Human Immunodeficiency Virus Type 1 Virological Sy
http://www.100md.com
病菌学杂志 2005年第18期
The Sir William Dunn School of Pathology, University of Oxford, Oxford OX13RE, United Kingdom
ABSTRACT
Human immunodeficiency virus type 1 (HIV-1) can spread directly between T cells by forming a supramolecular structure termed a virological synapse (VS). HIV-1 envelope glycoproteins (Env) are required for VS assembly, but their mode of recruitment is unclear. We investigated the distribution of GM1-rich lipid rafts in HIV-1-infected (effector) T cells and observed Env colocalization with polarized raft markers GM1 and CD59 but not with the transferrin receptor that is excluded from lipid rafts. In conjugates of effector T cells and target CD4+ T cells, GM1, Env, and Gag relocated to the cell-cell interface. The depletion of cholesterol in the infected cell dispersed Env and GM1 within the plasma membrane, eliminated Gag clustering at the site of cell-cell contact, and abolished assembly of the VS. Raft integrity is therefore critical for Env and Gag coclustering and VS assembly in T-cell conjugates.
TEXT
Efficient spread between T cells by the retroviruses human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 can take place in vitro via the formation of a virological synapse (VS) (3, 17). Previous studies have demonstrated that this mode of viral dissemination is productive and more efficient than that achieved by infection with cell-free virions (25, 31). HIV-1 dissemination via VS may contribute to the rapid spread of HIV-1 within secondary lymphoid tissues in vivo, particularly during early infection (5). Similarly, HIV-1-infected or uninfected but pulsed dendritic cells can efficiently transfer virus to T cells via a synapse (2, 21, 26), and this may represent an important pathway of viral dissemination after mucosal inoculation (28). Furthermore, the movement of HIV-1 across an adhesive junction may help to protect the virus from elements of the humoral immune response, such as antibodies and complement.
The HIV-1 VS assembles at the site of contact between an infected (effector) and receptor-expressing (target) cell and is exemplified by the recruitment of the viral receptors (CD4 and a chemokine receptor, CXCR4 or CCR5) and adhesion molecules on the target cell and of viral Env and Gag and cognate adhesion molecules on the effector cell (16, 17, 26). The assembly of these supramolecular complexes relies on engagement of the viral receptors by Env and on a functional actin cytoskeleton in the target cell (16). The recruitment of Env and Gag to the intercellular junction is followed by viral antigen transfer across the synapse into the target cell (16), which has been shown by others to lead to rapid de novo reverse transcription (8, 18, 31).
HIV-1 particles assemble under and bud through specific plasma membrane domains termed lipid rafts or related raft-like structures (6, 9, 15, 20, 22, 24). Rafts are biochemically and biophysically distinct regions of plasma membrane that are rich in cholesterol and sphingomyelin, maintain an ordered structure, and contain proteins important in immune cell signaling via the immunological synapse (IS) (13). Virion envelopes similarly contain a large proportion of cholesterol and sphingomyelin (1), which is important for the maintenance of virion structure and infectivity (7, 10). Virion assembly is targeted to rafts or raft-like regions of the plasma membrane via lipid interactions with acylated residues in viral Gag (6, 9, 15, 20, 22, 24) and Env (4, 29). What has not been investigated is the role of lipid rafts in the targeting of viral Env and Gag to the plasma membrane in infected T cells and in the subsequent assembly and function of the HIV-1 VS.
To localize HIV-1 Env and lipid rafts in the plasma membrane, we labeled T cells with the biotinylated cholera toxin ? subunit (B-Ctx) to detect the raft marker ganglioside GM1 and Env with the gp41-specific monoclonal antibody (MAb) 50-69 (16). B-Ctx and 50-69 binding was detected using streptavidin-fluorescein isothiocyanate (FITC) and anti-human-tetramethyl rhodamine isocyanate (TRITC), respectively, and immunofluorescence was analyzed by laser scanning confocal microscopy (LSCM). Unless marked, images are single x-y optical sections through the middle of the T cell. Jurkat CE6.1 cells show strong GM1 membrane staining distributed around the entire plasma membrane, resting primary CD4+ T cells show no detectable fluorescence, and 3-day phytohemagglutinin (PHA)-activated CD4+ T-cell blasts show patchy diffuse membrane staining (Fig. 1A). This pattern of labeling agrees with previous studies showing a redistribution of GM1 from intracellular stores to the plasma membrane upon the activation of resting T cells (32). The infection of Jurkat cells with CXCR4-tropic HIV-1LAI (JurkatLAI) led to the plasma membrane expression of Env, as detected by MAb 50-69. Env was generally cocapped with GM1 at one pole of the T cell, and both markers showed strong colocalization (Fig. 1B). This pattern of staining was observed whether labeling was carried out prior to or after fixation, excluding the possibility of ligand-induced capping. Similar results were obtained with PHA-activated primary CD4+ T cells infected for 5 days with HIV-1LAI (Fig. 1C). To confirm the lipid raft nature of the GM1-Env membrane compartment, we labeled the phosphatidylinositol-linked CD59 molecule that segregates into rafts. JurkatLAI cells showed tight polarized clustering of GM1, CD59, and Env (Fig. 2A), whereas minimal copolarization of Env and GM1 was observed with the transferrin receptor (TfR), which is excluded from rafts (Fig. 2B) (14). Although colocalization by immunofluorescence labeling and LSCM suggests a cosegregation of molecules, the resolution of this technique is not sufficient to unequivocally demonstrate this. We therefore analyzed JurkatLAI cells immunolabeled for Env and GM1 by cryo-electron microscopy. Figure 2C shows GM1 in the plasma membrane and colabeling of GM1 and Env in a budding virion, in accord with previous studies showing that HIV incorporates raft markers into the viral envelope (22, 30). In contrast, budding virions did not incorporate control markers such as LAMP-2 (data not shown).
The capping of GM1 in HIV-1-infected but not uninfected T cells implied that viral proteins were directing the aggregation of rafts in the plasma membrane. The most likely candidate for this is Gag, which was demonstrated by others using biochemical techniques to cluster rafts into large zones of Gag-raft aggregates, termed "barges" (20), with a higher density than that of noncoalesced rafts (9, 15, 24). We therefore analyzed the distribution of Gag in relation to GM1 staining in JurkatLAI cells. Gag colocalized strongly with Env and GM1 at one pole of the T cell (Fig. 3A).
In the IS, many proteins required for T-cell activation are recruited to the cell-cell contact surface in the context of lipid rafts (12). To investigate whether HIV-1 Gag and Env are similarly enriched in rafts at the VS, we formed conjugates and probed for GM1, Gag, and Env in effector cells and CD4 in target cells. Within 1 h, 47% of conjugates had formed a VS, as defined by copolarization to the site of cell-cell contact, of HIV antigens in the effector cell and CD4 in the target cell (Table 1 and Fig. 3B). Of these VS, the majority (63%) showed Env and Gag polarization to the VS in the context of GM1 (Table 1 and Fig. 3B and C). Furthermore, patches of Env and GM1 were observed in the target cell membrane (Fig. 3B), suggesting that fusion at the VS of virions with the receptor-expressing target cell had occurred, as we described previously (16).
To investigate whether lipid raft integrity is important in the assembly of the VS, we treated effector cells with 10 mM methyl-?-cyclodextrin (?-CD), an agent that depletes cholesterol from lipid bilayers, prior to washing and conjugate formation with target cells. Treatment of the effector cell with 10 mM ?-CD dispersed GM1 staining in the effector cell membrane, prevented CD4 polarization in the target cell, and unexpectedly, completely eliminated the detection of Env expression (Fig. 4A). This effect was dose dependent, with lower concentrations of ?-CD resulting in intermediate Env staining levels and the breaking up of Env clusters (Fig. 4B). This was the case whether Env was labeled with the gp41-specific 50-69 MAb, the gp120-specific 2G12 MAb, or polyclonal HIV immunoglobulin G (results not shown), implying that the loss of detectable staining was unlikely to be a result of conformational changes induced in Env. The loss of Env from the membrane by ?-CD-induced shedding was ruled out by measuring extracellular Env using a sensitive enzyme-linked immunosorbent assay (ELISA) (27): insignificant differences were observed (Fig. 4C) that could not explain the reduction in the detection of staining by confocal microscopy. Moreover, a flow cytometric analysis of surface Env expression showed only subtle differences between ?-CD-treated and untreated cells (Fig. 4D). The labeling of Env with various antibodies after cell fixation and permeabilization and analysis by either LSCM or flow cytometry did not reveal a difference in the level of intracellular Env, making increased internalization an unlikely explanation (data not shown). The loss of Env detection is most likely explained, therefore, by Env dispersion in the plane of the plasma membrane coordinate with the disruption of lipid rafts, with the resultant molecular dilution reducing Env labeling to below the levels that are readily detectable by LSCM. These results are in accord with studies demonstrating that HIV-1 virions treated with ?-CD retain Env despite losing their infectivity and some intravirion structures such as p24 and RNA (7, 10, 11).
In cultures of JurkatLAI cells, 80 to 100% of the cells are infected. Nevertheless, to confirm HIV infection of the effector cells in the absence of Env staining and to investigate the localization of Gag with respect to GM1 staining after ?-CD treatment, we costained conjugates for Gag, Env, and GM1. As before, Env staining was undetectable, whereas Gag was dispersed within the cytoplasm and completely uncoupled from GM1 staining, and GM1 was not polarized to the target-effector contact site (Fig. 4E). Neither capping of Env or Gag to the cell-cell contact site nor a transfer of Gag to the target cell was observed in target-effector conjugates, even after 3 h of incubation (results not shown). A statistical analysis of conjugates formed between target and effector cells showed an 88% reduction in VS formation (P = 0.0001) and an 89% reduction in GM1 polarization (P = 0.0003) after ?-CD treatment of the effector cells (Table 1). Based on these data, we suggest that the depletion of cholesterol in the effector cell membrane disrupts both the Gag association with rafts at the inner leaflet of the plasma membrane and gp41-raft localization, preventing Gag-Env interaction and abrogating the polarization of these molecules to the site of effector-target cell contact. This would impede virion assembly and budding and would prevent sufficient Env engagement of receptors on the target cell to drive VS assembly. We were unable to restore capped Env staining by the reconstitution of ?-CD-treated cells with exogenous cholesterol over a period of 24 h (data not shown). The disruption of Gag-Env clusters by raft dispersal may therefore be irreversible, or alternatively, reconstruction of Gag-Env assemblies in the plasma membrane is very slow following raft reconstitution. Moreover, it is possible that the disruption of HIV antigen localization is not solely a consequence of the dispersal of lipid rafts. ?-CD disruption of Gag targeting to the plasma membrane may take place via sequestration of the lipid phosphatidylinositol 4,5-bisphosphate (PIP2), which is normally concentrated in cholesterol-rich membrane domains and is involved in the organization of the actin cytoskeleton (19, 23). The perturbation of PIP2 levels by cholesterol depletion using, among other agents, ?-CD, causes Gag to be redirected away from the plasma membrane towards PIP2-enriched endosomal membranes (23). Since it is documented that cholesterol depletion can also disrupt the underlying actin cytoskeleton and influence the lateral mobility of membrane proteins (19), we cannot rule out the possibility that some effects of ?-CD treatment we observed were also the result of altered actin-mediated processes involved in HIV Env and Gag targeting and VS formation. However, we do not believe that this alters the principal interpretation of our data that correct raft structure and function, perhaps additionally influenced by components of the underlying cytoskeleton, are important in HIV Env and Gag localization and VS formation.
?-CD is toxic at high concentrations and could not reasonably be considered an antiviral strategy against HIV-1 cell-cell (and potentially cell-free) dissemination. However, the interruption of the HIV-1 Env and/or Gag association with lipid rafts may be effected by other, less toxic approaches that disrupt lipid rafts and/or alter PIP2 distribution and should be explored as an avenue to novel prophylaxis in the form of microbicides or as a therapeutic intervention in HIV-1 infection.
ACKNOWLEDGMENTS
This work was supported by grant G0400453 from the Medical Research Council, United Kingdom (MRC), grant G0100137 from the MRC and the UK Department for International Development, and a European Microbicide Project (EMPRO) consortium grant.
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ABSTRACT
Human immunodeficiency virus type 1 (HIV-1) can spread directly between T cells by forming a supramolecular structure termed a virological synapse (VS). HIV-1 envelope glycoproteins (Env) are required for VS assembly, but their mode of recruitment is unclear. We investigated the distribution of GM1-rich lipid rafts in HIV-1-infected (effector) T cells and observed Env colocalization with polarized raft markers GM1 and CD59 but not with the transferrin receptor that is excluded from lipid rafts. In conjugates of effector T cells and target CD4+ T cells, GM1, Env, and Gag relocated to the cell-cell interface. The depletion of cholesterol in the infected cell dispersed Env and GM1 within the plasma membrane, eliminated Gag clustering at the site of cell-cell contact, and abolished assembly of the VS. Raft integrity is therefore critical for Env and Gag coclustering and VS assembly in T-cell conjugates.
TEXT
Efficient spread between T cells by the retroviruses human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 can take place in vitro via the formation of a virological synapse (VS) (3, 17). Previous studies have demonstrated that this mode of viral dissemination is productive and more efficient than that achieved by infection with cell-free virions (25, 31). HIV-1 dissemination via VS may contribute to the rapid spread of HIV-1 within secondary lymphoid tissues in vivo, particularly during early infection (5). Similarly, HIV-1-infected or uninfected but pulsed dendritic cells can efficiently transfer virus to T cells via a synapse (2, 21, 26), and this may represent an important pathway of viral dissemination after mucosal inoculation (28). Furthermore, the movement of HIV-1 across an adhesive junction may help to protect the virus from elements of the humoral immune response, such as antibodies and complement.
The HIV-1 VS assembles at the site of contact between an infected (effector) and receptor-expressing (target) cell and is exemplified by the recruitment of the viral receptors (CD4 and a chemokine receptor, CXCR4 or CCR5) and adhesion molecules on the target cell and of viral Env and Gag and cognate adhesion molecules on the effector cell (16, 17, 26). The assembly of these supramolecular complexes relies on engagement of the viral receptors by Env and on a functional actin cytoskeleton in the target cell (16). The recruitment of Env and Gag to the intercellular junction is followed by viral antigen transfer across the synapse into the target cell (16), which has been shown by others to lead to rapid de novo reverse transcription (8, 18, 31).
HIV-1 particles assemble under and bud through specific plasma membrane domains termed lipid rafts or related raft-like structures (6, 9, 15, 20, 22, 24). Rafts are biochemically and biophysically distinct regions of plasma membrane that are rich in cholesterol and sphingomyelin, maintain an ordered structure, and contain proteins important in immune cell signaling via the immunological synapse (IS) (13). Virion envelopes similarly contain a large proportion of cholesterol and sphingomyelin (1), which is important for the maintenance of virion structure and infectivity (7, 10). Virion assembly is targeted to rafts or raft-like regions of the plasma membrane via lipid interactions with acylated residues in viral Gag (6, 9, 15, 20, 22, 24) and Env (4, 29). What has not been investigated is the role of lipid rafts in the targeting of viral Env and Gag to the plasma membrane in infected T cells and in the subsequent assembly and function of the HIV-1 VS.
To localize HIV-1 Env and lipid rafts in the plasma membrane, we labeled T cells with the biotinylated cholera toxin ? subunit (B-Ctx) to detect the raft marker ganglioside GM1 and Env with the gp41-specific monoclonal antibody (MAb) 50-69 (16). B-Ctx and 50-69 binding was detected using streptavidin-fluorescein isothiocyanate (FITC) and anti-human-tetramethyl rhodamine isocyanate (TRITC), respectively, and immunofluorescence was analyzed by laser scanning confocal microscopy (LSCM). Unless marked, images are single x-y optical sections through the middle of the T cell. Jurkat CE6.1 cells show strong GM1 membrane staining distributed around the entire plasma membrane, resting primary CD4+ T cells show no detectable fluorescence, and 3-day phytohemagglutinin (PHA)-activated CD4+ T-cell blasts show patchy diffuse membrane staining (Fig. 1A). This pattern of labeling agrees with previous studies showing a redistribution of GM1 from intracellular stores to the plasma membrane upon the activation of resting T cells (32). The infection of Jurkat cells with CXCR4-tropic HIV-1LAI (JurkatLAI) led to the plasma membrane expression of Env, as detected by MAb 50-69. Env was generally cocapped with GM1 at one pole of the T cell, and both markers showed strong colocalization (Fig. 1B). This pattern of staining was observed whether labeling was carried out prior to or after fixation, excluding the possibility of ligand-induced capping. Similar results were obtained with PHA-activated primary CD4+ T cells infected for 5 days with HIV-1LAI (Fig. 1C). To confirm the lipid raft nature of the GM1-Env membrane compartment, we labeled the phosphatidylinositol-linked CD59 molecule that segregates into rafts. JurkatLAI cells showed tight polarized clustering of GM1, CD59, and Env (Fig. 2A), whereas minimal copolarization of Env and GM1 was observed with the transferrin receptor (TfR), which is excluded from rafts (Fig. 2B) (14). Although colocalization by immunofluorescence labeling and LSCM suggests a cosegregation of molecules, the resolution of this technique is not sufficient to unequivocally demonstrate this. We therefore analyzed JurkatLAI cells immunolabeled for Env and GM1 by cryo-electron microscopy. Figure 2C shows GM1 in the plasma membrane and colabeling of GM1 and Env in a budding virion, in accord with previous studies showing that HIV incorporates raft markers into the viral envelope (22, 30). In contrast, budding virions did not incorporate control markers such as LAMP-2 (data not shown).
The capping of GM1 in HIV-1-infected but not uninfected T cells implied that viral proteins were directing the aggregation of rafts in the plasma membrane. The most likely candidate for this is Gag, which was demonstrated by others using biochemical techniques to cluster rafts into large zones of Gag-raft aggregates, termed "barges" (20), with a higher density than that of noncoalesced rafts (9, 15, 24). We therefore analyzed the distribution of Gag in relation to GM1 staining in JurkatLAI cells. Gag colocalized strongly with Env and GM1 at one pole of the T cell (Fig. 3A).
In the IS, many proteins required for T-cell activation are recruited to the cell-cell contact surface in the context of lipid rafts (12). To investigate whether HIV-1 Gag and Env are similarly enriched in rafts at the VS, we formed conjugates and probed for GM1, Gag, and Env in effector cells and CD4 in target cells. Within 1 h, 47% of conjugates had formed a VS, as defined by copolarization to the site of cell-cell contact, of HIV antigens in the effector cell and CD4 in the target cell (Table 1 and Fig. 3B). Of these VS, the majority (63%) showed Env and Gag polarization to the VS in the context of GM1 (Table 1 and Fig. 3B and C). Furthermore, patches of Env and GM1 were observed in the target cell membrane (Fig. 3B), suggesting that fusion at the VS of virions with the receptor-expressing target cell had occurred, as we described previously (16).
To investigate whether lipid raft integrity is important in the assembly of the VS, we treated effector cells with 10 mM methyl-?-cyclodextrin (?-CD), an agent that depletes cholesterol from lipid bilayers, prior to washing and conjugate formation with target cells. Treatment of the effector cell with 10 mM ?-CD dispersed GM1 staining in the effector cell membrane, prevented CD4 polarization in the target cell, and unexpectedly, completely eliminated the detection of Env expression (Fig. 4A). This effect was dose dependent, with lower concentrations of ?-CD resulting in intermediate Env staining levels and the breaking up of Env clusters (Fig. 4B). This was the case whether Env was labeled with the gp41-specific 50-69 MAb, the gp120-specific 2G12 MAb, or polyclonal HIV immunoglobulin G (results not shown), implying that the loss of detectable staining was unlikely to be a result of conformational changes induced in Env. The loss of Env from the membrane by ?-CD-induced shedding was ruled out by measuring extracellular Env using a sensitive enzyme-linked immunosorbent assay (ELISA) (27): insignificant differences were observed (Fig. 4C) that could not explain the reduction in the detection of staining by confocal microscopy. Moreover, a flow cytometric analysis of surface Env expression showed only subtle differences between ?-CD-treated and untreated cells (Fig. 4D). The labeling of Env with various antibodies after cell fixation and permeabilization and analysis by either LSCM or flow cytometry did not reveal a difference in the level of intracellular Env, making increased internalization an unlikely explanation (data not shown). The loss of Env detection is most likely explained, therefore, by Env dispersion in the plane of the plasma membrane coordinate with the disruption of lipid rafts, with the resultant molecular dilution reducing Env labeling to below the levels that are readily detectable by LSCM. These results are in accord with studies demonstrating that HIV-1 virions treated with ?-CD retain Env despite losing their infectivity and some intravirion structures such as p24 and RNA (7, 10, 11).
In cultures of JurkatLAI cells, 80 to 100% of the cells are infected. Nevertheless, to confirm HIV infection of the effector cells in the absence of Env staining and to investigate the localization of Gag with respect to GM1 staining after ?-CD treatment, we costained conjugates for Gag, Env, and GM1. As before, Env staining was undetectable, whereas Gag was dispersed within the cytoplasm and completely uncoupled from GM1 staining, and GM1 was not polarized to the target-effector contact site (Fig. 4E). Neither capping of Env or Gag to the cell-cell contact site nor a transfer of Gag to the target cell was observed in target-effector conjugates, even after 3 h of incubation (results not shown). A statistical analysis of conjugates formed between target and effector cells showed an 88% reduction in VS formation (P = 0.0001) and an 89% reduction in GM1 polarization (P = 0.0003) after ?-CD treatment of the effector cells (Table 1). Based on these data, we suggest that the depletion of cholesterol in the effector cell membrane disrupts both the Gag association with rafts at the inner leaflet of the plasma membrane and gp41-raft localization, preventing Gag-Env interaction and abrogating the polarization of these molecules to the site of effector-target cell contact. This would impede virion assembly and budding and would prevent sufficient Env engagement of receptors on the target cell to drive VS assembly. We were unable to restore capped Env staining by the reconstitution of ?-CD-treated cells with exogenous cholesterol over a period of 24 h (data not shown). The disruption of Gag-Env clusters by raft dispersal may therefore be irreversible, or alternatively, reconstruction of Gag-Env assemblies in the plasma membrane is very slow following raft reconstitution. Moreover, it is possible that the disruption of HIV antigen localization is not solely a consequence of the dispersal of lipid rafts. ?-CD disruption of Gag targeting to the plasma membrane may take place via sequestration of the lipid phosphatidylinositol 4,5-bisphosphate (PIP2), which is normally concentrated in cholesterol-rich membrane domains and is involved in the organization of the actin cytoskeleton (19, 23). The perturbation of PIP2 levels by cholesterol depletion using, among other agents, ?-CD, causes Gag to be redirected away from the plasma membrane towards PIP2-enriched endosomal membranes (23). Since it is documented that cholesterol depletion can also disrupt the underlying actin cytoskeleton and influence the lateral mobility of membrane proteins (19), we cannot rule out the possibility that some effects of ?-CD treatment we observed were also the result of altered actin-mediated processes involved in HIV Env and Gag targeting and VS formation. However, we do not believe that this alters the principal interpretation of our data that correct raft structure and function, perhaps additionally influenced by components of the underlying cytoskeleton, are important in HIV Env and Gag localization and VS formation.
?-CD is toxic at high concentrations and could not reasonably be considered an antiviral strategy against HIV-1 cell-cell (and potentially cell-free) dissemination. However, the interruption of the HIV-1 Env and/or Gag association with lipid rafts may be effected by other, less toxic approaches that disrupt lipid rafts and/or alter PIP2 distribution and should be explored as an avenue to novel prophylaxis in the form of microbicides or as a therapeutic intervention in HIV-1 infection.
ACKNOWLEDGMENTS
This work was supported by grant G0400453 from the Medical Research Council, United Kingdom (MRC), grant G0100137 from the MRC and the UK Department for International Development, and a European Microbicide Project (EMPRO) consortium grant.
REFERENCES
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