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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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The cytoplasmic domain of DC-SIGN is necessary for HIV-1 internalization. (A) Raji B cells expressing full-length DC-SIGN and cytoplasmic 35 amino acid–deleted DC-SIGNΔ35 bind HIV-1. Approximately 780 ng/ml HIV-1 GFP-labeled (blue peak) and a No-Env negative control (red peak) virions were incubated with Raji cells expressing full-length and the cytoplasmic mutant DC-SIGNΔ35 for 30 min at 4°C. Cells were then analyzed by flow cytometry for cell surface–bound virus. (B and C) Raji cells expressing full-length DC-SIGN internalize HIV-1. Raji cells were incubated with HIV-1-GFP virions at 37°C for 2 h, trypsinized, washed, and added to HeLa cells expressing CD4/CCR5 (MAGI-CCR5) to visualize not only internalization, but polarization. Unlabeled arrows indicate polarization of virus between cells. Cells were viewed using confocal microscopy (B, low magnification; C, high magnification) and analyzed using Leica software.
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fig2: The cytoplasmic domain of DC-SIGN is necessary for HIV-1 internalization. (A) Raji B cells expressing full-length DC-SIGN and cytoplasmic 35 amino acid–deleted DC-SIGNΔ35 bind HIV-1. Approximately 780 ng/ml HIV-1 GFP-labeled (blue peak) and a No-Env negative control (red peak) virions were incubated with Raji cells expressing full-length and the cytoplasmic mutant DC-SIGNΔ35 for 30 min at 4°C. Cells were then analyzed by flow cytometry for cell surface–bound virus. (B and C) Raji cells expressing full-length DC-SIGN internalize HIV-1. Raji cells were incubated with HIV-1-GFP virions at 37°C for 2 h, trypsinized, washed, and added to HeLa cells expressing CD4/CCR5 (MAGI-CCR5) to visualize not only internalization, but polarization. Unlabeled arrows indicate polarization of virus between cells. Cells were viewed using confocal microscopy (B, low magnification; C, high magnification) and analyzed using Leica software.

Mentions: To determine whether the cytoplasmic domain of DC-SIGN was required for internalization, HIV-1–GFP-labeled virions were incubated with Raji B cell lines expressing WT DC-SIGN or DC-SIGNΔ35. HIV-1–GFP-labeled virions were incubated at 4°C for 30 min to quantify cell surface binding. To assess internalization, cells were incubated with ∼780 ng/ml of HIV-1–GFP-labeled virions at 37°C for 2 h and treated with trypsin for 5 min before being added to HeLa cells expressing CD4/CCR5 (MAGI-CCR5). Raji cells expressing DC-SIGNΔ35 showed comparable cell surface expression and were able to bind GFP-labeled HIV-1 virions (Fig. 2 A). It has been shown previously (27) that HIV-1 pseudotypes bind to DC-SIGN and the other DC-SIGN mutants. However, in contrast to cells expressing WT DC-SIGN, cells with DC-SIGNΔ35 did not internalize HIV-1 (Fig. 1, B and C, left vs. right panel). Thus, internalization appears to be critical for mediating trans-enhancement of HIV infection.


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)

The cytoplasmic domain of DC-SIGN is necessary for HIV-1 internalization. (A) Raji B cells expressing full-length DC-SIGN and cytoplasmic 35 amino acid–deleted DC-SIGNΔ35 bind HIV-1. Approximately 780 ng/ml HIV-1 GFP-labeled (blue peak) and a No-Env negative control (red peak) virions were incubated with Raji cells expressing full-length and the cytoplasmic mutant DC-SIGNΔ35 for 30 min at 4°C. Cells were then analyzed by flow cytometry for cell surface–bound virus. (B and C) Raji cells expressing full-length DC-SIGN internalize HIV-1. Raji cells were incubated with HIV-1-GFP virions at 37°C for 2 h, trypsinized, washed, and added to HeLa cells expressing CD4/CCR5 (MAGI-CCR5) to visualize not only internalization, but polarization. Unlabeled arrows indicate polarization of virus between cells. Cells were viewed using confocal microscopy (B, low magnification; C, high magnification) and analyzed using Leica software.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2118718&req=5

fig2: The cytoplasmic domain of DC-SIGN is necessary for HIV-1 internalization. (A) Raji B cells expressing full-length DC-SIGN and cytoplasmic 35 amino acid–deleted DC-SIGNΔ35 bind HIV-1. Approximately 780 ng/ml HIV-1 GFP-labeled (blue peak) and a No-Env negative control (red peak) virions were incubated with Raji cells expressing full-length and the cytoplasmic mutant DC-SIGNΔ35 for 30 min at 4°C. Cells were then analyzed by flow cytometry for cell surface–bound virus. (B and C) Raji cells expressing full-length DC-SIGN internalize HIV-1. Raji cells were incubated with HIV-1-GFP virions at 37°C for 2 h, trypsinized, washed, and added to HeLa cells expressing CD4/CCR5 (MAGI-CCR5) to visualize not only internalization, but polarization. Unlabeled arrows indicate polarization of virus between cells. Cells were viewed using confocal microscopy (B, low magnification; C, high magnification) and analyzed using Leica software.
Mentions: To determine whether the cytoplasmic domain of DC-SIGN was required for internalization, HIV-1–GFP-labeled virions were incubated with Raji B cell lines expressing WT DC-SIGN or DC-SIGNΔ35. HIV-1–GFP-labeled virions were incubated at 4°C for 30 min to quantify cell surface binding. To assess internalization, cells were incubated with ∼780 ng/ml of HIV-1–GFP-labeled virions at 37°C for 2 h and treated with trypsin for 5 min before being added to HeLa cells expressing CD4/CCR5 (MAGI-CCR5). Raji cells expressing DC-SIGNΔ35 showed comparable cell surface expression and were able to bind GFP-labeled HIV-1 virions (Fig. 2 A). It has been shown previously (27) that HIV-1 pseudotypes bind to DC-SIGN and the other DC-SIGN mutants. However, in contrast to cells expressing WT DC-SIGN, cells with DC-SIGNΔ35 did not internalize HIV-1 (Fig. 1, B and C, left vs. right panel). Thus, internalization appears to be critical for mediating trans-enhancement of HIV infection.

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