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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

HIV-1 Nef, an essential factor in AIDS pathogenesis, boosts virus replication in vivo. As one of its activities in CD4+ T-lymphocytes, Nef potently retargets the Src family kinase (SFK) Lck but not closely related Fyn from the plasma membrane to recycling endosomes and the trans-Golgi network to tailor T-cell activation and optimize virus replication. Investigating the underlying mechanism we find Lck retargeting involves removal of the kinase from membrane microdomains. Moreover, Nef interferes with rapid vesicular transport of Lck to block anterograde transport and plasma membrane delivery of newly synthesized Lck. The sensitivity of Lck to Nef does not depend on functional domains of Lck but requires membrane insertion of the kinase. Surprisingly, the short N-terminal SH4 domain membrane anchor of Lck is necessary and sufficient to confer sensitivity to Nef-mediated anterograde transport block and microdomain extraction. In contrast, the SH4 domain of Fyn is inert to Nef-mediated manipulation. Nef thus interferes with a specialized membrane microdomain-associated pathway for plasma membrane delivery of newly synthesized Lck whose specificity is determined by the affinity of cargo for these sorting platforms. These results provide new insight into the mechanism of Nef action and the pathways used for SFK plasma membrane delivery.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Nef is a 25–34 kDa myristoylated accessory protein encoded by HIV-1, HIV-2 and SIV. Ex vivo, Nef enhances the single-round infectivity of virus particles and moderately accelerates virus spread over multiple rounds (Miller et al., 1994; Spina et al., 1994; Aiken and Trono, 1995; Schwartz et al., 1995). In vivo, Nef provides the virus with a strong replication advantage that is particularly pronounced during primary infection, where the presence of Nef elevates virus titres by more than two-logs (Kestler et al., 1991; Deacon et al., 1995; Kirchhoff et al., 1995). Consequently, Nef is critical for rapid disease progression in this physiological environment (Kestler et al., 1991; Deacon et al., 1995; Kirchhoff et al., 1995). Evidence for such a role of Nef as a pathogenicity factor is also provided by transgenic mice in which expression of Nef induces AIDS-like depletion of CD4+ T lymphocytes (Hanna et al., 1998; Rahim et al., 2009).

By virtue of interactions with host cell proteins, Nef induces a large variety of alterations in central intracellular transport and signalling pathways of HIV infected cells (Geyer et al., 2001; Kirchhoff et al., 2008). This includes modulating surface exposure of cell-surface receptors, such as MHC-I and II, CD4, chemokine receptors and co-stimulatory molecules such as CD80 and CD86, to evade host cell immune responses and prevent super-infection of infected cells respectively (Schwartz et al., 1996; Stumptner-Cuvelette et al., 2001; Michel et al., 2005; Chaudhry et al., 2007). Reduction of cell surface densities of these transmembrane receptors is achieved by distinct molecular mechanisms that affect endocytosis, anterograde transport, and/or stability of these molecules (Garcia and Miller, 1991; Schwartz et al., 1996; Stumptner-Cuvelette et al., 2001; Michel et al., 2005; Chaudhry et al., 2008). In addition, Nef affects the basal states of T cell activation and the responsiveness of T lymphocytes to TCR signalling (Baur et al., 1994; Simmons et al., 2001; 2005; Fackler and Baur, 2002) (see Abraham and Fackler, 2012 for review). This involves the inhibition of several TCR proximal signal transduction events such as induced actin remodelling, tyrosine phosphorylation and formation of signalling microcluster essential for signal initiation and transmission (Haller et al., 2006; 2007; Thoulouze et al., 2006; Abraham et al., 2012). Together these effects are thought to reduce activation-induced cell death and thus prolong survival of productively infected cells (Schindler et al., 2006). At the same time, Ras-Erk signalling in response to TCR engagement is specifically enhanced in infected T lymphocytes by Nef, an effect that appears to facilitate HIV-1 replication in primary human cells (Schrager et al., 2002; Pan et al., 2012). We recently reported that these selective effects of Nef on proximal and distal TCR signalling involve a common mechanism of host cell manipulation (Pan et al., 2012): As described first by Thoulouze and colleagues, Nef expression causes dramatic changes in the subcellular localization of the Src family kinase (SFK) Lck, which is retargeted from the plasma membrane to recycling endosomes (RE) and the trans-Golgi network (TGN) in the presence of the viral protein (Haller et al., 2006; Thoulouze et al., 2006; Arhel et al., 2009). Since Lck acts as master switch for TCR signalling at plasma membrane (PM) sites of TCR engagement (Salmond et al., 2009), this retargeting mechanism significantly contributes to the Nef-mediated disruption of early TCR signalling. Further characterization of the RE/TGN associated Lck pool revealed that Lck is catalytically active at these intracellular compartments and signals specifically to the Ras-Erk pathway to enhance virus replication (Pan et al., 2012). Despite assembly of RE/TGN associated active signalling complexes and removal of Lck from the PM, induction of the Ras-Erk pathway is not entirely uncoupled from TCR engagement in Nef expressing cells. This reflects the action of the SFK Fyn, which despite its close similarity with Lck, is not affected in localization and function by Nef (Pan et al., 2012). Nef thus employs a highly selective mechanism to tailor TCR signalling to the needs of HIV-1 by inducing specific changes in the intracellular transport of Lck. By which mechanism Nef affects the subcellular localization of Lck and how the viral protein distinguishes between Lck and Fyn in this context is unclear.

Plasma membrane localization of proteins is regulated by a complex equilibrium between a large array of specific intracellular transport pathways for anterograde delivery to and internalization from the PM. Most of the molecular principles that govern recognition of proteins as cargo for specific transport routes and their respective transport kinetics were established for transmembrane proteins. How the subcellular localization of peripheral membrane proteins is controlled is much less understood. SFKs constitute a class of such peripheral membrane proteins that exert important functions in fundamental cellular processes including growth, migration and survival (Parsons and Parsons, 2004). In CD4+ T lymphocytes, Lck and Fyn are the most abundant SFKs and both kinases act as key regulators of early TCR signal transduction (Salmond et al., 2009). SFKs are composed of four functional domains in which the SH1, SH2 and SH3 domains mediate kinase activity, recognition of phosphotyrosine residues, and protein interactions respectively (Boggon and Eck, 2004). The N-terminal SH4 domain determines the subcellular localization of SFKs and is responsible for their insertion into the inner leaflet of the PM. The first 18 amino acids of SH4 domains are sufficient for membrane anchoring, a process that strictly depends on lipid modification of specific receptor residues in the SH4 domain (McCabe and Berthiaume, 1999; Tournaviti et al., 2007). In the case of Lck and Fyn, this involves myristoylation at glycin 2 as well as palmitoylation of two acceptor cysteins. Triggered by cotranslational myristoylation, membrane insertion of SFK is thought to occur at the level of the Golgi (Wolven et al., 1997; Denny et al., 2000; Stegmayer et al., 2005; Tournaviti et al., 2007; 2009) and thus the site of initial palmitoylation (Rocks et al., 2010). Palmitoylation also mediates partitioning of SFKs into membrane microdomains (Webb et al., 2000; Sandilands et al., 2007). How SFKs are subsequently transported to the PM is largely unclear, but the degree of palmitoylation represents a critical determinant since dually palmitoylated SFKs use transport pathways distinct from those with one or no palmitoylation site (Sato et al., 2009). In the case of Lck, components of the transport machinery for PM targeting such as the protein MAL, the formin actin nucleator INF2, the adaptor protein Unc119, and the GTPase Rab11 have been identified (Gorska et al., 2004; 2009; 2010; Anton et al., 2008; Andres-Delgado et al., 2010). Whether Fyn relies on the same machinery and how these individual components facilitate PM targeting is unclear. Little investigated is also by which pathways SFKs are removed from the PM. As palmitoylation is reversible, rapid alterations in the palmitoylation status similar to those described for the Ras GTPase (Rocks et al., 2010) could be able to control SFK membrane association. For Lck, exchange rates between the PM and the cytosol in the millisecond range that are consistent with such a mechanism have been observed (Zimmermann et al., 2010). Whether and how Lck and Fyn are sorted from the PM to other subcellular compartments in a membrane-associated configuration is unclear.

The goal of the present study was to unravel which steps of Lck intracellular transport HIV-1 Nef affects to alter its steady state subcellular localization and function as well as to address how this mechanism allows Nef to distinguish between Lck and Fyn.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Nef causes accumulation of Lck at RE and the TGN and removes the kinase from lipid raft microdomains

As previously reported (Haller et al., 2006; Thoulouze et al., 2006; Pan et al., 2012), the Nef protein of the HIV-1 strain SF2 fused to GFP (Nef.GFP, a functional analogue of non-fusion Nef) retargets Lck in Jurkat T lymphocytes from the PM to REs and the TGN (see Fig. 1A–C for the effects of Nef on Lck subcellular localization, frequency of Lck retargeting and per cell magnitude of RE/TGN enrichment in the presence of Nef). While these earlier studies provided evidence that this retargeting induces a constitutively active signalling platform that facilitates HIV-1 replication, they did not provide insight into the mechanism by which Nef affects Lck trafficking and failed to explain why the subcellular localization of the closely related Src kinase Fyn is insensitive to Nef expression. Since Lck is incorporated into membrane microdomains by virtue of its myristoylated and palmitoylated N-terminal SH4 domain and Nef is known to alter membrane microdomain protein and lipid composition (Krautkramer et al., 2004; Simmons et al., 2005; Brugger et al., 2007; Rauch et al., 2008; Witte et al., 2008; Cui et al., 2012), we analysed whether Lck retargeting also affects its microdomain association. Detection of cross-linked, GM1 containing PM microdomains with fluorescently labelled cholera toxin (CTx) in Myc expressing control cells revealed a significant but incomplete co-distribution of Lck.GFP with the lipid raft marker (Fig. 1D, upper panel). Consistently and in line with reported results (Janes et al., 1999), flotation analysis for the detection of bulk membrane microdomains resistant to cold detergent extraction revealed an equivalent distribution of endogenous Lck as well as ectopically expressed Lck.GFP between soluble (S) and detergent-resistant membrane (DRM) fractions (Fig. 1E, left lanes). Expression of Nef.Myc did not alter the overall appearance of CTx cluster at the PM but caused a significant translocation of Lck.GFP from the PM to RE/TGN compartments. Importantly, the few Lck.GFP patches remaining at the PM in Nef expressing cells did not display any codistribution with CTx (Fig. 1D, lower panels). Similarly, Lck.GFP was quantitatively removed from DRM fractions in the presence of Nef (Fig. 1E, right lanes). DRM-associated Lck was detected as two distinct species that both migrated considerably slower than the single Lck band detected in soluble fractions. These differences in mobility might reflect specific post-translational modifications of Lck in DRMs such as phosphorylation and attempts to identify the molecular basis for this phenomenon are still ongoing. Of note, the presence of Nef does not affect overall membrane association of Lck but causes a redistribution of Lck from PM to RE/TGN membrane fractions (Pan et al., 2012). Nef-induced retargeting of Lck from the PM to RE/TGN is thus paralleled by a shift of the kinase from membrane microdomains to membrane fractions that are not resistant to extraction by cold detergent.

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Figure 1. HIV-1 Nef targets Lck to the trans-Golgi network (TGN) and recycling endosomes (RE). Analyses were conducted with Jurkat T cells transiently expressing GFP or Nef.GFP detecting endogenous Lck.

A. Representative confocal micrographs of Lck staining (red), GFP fluorescence and the merge channel. Scale bar = 10 μm.

B. Frequency of cells that display Lck RE/TGN accumulation. Depicted are mean values from three independent experiments ± SD with at least 100 cells analysed per condition.

C. Quantification of Lck distribution in single cells. Depicted are the percentages of the total per cell Lck signal detected in RE/TGN accumulation. Each symbol designates a value for an individual cell. Bars indicate the mean values of all cells analysed.

D. Clustering of lipid rafts. 24 h post-transfection, Jurkat T cells transiently coexpressing Lck.GFP with Myc or Nef.Myc were incubated with CTx conjugated with Alexa-555, and subsequently cross-linked with anti-CTx antibody. Cells were analysed by confocal microscopy and single representative sections are presented. The Nef.Myc positive cells were identified with Myc antibody staining in blue (not shown). The merge panel depicts the overlay of Lck (green) and Ctx (red) fluorescence signals.

E. DRM flotation analysis from Jurkat T cells co-transfected with Lck.GFP and RFP or Nef.RFP expression plasmids. The detergent-resistant membrane fraction (DRM, fraction 2) and the pooled non-raft fractions (S, fractions 7 and 8) were analysed by Western blotting for the distribution of Lck.GFP. TfR and Lat are markers for S and DRM fractions respectively.

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Clathrin-mediated endocytosis is dispensable for Lck retargeting by Nef

These effects by Nef on Lck localization could, in principle, result from enhanced internalization of PM-resident Lck pools or from a block of anterograde transport of Lck to the PM. To test this hypothesis we sought to interfere with clathrin-mediated internalization of cargo from the PM by expression of well-characterized dominant-negative variants of the GTP dynamin2 or the adaptor protein Eps15 (Fig. 2) in Jurkat T lymphocytes. Uptake of fluorescently labelled transferrin was used as control to monitor inhibition of clathrin-dependent endocytosis and indeed, transferrin internalization was only observed in cells expressing wt but not the dominant-negative mutants of Dyn2.GFP or Eps15.GFP. In cells expressing wt Dyn2 or Eps15 (indicated by green circles), expression of Nef.Myc (indicated by red asterisks) readily caused a redistribution of endogenous Lck from the PM to an enlarged intracellular membrane compartment. Expression of dn Dyn2 and in particular Eps15 caused slight alteration in the subcellular localization of Lck in the absence of Nef in that the kinase was detected in a more disperse intracellular compartment than observed without inhibition of clathrin-mediated internalization. This indicates that intracellular transport of Lck per se may involve pathways dependent on Dyn2 and Eps15. However, Nef still induced removal of Lck from the PM and enrichment in the less defined intracellular compartment in presence of these endocytosis inhibitors. While these results do not exclude a role for clathrin-independent internalization pathways, clathrin-mediated internalization does not appear as an essential step for Lck retargeting by Nef.

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Figure 2. Inhibition of clathrin- and/or dynamin-mediated endocytosis does not impair Lck retargeting by HIV-1 Nef. Jurkat T cells were transfected with expression plasmids for Nef.Myc together with plasmids encoding WT Dynamin.GFP, dominant-negative (DN) Dynamin.GFP (K44A), WT Eps15.GFP or DN Eps15.GFP. Transferrin 660 was fed to the cells for 30 min to monitor endocytosis. Successful transferrin uptake is reflected by bright intracellular fluorescence in large intracellular aggregates in the transferrin channel. Dotted lines in the transferrin channel indicate the position of GFP positive cells. Note that expression of DN dynamin or Eps15 fully abrogated transferrin uptake in transfected but not in untransfected neighbouring cells. Despite this block of endocytic uptake by DN dynamin or Eps15, RE/TGN targeting of Lck was still observed in cells positive for Nef.Myc (indicated by asterisks in the Lck channel).

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Nef blocks vesicular Lck movement

We next sought to use live-cell imaging to study Nef's effects on intracellular transport dynamics of Lck. Since this approach is technically challenging on suspension cells such as T lymphocytes, we asked whether Nef's effects on Lck subcellular localization are also observed in fibroblasts. To this end, the localization of Lck.Myc was analysed in Chinese hamster ovary (CHO) fibroblast clones inducible for expression of GFP or Nef.GFP (Stolp et al., 2009). While Lck displayed its typical PM localization in presence of GFP, Nef.GFP triggered in about 80% of cells analysed a potent relocalization to intracellular compartments that had previously been validated by immunoelectron microscopy to represent Golgi and RE membranes (Fig. 3A and B) (Pan et al., 2012). Lck retargeting by Nef in fibroblasts thus resembles in phenotype and magnitude what was observed in T lymphocytes and the CHO cell system was used further for live cell microscopy. To this end, RFP or Nef.RFP was expressed in CHO cells stably expressing Lck.GFP (Fig. 3C). In the presence of RFP, we observed highly dynamic movement of Lck and PM blebbing [the latter due to the presence of the SH4 domain in Lck (Tournaviti et al., 2007)] (Fig. 3C, upper panel, Movie S1). In sharp contrast, expression of Nef.RFP caused intracellular accumulation of Lck and virtually abrogated all dynamic transport events (Fig. 3C, lower panel, Movie S2). The motility of control proteins, such as ecotropic envelope of the Moloney murine leukaemia virus, was not affected by Nef (Movies S3 and S4).

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Figure 3. HIV-1 Nef blocks vesicular transport of Lck.

A. Analyses were conducted with stable CHO cells after induction of transgene expression (GFP or Nef.GFP) and transfection with an expression plasmid for Lck.Myc. Representative confocal micrographs of Lck.Myc staining, GFP fluorescence and the merge channel.

B. Frequency of cells that display Lck RE/TGN accumulation in (A). Depicted are mean values from three independent experiments ± SD with at least 100 cells analysed per condition.

C. Still images of the time-lapse Movies S1 and S2. CHO cells stably expressing Lck.GFP were transfected with expression constructs for RFP or Nef.RFP and subjected to spinning disk confocal live-cell imaging. The top panels depict the RFP distribution at the beginning of the movie. The red arrows indicate the trajectory of an individual Lck.GFP containing vesicle. Note that vesicles cover significant distances within 16 s in the presence of RFP, while they remain static when Nef.RFP is coexpressed. Scale bar = 10 μm.

D. Nef induces rapid retargeting of Lck. Shown are still pictures from movies (see Movie S5 for Nef.RFP). CHO cells stably expressing Lck.GFP were microinjected with expression plasmids for RFP or Nef.RFP and imaged for 120 min (1 frame 5 min−1). Fluorescent dextran-cascade blue was co-injected to identify microinjected cells prior to expression of the RFP fusion proteins encoded by the microinjected plasmids (see panel Dextran 30 min). The RFP channel is shown for time points at the beginning and the end of the sequence (RFP). The remaining panels (Lck.GFP) depict the distribution of Lck.GFP in the identical cells analysed for dextran and RFP at the indicated time points post-microinjection. Note that the Lck.GFP localization remains largely unaltered after expression of RFP, while Nef.RFP rapidly induces intracellular accumulation of Lck.GFP. Scale bar = 10 μm.

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We next sought to determine how quickly Nef can turn Lck's subcellular localization from the normal steady-state distribution to a pronounced enrichment at RE/TGN compartments. We therefore induced expression of an RFP control or Nef.RFP expression by microinjecting the respective expression plasmids into the CHO cells stably expressing Lck.GFP (Fig. 3D). Expectedly, expression of RFP did not impact significantly on the subcellular distribution of Lck.GFP that remained associated with the PM and intracellular vesicles and was found diffusely in the cytoplasm up to 110 min post-microinjection (Fig. 3D, upper panel). In contrast, microinjection of an expression plasmid for Nef.RFP caused rapid redistribution of Lck.GFP to a perinuclear compartment detectable as soon as 50 min post-injection. This Nef-induced retargeting of Lck.GFP was completed 110 min post-injection (Fig. 3D, lower panel and Movie S5). Together these results suggest that the subcellular localization of Lck is determined by highly dynamic intracellular transport events and that Nef action very rapidly leads to RE/TGN enrichment of the majority of the cellular Lck pools.

Interference of anterograde Lck transport by Nef

Since the results described above suggested that Nef may block anterograde transport of Lck, we next specifically followed the fate of newly synthesized Lck in the presence of Nef after microinjecting an expression plasmid for Lck.RFP into CHO cells that express GFP or Nef.GFP (Fig. 4). In GFP-positive control cells, Lck expression was detected at intracellular membranes and in the cytoplasm 1 h post-injection. This localization matured to the steady-state distribution at intracellular membranes and the cell surface within 4 h post-injection in over 40% of cells analysed (Fig. 4A, upper panel, see Fig. 4B for quantification), defining this as the minimal time for efficient PM delivery of newly synthesized Lck. In sharp contrast, Nef.GFP caused a pronounced perinuclear accumulation of newly synthesized Lck already early post-injection and subsequently prevented anterograde transport of this perinuclear pool of Lck to the PM in virtually all cells analysed (Fig. 4A, lower panel, see Fig. 4B for quantification). We concluded that Nef potently blocks the anterograde transport of newly synthesized Lck to the PM.

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Figure 4. HIV-1 Nef induces Lck RE/TGN accumulation by blocking the anterograde transport of Lck to the plasma membrane.

A. Representative micrographs of CHO cells stably expressing GFP or Nef.GFP after microinjection with an expression plasmid for Lck.RFP. Depicted are the localizations of Lck.RFP at the indicated times post-injection. Note that Nef.GFP prevents the transport of newly synthesized Lck.RFP to the PM. Dotted lines in the lower panel indicate cell circumferences. Scale bar = 10 μm.

B. Quantification of the experiment shown in (A). Microinjected cells fixed at the indicated time points were analysed by microscopy and Lck.RFP localization was categorized in predominantly present at the PM (PM) or in intracellular accumulations (accumulation). Cells with cytoplasmic distribution such as in the presence of GFP at 1 h were not considered for these two categories and are not plotted. Results are from the analysis of over 90 cells per time point and condition.

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Nef-mediated RE/TGN targeting of Lck depends on the Lck N-terminus

Our previous results indicated that Nef alters the subcellular localization of Lck in a manner depending on its own association with cellular membranes and via interactions with SH3 domains (Pan et al., 2012). To gain insight into the underlying mechanism, we next attempted to map the determinants in Lck that govern the sensitivity of its subcellular localization for the presence of Nef and analysed a panel of Lck mutants (Fig. 5). Since Nef was reported to physically associate with Lck via PxxP-SH3 interactions (Greenway et al., 1996), we determined if the SH3 domain of Lck was required for retargeting. Consistent with the finding that TGN localization of Nef itself is not required to induce intracellular accumulation of Lck (Pan et al., 2012), the W97A Lck mutant, which no longer undergoes SH3 domain-mediated protein interactions (Denny et al., 1999), was retargeted by Nef as efficiently as Lck WT (Fig. 5). The viral protein thus does not rely on colocalization or direct SH3 domain interaction with Lck to alter the subcellular localization of the kinase. Residues governing the function of Lck's SH2 domain [R154 (Xu and Littman, 1993)], its kinase activity [Y505, Y394 (Ostergaard et al., 1998), K273 (Xu and Littman, 1993)], or regulation thereof [S42, S59 (Winkler et al., 1993)] were also dispensable for its sensitivity to Nef. Only Lck mutants that lacked membrane anchoring due to loss of the myristoylation (G2A) site in the N-terminal SH4 domain (Resh, 1994) displayed a homogenous cytoplasmic subcellular distribution and failed to be retargeted by Nef.

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Figure 5. Molecular determinants of Nef-mediated RE/TGN targeting of Lck.

A. Schematic representation of Lck; SH, Src homology.

B. Representative confocal micrographs of Jurkat T cells transiently expressing the indicated Lck mutant proteins when coexpressed with RFP or Nef.RFP. Shown is the Lck channel and merged pictures of Lck.GFP (green) and RFP or Nef.RFP (red). Scale bar = 10 μm.

C. Frequency of cells that display Lck RE/TGN accumulation. Depicted are mean values from three independent experiments ± SD with at least 100 cells analysed per condition.

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Since these results suggested membrane anchorage of Lck as the sole determinant for Nef-mediated retargeting, we asked next whether the membrane anchor of Lck is also sufficient for recognition by the retargeting mechanism. To this end, the first 18 amino acids of Lck that comprise its membrane anchoring SH4 domain in the absence of additional protein interaction surfaces were expressed in fusion with GFP (Lck N18.GFP). In presence of an RFP control, the Lck SH4 domain efficiently targeted GFP to the PM and an intracellular compartment, resulting in a subcellular localization that was indistinguishable from that of the full-length kinase (Fig. 6B, upper left panels). Importantly, Nef.RFP potently targeted Lck N18.GFP to RE/TGN compartments in over 70% of cells analysed (Fig. 6B, upper right panels, Fig. 6C) and caused an approximately fivefold enrichment at this subcellular site (Fig. 6D). This retargeting of Lck N18.GFP was observed despite the persistence of PM-resident pools, which are typically removed by Nef in the case of Lck WT. This different response of Lck N18.GFP and Lck.GFP presumably reflects that the endocytosis signals that mediate internalization of Lck.GFP or not located in the SH4 domain and thus are not included Lck N18.GFP.

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Figure 6. HIV-1 Nef targets Lck.N18 but not Fyn.N18 to RE/TGN.

A. Sequence alignment of the first 18 amino acids of Lck and Fyn. G, myristoylation site; C, palmitoylation site; S, phosphorylation site.

B. Representative confocal micrographs of Jurkat T cells transiently expressing Lck.N18.GFP or Fyn.N18.GFP together with RFP or Nef.RFP. Shown are individual channels and merged pictures.

C. Frequency of cells that display RE/TGN accumulation. Depicted are mean values from three independent experiments ± SD with at least 100 cells analysed per condition.

D. Quantification of N18.GFP distribution in single cells. Depicted are the percentages of the total per cell N18.GFP signal detected in RE/TGN compartments. Each symbol designates a value for an individual cell. Bars indicate the mean values of all cells analysed.

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SH4 domain specificity governs membrane microdomain association and sensitivity to Nef-mediated RE/TGN targeting

Having defined the SH4 domain of Lck as necessary and sufficient for Nef-induced retargeting, we next analysed whether the specificity of this mechanism for Lck but not Fyn is also determined by the SH4 domain (Fig. 6A). The subcellular localization of a fusion protein of the first 18 amino acids of Fyn with GFP (Fyn N18.GFP) resembled that of Lck N18.GFP and localized to the PM and intracellular compartments in control cells. In contrast to Lck N18.GFP, however, Fyn N18.GFP was unaffected by Nef expression in the large majority of cells (Fig. 6B, lower panels, Fig. 6C and D). This result for Fyn N18.GFP largely reflected the scenario observed with full-length Fyn.GFP whose subcellular localization remained unaffected by Nef expression in over 75% of cells analysed and that was still present in considerable amounts at the PM in the minority of cells that displayed slight intracellular enrichment in the presence of the viral protein (Fig. S1). To further dissect the difference between the Lck and Fyn SH4 domains, functionally relevant residues in the SH4 domain were mutated in the context of full-length Lck.GFP (Fig. 7). Individual mutation of the two palmitoylation acceptor cysteins to serine (C3S or C5S) did not affect the subcellular localization of Lck.GFP in control cells and did not affect Nef's ability to remove Lck from the PM and trigger RE/TGN enrichment of the kinase (Fig. 7A and B). Simultaneous mutation of both palmitoylation acceptor sites (C3SC5S) resulted in less stringent PM tethering of Lck and largely abrogated retargeting by Nef. This phenotype resembled that observed with the myristoylation site mutant of Lck and reflects the reduced membrane affinity of the mutant Lck protein. While this confirms the requirement for membrane insertion for PM delivery of Lck, no further conclusions on the mechanism of Nef action could by drawn from this Lck mutant. In search for determinants that confer specificity between Lck and Fyn it has to be noted that a main difference between the Lck and Fyn SH4 domains consists of two potential phosphorylation sites (serines at position 6 and 7 present in Lck but not Fyn) (Fig. 6A). Mutating these sites individually did not significantly affect Lck's subcellular localization and sensitivity to retargeting by Nef. The S6AS7A double mutation, however, resulted in a protein distributed at the PM, intracellular compartments and the cytoplasm that was not further enriched at RE/TGN by Nef. While these results define the two serines in Lck as an essential determinant of sensitivity to Nef, introducing two serines into the Fyn SH4 domain failed to render it responsive to Nef expression (data not shown). The two serines in the Lck SH4 domain are therefore necessary but not sufficient to confer sensitivity to Nef.

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Figure 7. The SFK SH4 domain determines kinase sensitivity to Nef mediated RE/TGN retargeting and DRM extraction.

A. Representative confocal micrographs of Jurkat T cells transiently expressing the indicated Lck.GFP proteins together with RFP or Nef.RFP. Shown is the Lck channel and merged pictures of Lck.GFP (green) and RFP or Nef.RFP (red). Scale bar = 10 μm.

B. Frequency of cells that display Lck RE/TGN accumulation. Depicted are mean values from three independent experiments ± SD with at least 100 cells analysed per condition. DRM flotation analysis from Jurkat T cells transiently expressing the indicated proteins (C), or FnyN18-Lck.GFP, FynN18.GFP, LckN18.GFP or Lck.GFP when coexpressed with RFP or Nef.RFP (D). The detergent-resistant membrane fraction (DRM, fraction 2) and the pooled non-raft fractions (S, fractions 7 and 8) were analysed by Western blotting. TfR and Lat are markers for S and DRM fractions respectively.

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Since the above results together suggested the SH4 domain as critical target of Nef action in SFK transport, we next replaced the Lck SH4 domain with that of Fyn in the context of full-length Lck (FynN18-Lck). In the absence of Nef, the subcellular localization of this FynN18-Lck chimera was indistinguishable from that of Fyn N18.GFP. Moreover, FynN18-Lck was entirely resistant to relocalization by Nef. These results reveal that the SH4 domain is sufficient to govern functional membrane interactions in the context of full-length SFKs and define the SH4 domain as the sole determinant for the selection of cargo to the Nef sensitive anterograde transport pathway.

Since Nef-mediated retargeting of Lck is paralleled by removal of the kinase from membrane microdomains (see Fig. 1), we probed all Lck mutants tested above for their association with DRMs by membrane flotation (Fig. 7C). In the absence of Nef, wt Lck and the Nef-sensitive S6A and S7A mutants were detected in DRM fractions with similar efficiency. The wt SH4 domains of Lck and Fyn targeted GFP to DRMs with similar efficiency and also replacing the Lck SH4 domain with that of Fyn in the context of the full-length kinase did not abolish DRM association. In contrast and reflecting their reduced overall membrane affinity, the Nef-insensitive mutants C3SC5S and S6AS7A lacked detectable DRM association. Since these results were compatible with a scenario in which microdomain insertion represents a prerequisite for retargeting by Nef, we further analysed the connection between microdomain association and retargeting by testing the effects of Nef on DRM association of Lck N18.GFP and Fyn N18.GFP (Fig. 7D). In line with its ability to reflect key aspects of Nef-mediated retargeting, Lck N18.GFP was extracted from DRMs by Nef as efficiently as full-length Lck. In contrast, the distribution of Fyn N18.GFP between soluble and DRM resistant fractions was unaffected by Nef expression and similar results were obtained with FynN18-Lck.GFP. RE/TGN retargeting of Lck by Nef thus appears to be tightly linked to removal of the kinase from membrane microdomains. Together these results imply that Nef disrupts anterograde transport of specialized membrane microdomains to which selected Src kinases associate as cargo based on the specificity of their respective SH4 domain.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In HIV-1 infected T lymphocytes, the viral protein Nef selectively modulates intracellular transport of the SFK Lck but not its close relative Fyn to create T cell activations states optimal for HIV-1 propagation. The goal of this study was to gain insight into (i) the SFK transport processes that are inhibited by Nef and (ii) the determinants that distinguish Lck and Fyn with respect to their sensitivity to Nef-retargeting. The results presented provide evidence for a potent block of Nef on anterograde transport of newly synthesized Lck to the PM that leads to an accumulation of the SFK in RE/TGN compartments. This shift in intracellular trafficking of Lck is paralleled by the exclusion of Lck from DRM microdomains, while overall membrane association is preserved. Surprisingly, the SH4 domain membrane anchor of Lck was identified as necessary and sufficient for these Nef-induced changes in intracellular transport and membrane partitioning properties of Lck. These findings support the model that PM delivery of Lck is achieved by incorporation into specialized membrane microdomain platforms at the levels of the Golgi that mediate forward transport to the PM. In this scenario, Nef prevents Lck sorting via these microdomains to preclude PM delivery of the kinase, thereby causing enrichment of Lck at RE and the TGN.

The results presented identify a potent block of Nef on the anterograde transport of Lck. Since de novo expression of Nef resulted in the quantitative retargeting of Lck from the PM to RE/TGN compartments within 110 min, the turnover of PM Lck would have to be extremely high in order to allow this mechanism to cause such drastic changes in overall subcellular localization. Indeed, PM association of Lck is subject to rapid on and off rates (Zimmermann et al., 2010); however, due to the availability of cytosolic Lck for reinsertion, this does not necessarily result in net removal of Lck from the PM. It is thus plausible that active transport pathways remove Lck from the PM. Our results indicate that this internalization pathway is distinct from clathrin-mediated endocytosis and preliminary results indicate that also inhibition of the Arf6 GTPase, which is involved in a broad range of endocytic uptake pathways, does not prevent Nef-induced RE/TGN targeting of Lck (data not shown). However, more precise estimation of the relative contribution of internalization to the effects of Nef on Lck trafficking will have to await the identification of the specific internalization route at work. Irrespective of its nature, the Lck internalization pathway will likely be coupled to recycling pathways back to the PM. At this recycling step, Nef may interfere with PM transport via the same mechanism used for newly synthesized Lck. As the most abundant early viral protein expressed upon HIV-1 infection of CD4+ T lymphocytes, this combined inhibition of biosynthetic and recycling anterograde transport would enable Nef to rapidly and efficiently retarget all pools of Lck to optimize virus replication. Similar mechanisms are employed by the HIV-1 protein Vpu to reduce plasma membrane delivery and thus overall cell surface levels of the host cell restriction factor CD317/tetherin (Dube et al., 2009; Schmidt et al., 2011; Sauter et al., 2012). Anterograde host cell transport routes thus emerge as a central target of pathophysiological alterations in the context of HIV-1 infection.

The precise molecular mechanism by which Nef exerts this novel activity on PM delivery of Lck remains to be delineated. Previous mapping of the determinants in Nef that mediate Lck retargeting revealed that it does not rely on physical contact of Nef with Lck and does not even require localization of the viral protein to Lck-positive compartments (Witte et al., 2008; Pan et al., 2012). In an indirect manner, Nef may thus target any component of the active transport machinery that facilitates Lck PM delivery such as Unc119, Rab11, IFN2 or MAL. Notably, coexpression of Nef with the Unc119 adaptor released Lck from RE/TGN compartments into the cytosol, but did not allow for proper PM targeting of the kinase (Pan et al., 2012). This suggests that protein components of the transport machinery can recognize Lck as cargo in the presence of Nef. Furthermore, Nef action on this machinery is unlikely to account for the induced changes in microdomain partitioning of Lck. We therefore favour a model in which the prevention of microdomain insertion of Lck is the critical step by which Nef prevents PM delivery of the kinase. In this scenario, in the presence of Nef, non-microdomain associated Lck is still recognized by the Unc119 machinery, but is not targeted to the PM as this delivery depends on insertion in these specialized microdomains. How then does Nef prevent microdomain insertion of Lck following its biosynthesis? Given that the effect of Nef on Lck is indirect and does not involve loss of membrane association of the kinase, we suggest that Nef may be acting on the level of the microdomain. Nef is known to alter protein and lipid composition of membrane microdomains (Krautkramer et al., 2004; Simmons et al., 2005; Brugger et al., 2007; Rauch et al., 2008; Cui et al., 2012). These proteomic and lipidomic analyses were conducted with bulk preparations of DRMs and some of the effects observed were relatively subtle. Conceivably, effects of Nef may be much more pronounced when analysing specifically the specialized microdomains devoted to PM delivery of Lck. Such alterations may well reduce the affinity of Lck for these domains, actively prevent its inclusion, or interfere with the formation of these specialized domains altogether. Determination of the proteome and lipidome of these microdomains represents an important future task and Nef will serve as useful tool to define components with direct functional relevance for Lck PM delivery.

One important result of this study is that the SH4 domain of Lck not only determines the sensitivity of the SFK to Nef-mediated anterograde transport block and reduction of microdomain association, it also defines the specificity between Lck and Fyn, the latter of which is inert to effects of Nef. These findings further support the concept that cargo selection of the Nef-sensitive transport occurs at the level of membrane microdomain insertion and predicts that Lck and Fyn are transported to the PM via separate membrane microdomain dependent pathways. This scenario is consistent with previous reports on fundamental differences in both transport routes (Sato et al., 2009) and the recent observation that Lck and Fyn partition into distinct membrane microdomains (Ballek et al., 2012). The concept of specialized membrane microdomains as specific anterograde transport platforms, however, raises the interesting questions how SH4 domains can probe the environment to govern targeting to or exclusion from specific microdomains and how two closely related SH4 domains such as those of Lck and Fyn can exert opposing activities. Acylation is clearly a prerequisite for efficient membrane interactions and microdomain insertion of both SH4 domains. Since myristoylation and palmitoylation patterns are identical between Lck and Fyn and Nef does not alter the steady-state acylation level of Lck (data not shown), these modifications are unlikely to explain the different biological properties of Lck and Fyn SH4 domains. Another key difference may consist of the presence of two potential phosphoacceptor serines in the Lck SH4 domain that are absent in Fyn. Notably, phosphorylation of N-terminal serines by protein kinase C (PKC) isoforms enhances membrane interactions and biological activity of Lck (Yasuda et al., 2000) as well as Nef (Wolf et al., 2008). We recently identified PKCalpha as a critical component of PM delivery of the SH4 domains of the Leishmania protein HASPB and the SFK Yes (Ritzerfeld et al., 2011), suggesting PKC-mediated phosphorylation as a general regulatory mechanism of SH4 domain membrane interactions and intracellular transport. Moreover, SH4 domain trafficking appears to be regulated by dynamic phosphorylation/dephosphorylation cycles (Tournaviti et al., 2009) and the outcome of this regulation may be SH4 domain specific as a function of, e.g. the specific micro-environment. It would thus be plausible that phosphorylation enables the Lck SH4 domain to recognize the specific lipid and protein composition of membrane microdomains devoted for PM transport, while the Fyn SH4 domain couples to a distinct transport pathway that is preferentially recognized by unphosphorylated SH4 domains. The fact that the two phosphoacceptors in the Lck SH4 domain were found to be critical for membrane association per se and their insertion into the Fyn SH4 domain failed to render it sensitive to Nef prevents us from drawing firm conclusions on the role of phosphorylation for Nef-mediated Lck retargeting. The Fyn SH4 domain can be methylated at two lysine residues that are absent from the Lck domain (Liang et al., 2004), potentially providing an additional mechanism for the generation of SH4 domains with distinct biophysical properties. Finally, Nef also triggers TGN retargeting of the myeloid-specific SFK Hck (Hung et al., 2007; Hiyoshi et al., 2008; 2012), suggesting that similar mechanisms as employed for Lck are at work. Nef affects preferentially the localization of the cytoplasmatic p56 Hck isoform that is myristoylated and palmitoylated while effects are less pronounced on the lysosomal p59Hck isoform whose SH4 domain is distinct from that pf p56Hck and lacks a palmitoylation site (see Guiet et al., 2008 for review on Hck isoforms). The Hck p56 SH4 domain contains potential phosphorylation and methylation sites but whether they are functional and subject to regulation by Nef remains to be determined. Dissecting the precise molecular determinants that govern the specificity for the distinct anterograde transport pathways of individual SFKs represents an important next goal towards the identification of these transport pathways and the inhibitory mechanism of Nef on Lck PM delivery.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Cells, reagents, and plasmids

Jurkat T cells and CHO were cultivated in RPMI 1640 and DMEMα plus GlutaMAX-I, supplemented with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin (all from Invitrogen). Transferrin from human serum conjugated with Alexa-647, cascade-blue fluorescent Dextran and Cholera toxin (CTx) conjugated with Alexa-555 fluorescent dye were purchased from Invitrogen. The following antibodies were used: mouse anti-Lck (clone 3A5) (Santa Cruz), rabbit anti-c-Myc (Santa Cruz), rabbit anti-Lat (Upstate Biotechnology), rabbit anti-CTx (Sigma), mouse anti-transferrin Receptor (ZYMED Laboratories) and mouse anti-GFP (Sigma). Secondary goat anti-mouse Alexa Fluor 568 and anti-rabbit Alexa Fluor 350-conjugated antibodies were purchased from Invitrogen. The expression constructs for HIV-1 SF2 Nef WT fused to GFP or RFP as well as the respective plasmids encoding for GFP or RFP were described earlier (Haller et al., 2006; Rauch et al., 2008). Lck-K273AY505F and Lck-Y505F plasmids were provided by Rafick-Pierre Sekaly (Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada) (Sharif-Askari et al., 2007). Lck-W97A and Lck-R154K expression plasmids were received from Marietta L. Harrison (Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University) (Janes et al., 1999). All the other Lck mutants were cloned into pEGFP-N1 by site-directed mutagenesis. To obtain FynN18-Lck.GFP, nucleotides 1–54 of lck were replaced with nucleotides 1–54 of fyn.

Transfection of Jurkat cells

Jurkat T cells (5 × 106–1 × 107) were transfected with 15–80 μg of total plasmid DNA via electroporation (850/950 μF, 250 V; Bio-Rad GenePulser).

Immunofluorescence analyses

For T cells, microscope cover glasses (Marienfeld) were prepared by incubation in 0.01% poly-l-lysine (Sigma) solution at 4°C overnight. Cells were plated on the cover glasses, incubated for 5 min, and subsequently fixed for at least 15 min, by directly adding PBS-3% paraformaldehyde. For CHO cells, the cells were fixed with PBS-3% paraformaldehyde at 24 h post-transfection or after the indicated time points post-microinjection. After permeabilization with PBS–0.1% Triton X-100 for 1–5 min, the cells were blocked with PBS–1% bovine serum albumin for 30 min. Indirect immunofluorescence was performed by incubating cells with primary antibodies for 2 h at RT. (anti-Lck 1:50, anti-Myc 1:200). After being washed with PBS, fluorochrome-labelled secondary antibodies (1:2000) were added for 1 h. Cover glasses were mounted in LinMount (Linaris) or ProLong® Gold Antifade Reagent and analysed with a six-line spinning disk confocal microscope (Perkin-Elmer). Images were taken by using a 100× oil immersion objective lens and processed by using Adobe Photoshop.

Image quantification

For quantification of frequencies of observed phenotypes such as intracellular accumulation or cytoplasmic dispersion, transfected cells were judged microscopically for presence or absence of the respective phenotype. More than 100 positive and randomly chosen cells were counted per experiment by two independent members of the lab without disclosing the identity of the samples. For intensity quantification, pixel intensities of images taken with identical parameters and exposure times were measured by ImageJ software. For quantification of total per cell levels, maximum projection pictures were taken and the integrated total pixel intensity of transfected cells was plotted relative to the average intensity of 10 untransfected neighbouring cells following subtraction of unspecific background signals. For quantification of subcellular localization, areas of interest were defined using the ImageJ software as described (Pan et al., 2012). Signal intensities in areas of interest were plotted relative to the total per cell signal following background subtraction.

Microinjection

CHO cells, which were grown on cover glasses and induced for transgene expression, were microinjected into their nuclei with an AIS 2 microinjection apparatus using pulled borosilicate glass capillaries in principle as reported (Fackler et al., 1999; Schmidt et al., 2011). Plasmids encoding Lck.RFP, Nef.RFP or RFP were mixed in water at concentrations of 10 ng μl−1. Following microinjection, cells were cultured for various times to allow protein expression and trafficking. At the indicated time points cells were either fixed with PBS-4% paraformaldehyde and subjected to microscopic analysis or imaged live. For live cell imaging, microinjected cells were identified by cascade-blue fluorescent dextran (Invitrogen) which was added to the microinjection solution.

Lipid raft clustering

The raft clustering was performed using modifications of a previously described protocol (Giese et al., 2006). Jurkat T cells were cotransfected with pEGFP-N1-Lck and pEF-Myc or pEF-Nef.Myc plasmids. 24 h post-transfection, the cells were incubated with Alexa 555-conjugated CTx (25 μg ml−1) in 0.1% BSA/PBS for 30 min on ice after washing with PBS for 3 times. The cross-linking was performed by incubation with anti-CTx antibody (1:200) for 20 min on ice. After 8–10 min incubation at 37°C, cells were seeded on poly-l-lysine-coated glass coverslips, fixed with PBS 3% paraformaldehyde, washed and mounted with LinMount (Linaris). Fluorescence microscopy images were acquired with a six-line spinning disk confocal microscope (Perkin-Elmer). Images were taken by using a 100× oil immersion objective lens and processed by using Adobe Photoshop.

Detergent resistant membrane flotation

Jurkat Tag cells were transfected with Lck.GFP mutants and Nef.RFP expressing plasmids. At 48 h post-transfection, cells were lysed in ice-cold TXNE (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, protease inhibitors) and incubated for 20 min on ice. After homogenization by 60 passages through a pipette tip, lysates were adjusted to 60% Optiprep (Life Technologies, Karlsruhe, Germany) and deposited in SW60 centrifuge tubes. Samples were overlaid with 2.5 ml of 28% Optiprep in TXNE and with 0.6 ml of TXNE and then centrifuged for 3 h at 35 000 r.p.m. at 4°C. 500 μl fractions were collected from the top with the second and the last two fractions corresponding to the DRM and soluble (S) fractions respectively. Aliquots (15 μl) of both fractions were analysed by Western blotting.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We are grateful to the Nikon Imaging Center for access and service, to Nadine Tibroni for expert technical help, and to Rafick-Pierre Sekaly and Marietta L. Harrison for the kind gift of reagents. This project is supported by the Deutsche Forschungsgemeinschaft (TRR83 project 5 to WN and project 15 to OTF). OTF and WN are members of the cluster of excellence EXC81.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
cmi12148-sup-0001-si.eps2155K

Fig. S1. HIV-1 Nef does not affect subcellular localization of full-length Fyn.GFP.

A. Representative confocal micrographs of Jurkat T cells transiently expressing full-length Lck.GFP or Fyn.GFP together with RFP or Nef.RFP. Shown are individual channels and merged pictures.

B. Frequency of cells that display RE/TGN accumulation. Depicted are mean values from three independent experiments ± SD with at least 100 cells analysed per condition.

cmi12148-sup-0002-si.avi9942K

Movie S1. CHO cells which stably express Lck.GFP were transfected with a RFP expression plasmid. Lck vesicular and tubular movements were recorded for 3 min, 1 frame s−1, and is shown in a 16 s movie.

cmi12148-sup-0003-si.avi8064K

Movie S2. CHO cells which stably express Lck.GFP were transfected with a Nef.RFP plasmid. Lck movement was recorded for 3 min, 1 frame s−1, and is shown in a 16 s movie.

cmi12148-sup-0004-si.avi1017K

Movie S3. CHO cells which stably express GFP were transfected with an expression plasmid for MLV-Env.mCherry. MLV-Env trafficking was recorded for 3 min, 1 frame s−1, and is shown in a 16 s movie.

cmi12148-sup-0005-si.avi919K

Movie S4. CHO cells which stably express Nef.GFP were transfected with an expression plasmid for MLV-Env.mCherry. MLV-Env trafficking was recorded for 3 min, 1 frame s−1, and is shown in a 16 s movie.

cmi12148-sup-0006-si.AVI4106K

Movie S5. CHO cells which stably express Lck.GFP were microinjected with a solution containing a Nef.RFP expression plasmid solution and Dextran-cascade blue. The Dextran-cascade blue indicated the microinjected cells. The Lck.GFP in positive cells was monitored for 2 h, 1 frame 5 min−1 and is shown in a 8 s movie.

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