washed in PBT and cytosolic extracts from either Sf9 cells infected with AcCD4, AcCD4(LL-AA) or AcCD4stop394, or HeLa cells expressing CD4, CD4(LL-AA) or CD4stop399, diluted in GLB supplemented with protease inhibitors (50 µl per well) were added for 2 h (Sf9) or overnight (HeLa) at 4 °C. After three washes with PBT, CD4 was detected using the CD4 monoclonal antibody Q4120 (obtained from the MRC AIDS Reagent Repository, NIBSC, UK) (1 : 1000 in PBTM) for 1 h followed by a horseradish peroxidase-conjugated anti-mouse antibody (Sigma, 1 : 500 in PBTM) for a further hour. Bound antibody was visualized using o-phenylenediamine (Dako) and quantified at 490 nm with a reference filter at 630 nm using an MRX microplate reader (Dynex).
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RESULTS |
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Nef forms a stable complex with CD4(LL-AA) in mammalian cells
We and others have previously hypothesized that the failure to detect a NefCD4 complex in mammalian cells might be due to the transient nature of the complex upon CD4 internalization and degradation in lysosomes the complex would be disrupted (Harris & Neil, 1994; Rossi et al., 1996
). Because CD4(LL-AA) was able to bind Nef both in insect cells and in vitro, and was also refractory to Nef-mediated endocytosis, we investigated whether a stable complex between Nef and CD4(LL-AA) could be detected in mammalian cells. HeLa CD4(LL-AA) cells were therefore transfected with pSG5 vectors expressing NefGST, a non-myristoylated derivative [Nef(G2S)GST] or myristoylated GST (myrGST). Lysates were incubated with glutathioneagarose beads and precipitated protein was analysed by immunoblotting for the presence of either Nef or CD4. Fig. 5(b)
clearly shows that NefGST was able to precipitate CD4(LL-AA) (lane 8), but failed to form stable complexes with either wild-type CD4 or CD4stop399 (lanes 4, 6). As expected neither Nef(G2S)GST nor myrGST bound to CD4(LL-AA) (lanes 9, 10). Appropriate expression of either CD4 or the GST fusions was verified by immunoblotting of lysates with antibodies to either GSTNef or CD4 (Fig. 5a
, upper and lower panels respectively). As described above, wild-type CD4 is clearly expressed at a lower level than both the CD4stop399 and CD4(LL-AA) mutants; however, the ability to detect a complex with NefGST is not a consequence of the higher level of expression of CD4(LL-AA), as overexposure of the immunoblot failed to reveal a signal for wild-type CD4 in lane 4 (Fig. 5b
, lower panel) (data not shown). We conclude that a CD4 mutant that is refractory to endocytosis is capable of forming a stable complex with Nef in mammalian cells the data presented in Fig. 5
provide the first experimental evidence for such interaction.
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DISCUSSION |
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Our data confirm the previous observation that the di-leucine motif in CD4 is essential for both PMA- (Pitcher et al., 1999), and Nef- (Aiken et al., 1994
) mediated down-modulation. Taken together with the demonstration of constitutive internalization of a chimeric molecule in which the cytoplasmic tail of CD4 was replaced by Nef (Mangasarian et al., 1997
), a widely accepted interpretation is that Nef binds to the di-leucine motif and then directly associates with the endocytotic machinery to mediate internalization of the NefCD4 complex. Our data imply that this interpretation will have to be reassessed we propose that Nef binds to the cytoplasmic tail of CD4 and facilitates the interaction between the CD4 di-leucine motif and adaptor proteins. This could occur either by the association between Nef and clathrin-coated pits to juxtapose CD4 and the endocytotic machinery, or by the induction of a conformational change in the cytoplasmic tail to expose the di-leucine motif. A number of previous observations lend support to this hypothesis: firstly the binding of Nef to components of clathrin-coated pits (Greenberg et al., 1997
; Lu et al., 1998
); secondly the changes in secondary structure of CD4 peptides upon Nef binding as measured by CD spectra (Preusser et al., 2002
); and lastly the fact that Nef-mediated CD4 down-modulation is independent of serine phosphorylation in the cytoplasmic tail (Garcia & Miller, 1991
). Alternatively, the presence of the CD4 di-leucine motif might induce a conformational change in Nef, thus facilitating binding of Nef to endocytotic proteins. This hypothesis is supported by the observation that although a CD4Nef chimera (with Nef replacing the cytoplasmic tail) was constitutively endocytosed, this molecule was unable to mediate endocytosis of a chimeric protein consisting of CD8 extracellular and transmembrane domains fused to the CD4 cytoplasmic tail containing the LL-AA mutation (Mangasarian et al., 1997
). It is important to note that the structure and function of Nef may be dramatically influenced by the presence of a trans-membrane domain at the N terminus instead of the myristate group as, in comparison to native myristoylated Nef, a CD4Nef chimera will be unable to dynamically associate with the membrane and may be subject to abnormal structural constraints. Further experiments are clearly needed to fully characterize the mechanism of Nef-mediated down-modulation.
The existence of a complex between Nef and CD4 in mammalian cells has been frequently alluded to, and indeed has become part of the accepted dogma used to explain the mechanism of Nef-mediated CD4 down-modulation. However, to date there has been no experimental evidence in support of this hypothesis. The data presented in Fig. 5 thus provide the first proof that Nef can physically associate with CD4 in a mammalian context. The observation that CD4(LL-AA) binds to Nef in HeLa cells also supports the conjecture that the complex is normally transient and is disrupted during endocytosis. It raises the possibility that a complex between Nef and wild-type CD4 might be stabilized (and therefore identified) in cells treated with pharmacological inhibitors of endocytosis, or transfected with dominant-negative mutants of endocytotic proteins (e.g. dynamin). Such experiments are currently in progress in our laboratory and we expect will shed further light on the mechanism of Nef-mediated CD4 down-modulation.
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ACKNOWLEDGEMENTS |
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Received 5 April 2003;
accepted 10 July 2003.