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- Materials and methods
- Supporting Information
The HIV-1 Nef protein perturbs the trafficking of membrane proteins such as CD4 by interacting with clathrin–adaptor complexes. We previously reported that Nef alters early/recycling endosomes, but its role at the plasma membrane is poorly documented. Here, we used total internal reflection fluorescence microscopy, which restricts the analysis to a ∼100 nm region of the adherent surface of the cells, to focus on the dynamic of Nef at the plasma membrane relative to that of clathrin. Nef colocalized both with clathrin spots (CS) that remained static at the cell surface, corresponding to clathrin-coated pits (CCPs), and with ∼50% of CS that disappeared from the cell surface, corresponding to forming clathrin-coated vesicles (CCVs). The colocalization of Nef with clathrin required the di-leucine motif essential for Nef binding to AP complexes and was independent of CD4 expression. Furthermore, analysis of Nef mutants showed that the capacity of Nef to induce internalization and downregulation of CD4 in T lymphocytes correlated with its localization into CCPs. In conclusion, this analysis shows that Nef is recruited into CCPs and into forming CCVs at the plasma membrane, in agreement with a model in which Nef uses the clathrin-mediated endocytic pathway to induce internalization of some membrane proteins from the surface of HIV-1-infected T cells.
The nef gene product of HIV-1 is a 27-kDa protein that associates with cell membranes through N-terminal myristoylation. Like other HIV-1 accessory proteins, Nef is essential for maximal viral replication in vivo and markedly contributes to the pathogenesis of AIDS. In vitro, Nef modulates T-cell activation and alters the intracellular distribution of a number of cellular proteins. These effects facilitate viral replication, in part by enhancing the infectivity of virions [reviewed by Fackler and Baur and Wei et al. (1,2)].
The positive influence of Nef on viral replication and infectivity is a multifactorial process but relates, at least in part, to the disturbance of the trafficking of membrane proteins within the endocytic pathway. Nef both misdirects CD4, the primary receptor for HIV, from the trans Golgi network (TGN) to endosomes and stimulates its internalization from the cell surface, resulting in an accumulation of CD4 in the early/sorting endosomal compartment. CD4 is ultimately targeted to lysosomes where it is degraded. Together, these Nef-mediated effects drastically reduce the level of cell surface-associated CD4 molecules at steady state, a process collectively referred as ‘CD4 downregulation’ (3–6).
These observations led to the hypothesis that Nef functions as a specific connector between the cytoplasmic domain of CD4 and the endosomal and TGN-associated protein sorting machineries [reviewed by Piguet et al. (7)]. However, it was subsequently reported that Nef also affected the steady-state distribution and endocytosis of the class I major histocompatibility complex (MHC) molecules (5). More recently, the list of cellular membrane-associated proteins whose intracellular trafficking is altered by Nef has expanded to include mature and immature class II MHC, the costimulatory CD28 molecules, the lectin DC-SIGN (dendritic cell-specific ICAM-grabbing non integrin) expressed on dendritic cells, the CCR5 and CXCR4 chemokine coreceptors and also the transferrin receptor (TfR) (8–13). This increasing number of proteins whose trafficking is altered during infection suggests that Nef is unlikely to be a specific adaptor but rather exerts a more general effect on the endocytic system.
The mechanisms responsible for these widespread Nef-mediated effects are not fully understood but are likely related to the ability of Nef to interact with essential components of the machineries involved in the budding of transport vesicles and protein sorting. Indeed, Nef directly interacts with the clathrin–adaptor protein (AP) complexes that govern clathrin polymerization on specific membranes (TGN, endosomes or plasma membrane) and cargo selection for vesicular transport (4,5,14,15). Nef also interacts directly with the catalytic subunit of the vacuolar adenosine triphosphatase required for acidification of endosomes, and with the PACS-1 protein (phosphofurin acidic cluster sorting protein-1) involved in TGN localization of membrane proteins containing acidic cluster motifs (16–18).
Four distinct types of AP complexes (AP-1 to AP-4) have been characterized and consist of heterotetramers composed of two large subunits (adaptins: α, δ, γ or ε, and β1-4), one medium chain (μ1–4) and one small subunit (σ1–4). With the exception of AP-2, the association of AP complexes with membranes is regulated by ADP ribosylation factor 1 (ARF1). While AP-1, AP-3 and AP-4 mediate transport between the TGN and the endosomes or lysosomes, AP-2 is specifically localized to plasma membrane where it plays a central role in both the assembly and the function of clathrin-coated pits (CCPs) (19). The sorting function of AP complexes is related to the recognition of specific signals present in the cytoplasmic domain of transmembrane trafficking proteins. These signals are based on tyrosine or leucine residues that conform to the sequences YXX Φ or E/DXXXLΦ [where Φ is a bulky hydrophobic residue; reviewed by Bonifacino and Traub (20)].
Interestingly, HIV-1 Nef contains a canonical leucine-based AP-binding motif located within an unstructured loop found in the C-terminal part of the protein. The presence of this di-leucine motif is critical for the Nef effects on CD4 and some other receptors but not for its effects on MHC-I molecules (14,15,17,18,21–24). It is also required for the maximal infectivity of HIV-1 virions (13,25,26), indicating that the positive influence of Nef on viral infectivity is related, at least in part, to its general impact on the endosomal system. Although the specific contributions and the dynamics of the association of Nef with the individual AP complexes are still unclear, previous in vitro studies suggested that the HIV-1 Nef protein preferentially interacts with AP-1 and AP-3 complexes (24,26,27). In living cells, Nef recruits and stabilizes the association of AP-1 and AP-3 on endosomal membranes by an ARF1-independent mechanism (27), resulting in morphological and functional alterations of the early/sorting endosomal compartments (13,28–30). In contrast, the specific role of Nef at the plasma membrane and how this myristoylated protein reaches early endosomes are poorly documented. Paradoxically, although HIV-1 Nef interacts at best weakly with the intact AP-2 complex (5,23,24,26,31), its ability to accelerate the rate of internalization of CD4 from the cell surface suggests a role for clathrin-mediated endocytosis in Nef action (32).
To clarify the action of Nef at the cell surface in relation to the clathrin-dependent endocytic pathway, we investigated the dynamic behavior of Nef at the plasma membrane of living cells using total internal reflection fluorescence microscopy (TIR-FM). This technique restricts the morphologic analysis of the samples to the adherent surface of the cells and has been recently extensively used to study the dynamics of clathrin and clathrin-associated proteins (33,34). Here, TIR-FM revealed the specific localization of Nef in CCPs and in forming clathrin-coated vesicles (CCVs) at the plasma membrane and was further used to characterize the mechanisms responsible for this localization using Nef mutants and CD4-expressing cell lines. Finally, the results obtained by TIR-FM were challenged by testing the effect of the same Nef variants on CD4 endocytosis in T lymphocytes.
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- Materials and methods
- Supporting Information
The use of TIR-FM enables the first detailed dynamic analysis of the HIV-1 Nef protein at the plasma membrane of living cells. TIR-FM imaging shows that Nef is concentrated at the plasma membrane in spots that colocalize with clathrin-positive structures, and this distribution is independent of CD4 expression. Nef is distributed in both static and disappearing CS, which correspond to CCPs and forming vesicles (CCVs), respectively (33,34). In addition, the distribution of Nef in CCP/CCV correlates with its ability to stimulate CD4 internalization in T cells and is dependent on the presence of a functional leucine-based motif involved in Nef binding to AP complexes. Because this motif is also required for the maximal infectivity of HIV-1 virions (13,25,26), these results imply that the targeting of Nef to CCPs and CCVs participates in the Nef-mediated enhancement of viral infectivity.
Our results show that the HIV-1 Nef–GFP fusion is specifically enriched within clathrin-coated structures at the plasma membrane because the great majority if not all Nef-positive spots (>90%) detected by TIR-FM contain clathrin. While the majority (60%) of the clathrin structures were also positive for Nef, it is not clear whether the lack of a Nef–GFP signal in the remaining 40% results from insufficient sensitivity of detection or is related to a heterogeneous population of CS. Although previous studies have suggested that Nef may be partially associated with detergent-resistant membranes or ‘rafts’ at the plasma membrane, we failed to observe any overlap between Nef and caveolin-1 (data not shown), a marker of a raft-dependent non-clathrin pathway. This result is in agreement with a recent study, showing that functions of Nef at the plasma membrane do not depend on raft structures (45).
Our analysis of video streams showed that Nef is found in the three different populations of CS observable in live cells by TIR-FM. It is present in most (91.5%) of static CS that were previously characterized as CCPs that contain both clathrin and AP-2 complex (33,34). Nef is also found in 50.6% of the disappearing CS that correspond to forming CCVs. Although this value is likely underestimated by the observation that the intensity of clathrin is dimmer in the disappearing spots than in the static spots (37), this indicates that Nef is also incorporated into forming vesicles. The presence of Nef in only half of the disappearing CS is not dissimilar to what we have recently found for epsin (unpublished observation), an adaptor for clathrin involved in CCV formation at the plasma membrane (46). Finally, Nef is present in laterally mobile CS; the function of such structures is not clear, but they may correspond to endosomal-associated clathrin coats [(33) and unpublished results]. Therefore, the detection of Nef–GFP within these laterally mobile structures may correspond to Nef on endosomes, where it is associated with AP-1 and AP-3 clathrin–adaptor complexes (27).
The observation that Nef was efficiently targeted to both CCPs and CCVs in HeLa cells suggested that CD4 was not directly involved in this process. Indeed, the hypothesis that CD4 would play an active role came from the model in which Nef functions as a specific ‘connector’ or ‘adaptor’ for CD4 driving its clathrin-dependent internalization through direct interaction with the CD4 cytoplasmic domain and the endocytic machinery [reviewed by Piguet and Trono (41)]. The results we obtained in this study clearly show that Nef was targeted to CCPs in the absence of CD4 (HeLa cells, Figure 2) and that Nef was similarly distributed in CCPs in cells stably expressing wild-type CD4 or a mutant form of CD4 lacking its cytoplasmic domain (Figure 8). These results paralleled those found for the Nef-induced perturbations of the early/sorting endosomal compartments that are also independent of CD4 expression and can be fully reproduced in cells that do not express CD4 (see below). Altogether, these results stress for a clear independence of Nef functions on the endocytic pathway relative to CD4 expression, in agreement with the increasing number of studies showing that Nef is able to perturb the trafficking of many different plasma membrane-associated proteins (8–13).
The accumulation of Nef in CS required the di-leucine motif found in the C-terminal solvent-exposed loop, which is also required for efficient binding to AP complexes. This latter result is in agreement with previous studies, which identified the di-leucine motif of Nef as the determinant required for efficient binding to AP complexes and colocalization with AP-2 in fixed cells (14,15). Our results show that this motif can be functionally replaced by leucine-based AP-binding motifs from heterologous cellular proteins but not by tyrosine-based motifs, even if the latter are also able to interact with AP complexes (26). This functional difference among endocytic motifs may be related to their different target subunits within AP complexes. Tyrosine-based motifs bind to the medium μ subunits (20), whereas leucine-based motifs were reported to bind to the γ/σ1 or δ/σ3 hemicomplexes of AP-1 and AP-3 complexes (24,26) and/or to the β-adaptins (15,47). The finding that only leucine motifs are able to target Nef into CCPs suggests that binding to β2-adaptin or to the α/σ2 hemicomplex of AP-2 is necessary for localization in CCPs. Interestingly, both ARH (autosomal recessive hypercholesterolemia protein) and β-arrestins, APs specific for low-density lipoprotein (LDL) receptor family members and G protein-coupled receptors, respectively, are also targeted to CCPs through binding to β2-adaptin (48,49). However, Nef is also able to interact directly with the μ chains of AP complexes in a di-leucine independent manner. Thus, Nef is unusual in that it possesses distinct interfaces to mediate interactions with different subunits of the heterotetrameric AP complexes. Additional investigations are needed to understand the specific contributions and dynamics of these interactions.
Of note, Nef variants with heterologous leucine-based motifs are efficiently targeted to CCPs, but they are not fully functional (Figures 6 and 7). These results suggest that the endogenous ENTSLL signal of HIV-1 Nef may have attributes in addition to CCP targeting that confer maximal effects on CD4. Interestingly, the Nef variant containing the ERQPLL motif from tyrosinase is less prevalent than wild-type Nef in disappearing CS but is more prevalent in laterally mobile CS (Figure S2). Because disappearing CS have been characterized as forming CCVs, this observation may explain why this Nef variant was slightly less efficient than the wild-type for CD4 internalization.
Nef profoundly disturbs the morphology and function of the early endosomal/recycling compartment (28–30). These effects may relate to its ability to recruit and stabilize the association of the clathrin-associated AP-1 and AP-3 complexes on endosomal membranes (27). Similarly, Nef could stabilize clathrin-coated structures containing AP-2 complexes at the plasma membrane, leading to an increase in the total number of CCPs. However, we found that the number of CS observable by TIR-FM at the cell surface was not significantly increased by the expression of Nef in HeLa cells that express or do not express CD4 (Figure S3). These results are partially in contrast to those of a previous study, which showed that the expression of Nef together with CD4, but not that of Nef alone, leads to an increase in the number of CCPs in B lymphocytes, which normally do not express CD4 (50). The difference in the effects of CD4 expression on Nef-induced formation of CCPs may be due to the different cell types used in the two studies (HeLa versus B lymphocytes). However, the results show that Nef is not able per se to drive CCP assembly at the plasma membrane.
The results also highlight an important difference between the effect of Nef at the plasma membrane and on endosomes because Nef markedly recruits both AP-1 and AP-3 adaptors (27) and clathrin (Figure 1) on endosomal membranes in a CD4-independent manner. Together with the observation that Nef expression does not affect the clathrin-dependent internalization of TfR even in CD4-positive T cells (13), these results suggest that Nef does not perturb the dynamics of the clathrin-mediated pathway at the plasma membrane but rather uses this pathway to trigger internalization of specific surface receptors. In contrast, the Nef-induced membrane stabilization of AP-1 and AP-3 on early/sorting endosomes severely perturbs the function of the recycling compartment and leads to an inhibition of the recycling of CD4 and TfR to the cell surface (6,13). Interestingly, HIV-1 Nef binds very weakly to intact AP-2, whereas a relatively robust binding to intact AP-1 and AP-3 has been detected (5,14,15,24,51). These features may explain the differential impact of Nef on the AP-2 and AP-1/AP-3 pathways and suggest that Nef associates with these complexes through distinct molecular mechanisms.
The role of CCP localization on the function of Nef was investigated by testing the impact of Nef variants on CD4 endocytosis. In T lymphocytes, CD4 is primarily present at the cell surface and is only slowly internalized, but the expression of Nef strongly stimulates the endocytosis of CD4. As expected, the ability of Nef variants to reduce CD4 expression at the cell surface and to stimulate the internalization of CD4 correlated closely with their ability to accumulate in CCPs, suggesting that localization of Nef in CCPs and forming CCVs is required to induce the internalization of CD4. These results are in agreement with a recent study showing that Nef-induced downmodulation of CD4 in T lymphoid cells can be related to the clathrin-mediated endocytic pathway and is directly dependant on AP-2 expression (32).
The current molecular model for the action of Nef on CD4 suggests that Nef acts as a connector between the CD4 and the clathrin-associated AP-2 complex at the plasma membrane [reviewed by Piguet et al. (7)]. This connection would result in the targeting of CD4 to CCPs for rapid internalization via CCVs. Thus, Nef is expected to play a role similar to that of cargo-specific adaptors such as β-arrestin and ARH, which connect G protein-coupled receptors or LDL receptors to AP-2 (48,49). In this connector model, two distinct regions of Nef are involved in the recruitment of CD4 and in the binding to AP complexes. Whereas the recruitment of AP complexes is mediated by the C-terminal di-leucine motif of Nef (see above), the putative CD4-binding site is found in the N-terminal part of the protein and includes residue W57 (42). The fact that W57 mutants of Nef were affected in their ability to downregulate CD4 was then interpreted as an argument in favor of the connector model (7). However, our present observations show that the W57R mutant is not concentrated in clathrin-coated structures at the plasma membrane (Figure 6), in agreement with a previous study showing that mutation of W57 impaired colocalization of Nef with AP-2 complexes in fixed cells (35). Together, these results indicate that mutation of the W57 affects the interaction of Nef with both CD4 and the clathrin-dependent machinery. It remains to be determined if the W57 residue is directly involved in the interaction with AP complexes or if it is important for the general folding of the molecule as it was suggested by structural studies (52). The observation that the W57 mutants of Nef failed to localize in CCPs stresses that the results obtained with these mutants could not be simply interpreted as a consequence of a specific loss of interaction with CD4. However, the finding that the W57 residue of Nef is also important for efficient targeting in CCPs does not definitively argue against the Nef connector model but indicates that more specific Nef mutants are needed to properly evaluate this model.
The connector model has recently been challenged by the observations that the leucine motif of CD4, although required for Nef-induced internalization (3), is not required for the binding to Nef (53,54). These observations suggest an alternative model in which the leucine motif of CD4 participates directly in the binding to AP complexes during Nef-mediated downregulation. Interestingly, a recent study showed that Nef induces the relocalization of the CD4-associated kinase Lck from the plasma membrane toward intracellular compartments (55). One intriguing possibility is then that Nef may induce dissociation of CD4 from Lck, therefore allowing its internalization. However, the fact that the mislocalization of Lck was also observed with the LL/AA mutant of Nef (55) suggests that mislocalization of Lck and CD4 downregulation activities of Nef are not related events. Finally, in an alternative model, Nef functions as a general ‘troublemaker’ of the endocytic pathway affecting the intracellular trafficking of not only membrane proteins including of course CD4 but also many other plasma membrane-associated proteins (see introductory paragraphs). The latter model being not exclusive of the connector one, the way Nef is exactly acting on CD4 appears then far from being clarified.
Another possibility that is not mutually exclusive with the ‘connector’ function is that Nef may use the classical clathrin-dependent internalization pathway to reach the early/sorting endosomes. Once within the endosomal system, Nef induces morphological and functional disturbances that affect not only early/recycling endosomes but also late endosomes and multivesicular bodies (28–30). How Nef trafficks within the endosomal system to reach the perinuclear endosomal region where it is concentrated at steady state is not understood. In this regard, chimeras in which Nef is fused to the extracellular and transmembrane domains of an integral membrane protein, such as CD4 or CD8, are constitutively internalized, and the leucine motif of Nef is critical for this internalization (4,6,16,29). Furthermore, the ENTSLL sequence of HIV-1 Nef functions as an endocytic signal when appended to the cytoplasmic tail of a heterologous transmembrane protein (14,25). These findings provide definitive evidence that the leucine-based motif of Nef acts as an internalization signal per se. Here, the detection of Nef–GFP in at least 50% of forming CCVs likely en route to the early/sorting endosomes, confirms that Nef present at the plasma membrane uses the clathrin-mediated pathway to concentrate in the endosomal compartments, where it may promote the formation of a platform for assembly of HIV-1 virions by inducing an expansion of multivesicular endosomal structures.
In conclusion, our data show that Nef is present at the cell surface in both CCPs and forming vesicles. This distribution correlates with the Nef-induced stimulation of CD4 endocytosis and is strictly dependent on the di-leucine AP-binding motif of Nef. These findings confirm a model in which Nef initially uses the clathrin-mediated endocytic pathway to induce the rapid internalization of some transmembrane proteins, such as CD4, from the surface of HIV-1-infected cells.
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- Materials and methods
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Figure 1. Nef-DKQTLL mutant colocalizes with clathrin at the plasma membrane. HeLa cells transiently expressing the DKQTLL Nef substitution mutant (A) and DsRed-clathrin (B) were imaged by TIR-FM as in Fig. 2. Insets show higher magnification of a representative area and arrows stress colocalizing spots. Scale bar, 10 μm.
Figure 2. Distribution of the Nef-ERQPLL mutant within dynamic clathrin spot populations. Live HeLa cells expressing either wild-type (black bars) or ERQPLL (grey bars) GFP-tagged Nef fusions in combination with DsRed-clathrin were imaged by TIR-FM. The presence of Nef-GFP inside 40 CS (from more than 5 cells) from each CS population was determined using MetaMorph. Results are expressed as the percentage of clathrin spots of each population that contain Nef (see Fig. 3).
Figure 3: Expression of Nef does not increase the number of clathrin spots at the plasma membrane. HeLa cells transiently expressing wild-type (blue) or LL/AA (white) Nef GFP fusion together with dsRed-clathrin, or CD4-wt (red) or CD4- D cyt (grey) HeLa cell lines expressing Nef-GFP fusions together with DsRed-clathrin were imaged by TIR-FM. For each cell (n=4), the total number of clathrin spots present at the adherent membrane was quantified and then normalized to arbitrary surface unit.
Video 1: Time-lapse movie showing a disappearing spot imaged by TIR-FM which contained both clathrin (top) and Nef (bottom). ~ 0.3 s/frame.
Video 2: Time-lapse movie showing a laterally mobile spot imaged by TIR-FM which contained both clathrin (top) and Nef (bottom). ~ 0.3 s/frame.
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