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Leukocyte-specific protein 1 interacts with DC-SIGN and mediates transport of HIV to the proteasome in dendritic cells.

Smith AL, Ganesh L, Leung K, Jongstra-Bilen J, Jongstra J, Nabel GJ - J. Exp. Med. (2007)

Bottom Line: LSP1 is an F-actin binding protein involved in leukocyte motility and found on the cytoplasmic surface of the plasma membrane.LSP1 diverts HIV-1 to the proteasome.Down-regulation of LSP1 with specific small interfering RNAs in human DCs enhanced HIV-1 transfer to T cells, and bone marrow DCs from lsp1(-/-) mice also showed an increase in transfer of HIV-1(BaL) to a human T cell line.

View Article: PubMed Central - PubMed

Affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Dendritic cells (DCs) capture and internalize human immunodeficiency virus (HIV)-1 through C-type lectins, including DC-SIGN. These cells mediate efficient infection of T cells by concentrating the delivery of virus through the infectious synapse, a process dependent on the cytoplasmic domain of DC-SIGN. Here, we identify a cellular protein that binds specifically to the cytoplasmic region of DC-SIGN and directs internalized virus to the proteasome. This cellular protein, leukocyte-specific protein 1 (LSP1), was defined biochemically by immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. LSP1 is an F-actin binding protein involved in leukocyte motility and found on the cytoplasmic surface of the plasma membrane. LSP1 interacted specifically with DC-SIGN and other C-type lectins, but not the inactive mutant DC-SIGNDelta35, which lacks a cytoplasmic domain and shows altered virus transport in DCs. LSP1 diverts HIV-1 to the proteasome. Down-regulation of LSP1 with specific small interfering RNAs in human DCs enhanced HIV-1 transfer to T cells, and bone marrow DCs from lsp1(-/-) mice also showed an increase in transfer of HIV-1(BaL) to a human T cell line. Proteasome inhibitors increased retention of viral proteins in lsp1(+/+) DCs, and substantial colocalization of virus to the proteasome was observed in wild-type compared with LSP1-deficient cells. Collectively, these data suggest that LSP1 protein facilitates virus transport into the proteasome after its interaction with DC-SIGN through its interaction with cytoskeletal proteins.

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LSP1 traffics HIV-1 to the proteasome. (A) HIV-1 degradation is decreased in lsp1−/− DCs, and proteasome inhibitors block HIV-1 degradation in the presence of LSP1. Mature BMDCs from lsp1−/− and wt mice were pulsed with HIV-1–GFP for 2 h at 37°C, washed with PBS, trypsinized, and lysed with p24 lysis buffer reagent (left). Mature BMDCs from wt (lsp1+/+) were pretreated for 1 h with MG132 (proteasome inhibitor), chloroquine, or bafilomycin A-1 (lysosomal inhibitors) in RPMI or RPMI alone in a 96-well tissue culture dish (5 × 104 cells). Cells were then pulsed with HIV-1–GFP for 2 h at 37°C in the presence of inhibitors, washed with PBS, replaced with media containing the respective inhibitors, and incubated for an additional 1 h. Cells were then trypsinized, washed, and lysed with p24 lysis buffer reagent (right). p24 Gag concentrations were assayed by ELISA. These data are representative of the 1-h time point from multiple experiments performed in triplicate. The percentage values were calculated by setting the amount of p24 measured immediately after removing the virus from the cells, the 0-h time point, to 100%. The differences in p24 after several hours were then measured and compared with the 0-h time point. (B and C) HIV-1 shows greater colocalization with the proteasome in DCs expressing LSP1. (B) Mature BMDCs from lsp1−/− (−/−) and wt (+/+) mice were pulsed with HIV-1–GFP (green) for 30 min at 37°C. For proteasome inhibitor studies, BMDCs from lsp1−/− and wt mice were incubated with the proteasomal inhibitor MG132 in 5 μg/ml RPMI or RPMI alone for 1 h before infection by HIV-1–GFP (green) for 30 min. Cells were washed, fixed, and stained with a mAb to the 20S proteasome subunit a-4 (red) and viewed using confocal microscopy. HIV-1 that colocalizes with proteasomes appears yellow (overlay). (C) HIV-1 colocalization with proteasomes is representative of a percentage of HIV-1 that colocalizes with a percentage of proteasomes above a measurable threshold. Percentage colocalization was calculated using the Leica confocal software. Three images/sample were used, and error bars were assigned accordingly.
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fig7: LSP1 traffics HIV-1 to the proteasome. (A) HIV-1 degradation is decreased in lsp1−/− DCs, and proteasome inhibitors block HIV-1 degradation in the presence of LSP1. Mature BMDCs from lsp1−/− and wt mice were pulsed with HIV-1–GFP for 2 h at 37°C, washed with PBS, trypsinized, and lysed with p24 lysis buffer reagent (left). Mature BMDCs from wt (lsp1+/+) were pretreated for 1 h with MG132 (proteasome inhibitor), chloroquine, or bafilomycin A-1 (lysosomal inhibitors) in RPMI or RPMI alone in a 96-well tissue culture dish (5 × 104 cells). Cells were then pulsed with HIV-1–GFP for 2 h at 37°C in the presence of inhibitors, washed with PBS, replaced with media containing the respective inhibitors, and incubated for an additional 1 h. Cells were then trypsinized, washed, and lysed with p24 lysis buffer reagent (right). p24 Gag concentrations were assayed by ELISA. These data are representative of the 1-h time point from multiple experiments performed in triplicate. The percentage values were calculated by setting the amount of p24 measured immediately after removing the virus from the cells, the 0-h time point, to 100%. The differences in p24 after several hours were then measured and compared with the 0-h time point. (B and C) HIV-1 shows greater colocalization with the proteasome in DCs expressing LSP1. (B) Mature BMDCs from lsp1−/− (−/−) and wt (+/+) mice were pulsed with HIV-1–GFP (green) for 30 min at 37°C. For proteasome inhibitor studies, BMDCs from lsp1−/− and wt mice were incubated with the proteasomal inhibitor MG132 in 5 μg/ml RPMI or RPMI alone for 1 h before infection by HIV-1–GFP (green) for 30 min. Cells were washed, fixed, and stained with a mAb to the 20S proteasome subunit a-4 (red) and viewed using confocal microscopy. HIV-1 that colocalizes with proteasomes appears yellow (overlay). (C) HIV-1 colocalization with proteasomes is representative of a percentage of HIV-1 that colocalizes with a percentage of proteasomes above a measurable threshold. Percentage colocalization was calculated using the Leica confocal software. Three images/sample were used, and error bars were assigned accordingly.

Mentions: Incoming HIV-1 particles in DCs are bound and internalized by various DC-SIGN–dependent and –independent pathways. Immediately after internalization, most of the virions are degraded in an acidic lysosomal compartment. A fraction of the virus that escapes degradation is retained in endocytic compartments and is either transmitted to permissive CD4+ lymphocytes or degraded by the proteasome (30, 31). To investigate the role of LSP1 in DC processing and transport of HIV-1, HIV-1–GFP was incubated with DCs in the presence or absence of proteasome or lysosome inhibitors. Because of the inability to confirm the complete silencing of LSP1 and the limited window of knockdown in human mDCs, the LSP1 knockout mice provide the best model system to study the degradative pathway in the total absence of LSP1. Differentiated BMDCs were incubated with the lysosomal inhibitors chloroquine (100 μM), the endosome acidification inhibitor bafilomycin A1 (10 μg/ml), or the proteasomal inhibitor MG132 in RPMI (5 μg/ml) or RPMI alone for 1 h before transduction with concentrated HIV-1–GFP for 2 h. Cells were washed extensively and incubated with the respective inhibitors. Before lysis, cells were treated with trypsin and washed to remove virus bound to the cell surface. Total protein isolated at various time points after the 2-h transduction (0 h, 1 h, and 3 h) was assayed by ELISA for p24 Gag. Results at 1 h showed that more retention of HIV-1 occurred in lsp1−/− BMDCs when compared with lsp1+/+ controls (Fig. 7 A, left). The p24 values at other times showed a similar trend as the 1-h time point, but the effect was maximal at 1 h. In lsp1+/+ control cells treated with the proteasome inhibitor MG132, HIV-1 retention was higher compared with chloroquine-, bafilomycin-, or vehicle-treated lsp1+/+ cells (Fig. 7, middle) and were similar to lsp1−/− BMDCs when treated with the same inhibitors (Fig. 7, right), suggesting that LSP1 may be involved in the shuttling of virus to the proteasome for degradation.


Leukocyte-specific protein 1 interacts with DC-SIGN and mediates transport of HIV to the proteasome in dendritic cells.

Smith AL, Ganesh L, Leung K, Jongstra-Bilen J, Jongstra J, Nabel GJ - J. Exp. Med. (2007)

LSP1 traffics HIV-1 to the proteasome. (A) HIV-1 degradation is decreased in lsp1−/− DCs, and proteasome inhibitors block HIV-1 degradation in the presence of LSP1. Mature BMDCs from lsp1−/− and wt mice were pulsed with HIV-1–GFP for 2 h at 37°C, washed with PBS, trypsinized, and lysed with p24 lysis buffer reagent (left). Mature BMDCs from wt (lsp1+/+) were pretreated for 1 h with MG132 (proteasome inhibitor), chloroquine, or bafilomycin A-1 (lysosomal inhibitors) in RPMI or RPMI alone in a 96-well tissue culture dish (5 × 104 cells). Cells were then pulsed with HIV-1–GFP for 2 h at 37°C in the presence of inhibitors, washed with PBS, replaced with media containing the respective inhibitors, and incubated for an additional 1 h. Cells were then trypsinized, washed, and lysed with p24 lysis buffer reagent (right). p24 Gag concentrations were assayed by ELISA. These data are representative of the 1-h time point from multiple experiments performed in triplicate. The percentage values were calculated by setting the amount of p24 measured immediately after removing the virus from the cells, the 0-h time point, to 100%. The differences in p24 after several hours were then measured and compared with the 0-h time point. (B and C) HIV-1 shows greater colocalization with the proteasome in DCs expressing LSP1. (B) Mature BMDCs from lsp1−/− (−/−) and wt (+/+) mice were pulsed with HIV-1–GFP (green) for 30 min at 37°C. For proteasome inhibitor studies, BMDCs from lsp1−/− and wt mice were incubated with the proteasomal inhibitor MG132 in 5 μg/ml RPMI or RPMI alone for 1 h before infection by HIV-1–GFP (green) for 30 min. Cells were washed, fixed, and stained with a mAb to the 20S proteasome subunit a-4 (red) and viewed using confocal microscopy. HIV-1 that colocalizes with proteasomes appears yellow (overlay). (C) HIV-1 colocalization with proteasomes is representative of a percentage of HIV-1 that colocalizes with a percentage of proteasomes above a measurable threshold. Percentage colocalization was calculated using the Leica confocal software. Three images/sample were used, and error bars were assigned accordingly.
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Related In: Results  -  Collection

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fig7: LSP1 traffics HIV-1 to the proteasome. (A) HIV-1 degradation is decreased in lsp1−/− DCs, and proteasome inhibitors block HIV-1 degradation in the presence of LSP1. Mature BMDCs from lsp1−/− and wt mice were pulsed with HIV-1–GFP for 2 h at 37°C, washed with PBS, trypsinized, and lysed with p24 lysis buffer reagent (left). Mature BMDCs from wt (lsp1+/+) were pretreated for 1 h with MG132 (proteasome inhibitor), chloroquine, or bafilomycin A-1 (lysosomal inhibitors) in RPMI or RPMI alone in a 96-well tissue culture dish (5 × 104 cells). Cells were then pulsed with HIV-1–GFP for 2 h at 37°C in the presence of inhibitors, washed with PBS, replaced with media containing the respective inhibitors, and incubated for an additional 1 h. Cells were then trypsinized, washed, and lysed with p24 lysis buffer reagent (right). p24 Gag concentrations were assayed by ELISA. These data are representative of the 1-h time point from multiple experiments performed in triplicate. The percentage values were calculated by setting the amount of p24 measured immediately after removing the virus from the cells, the 0-h time point, to 100%. The differences in p24 after several hours were then measured and compared with the 0-h time point. (B and C) HIV-1 shows greater colocalization with the proteasome in DCs expressing LSP1. (B) Mature BMDCs from lsp1−/− (−/−) and wt (+/+) mice were pulsed with HIV-1–GFP (green) for 30 min at 37°C. For proteasome inhibitor studies, BMDCs from lsp1−/− and wt mice were incubated with the proteasomal inhibitor MG132 in 5 μg/ml RPMI or RPMI alone for 1 h before infection by HIV-1–GFP (green) for 30 min. Cells were washed, fixed, and stained with a mAb to the 20S proteasome subunit a-4 (red) and viewed using confocal microscopy. HIV-1 that colocalizes with proteasomes appears yellow (overlay). (C) HIV-1 colocalization with proteasomes is representative of a percentage of HIV-1 that colocalizes with a percentage of proteasomes above a measurable threshold. Percentage colocalization was calculated using the Leica confocal software. Three images/sample were used, and error bars were assigned accordingly.
Mentions: Incoming HIV-1 particles in DCs are bound and internalized by various DC-SIGN–dependent and –independent pathways. Immediately after internalization, most of the virions are degraded in an acidic lysosomal compartment. A fraction of the virus that escapes degradation is retained in endocytic compartments and is either transmitted to permissive CD4+ lymphocytes or degraded by the proteasome (30, 31). To investigate the role of LSP1 in DC processing and transport of HIV-1, HIV-1–GFP was incubated with DCs in the presence or absence of proteasome or lysosome inhibitors. Because of the inability to confirm the complete silencing of LSP1 and the limited window of knockdown in human mDCs, the LSP1 knockout mice provide the best model system to study the degradative pathway in the total absence of LSP1. Differentiated BMDCs were incubated with the lysosomal inhibitors chloroquine (100 μM), the endosome acidification inhibitor bafilomycin A1 (10 μg/ml), or the proteasomal inhibitor MG132 in RPMI (5 μg/ml) or RPMI alone for 1 h before transduction with concentrated HIV-1–GFP for 2 h. Cells were washed extensively and incubated with the respective inhibitors. Before lysis, cells were treated with trypsin and washed to remove virus bound to the cell surface. Total protein isolated at various time points after the 2-h transduction (0 h, 1 h, and 3 h) was assayed by ELISA for p24 Gag. Results at 1 h showed that more retention of HIV-1 occurred in lsp1−/− BMDCs when compared with lsp1+/+ controls (Fig. 7 A, left). The p24 values at other times showed a similar trend as the 1-h time point, but the effect was maximal at 1 h. In lsp1+/+ control cells treated with the proteasome inhibitor MG132, HIV-1 retention was higher compared with chloroquine-, bafilomycin-, or vehicle-treated lsp1+/+ cells (Fig. 7, middle) and were similar to lsp1−/− BMDCs when treated with the same inhibitors (Fig. 7, right), suggesting that LSP1 may be involved in the shuttling of virus to the proteasome for degradation.

Bottom Line: LSP1 is an F-actin binding protein involved in leukocyte motility and found on the cytoplasmic surface of the plasma membrane.LSP1 diverts HIV-1 to the proteasome.Down-regulation of LSP1 with specific small interfering RNAs in human DCs enhanced HIV-1 transfer to T cells, and bone marrow DCs from lsp1(-/-) mice also showed an increase in transfer of HIV-1(BaL) to a human T cell line.

View Article: PubMed Central - PubMed

Affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Dendritic cells (DCs) capture and internalize human immunodeficiency virus (HIV)-1 through C-type lectins, including DC-SIGN. These cells mediate efficient infection of T cells by concentrating the delivery of virus through the infectious synapse, a process dependent on the cytoplasmic domain of DC-SIGN. Here, we identify a cellular protein that binds specifically to the cytoplasmic region of DC-SIGN and directs internalized virus to the proteasome. This cellular protein, leukocyte-specific protein 1 (LSP1), was defined biochemically by immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. LSP1 is an F-actin binding protein involved in leukocyte motility and found on the cytoplasmic surface of the plasma membrane. LSP1 interacted specifically with DC-SIGN and other C-type lectins, but not the inactive mutant DC-SIGNDelta35, which lacks a cytoplasmic domain and shows altered virus transport in DCs. LSP1 diverts HIV-1 to the proteasome. Down-regulation of LSP1 with specific small interfering RNAs in human DCs enhanced HIV-1 transfer to T cells, and bone marrow DCs from lsp1(-/-) mice also showed an increase in transfer of HIV-1(BaL) to a human T cell line. Proteasome inhibitors increased retention of viral proteins in lsp1(+/+) DCs, and substantial colocalization of virus to the proteasome was observed in wild-type compared with LSP1-deficient cells. Collectively, these data suggest that LSP1 protein facilitates virus transport into the proteasome after its interaction with DC-SIGN through its interaction with cytoskeletal proteins.

Show MeSH
Related in: MedlinePlus