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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 down-regulation causes increased HIV-1 transfer in human DCs. (A) LSP1 expression in human DCs. Cell lysates prepared from immature or poly:IC-matured mDCs isolated from human elutriated monocytes were assayed for LSP1 expression by a polyclonal anti-LSP1 antibody. Expression was measured against control cell lysates from 293T transfected with the LSP1 gene. (B) Specific siRNAs for human LSP1 cause a down-regulation of LSP1 expression. Raji–DC-SIGN cells were transfected with the indicated LSP1, and control siRNA duplexes were introduced by nucleofection. 24 h after transfection, cell lysates (20 μg of total protein) were assayed for LSP1 expression by immunoblot (left). The film images were digitized using an Epson scanner and quantified using Bio-Rad Quantity One software. The numbers obtained for LSP1 were corrected using the numbers obtained from actin and plotted relative to control in a Microsoft Excel graph as indicated (right). (C and D) Down-regulation of human LSP1 in human DCs and Raji DC-SIGN cell line causes an increase in HIV-1 transfer to T cells. Mature human DCs were transiently transfected by GeneSilencer with siRNA for LSP1 (siA and siC) or scrambled siRNA (control). To label transfected cells, unrelated Cy5 siRNA was mixed with both control siRNA and LSP1 siRNAs at a ratio of 4:1. 8 h after the siRNA transfection, the cells were sorted for the Cy5 label and plated onto 96-well plates. 24 h after transfection, cells were pulsed with luciferase expressing 780 ng/ml HIV-1ADA for 2 h at 37°C, washed five times, and incubated with A3R5 T cells. Cells were lysed 72 h after transfection and analyzed for luciferase activity. These experiments were performed in three independent HIV-1− donors in triplicate samples, and two such experiments are represented in the figure.
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fig5: LSP1 down-regulation causes increased HIV-1 transfer in human DCs. (A) LSP1 expression in human DCs. Cell lysates prepared from immature or poly:IC-matured mDCs isolated from human elutriated monocytes were assayed for LSP1 expression by a polyclonal anti-LSP1 antibody. Expression was measured against control cell lysates from 293T transfected with the LSP1 gene. (B) Specific siRNAs for human LSP1 cause a down-regulation of LSP1 expression. Raji–DC-SIGN cells were transfected with the indicated LSP1, and control siRNA duplexes were introduced by nucleofection. 24 h after transfection, cell lysates (20 μg of total protein) were assayed for LSP1 expression by immunoblot (left). The film images were digitized using an Epson scanner and quantified using Bio-Rad Quantity One software. The numbers obtained for LSP1 were corrected using the numbers obtained from actin and plotted relative to control in a Microsoft Excel graph as indicated (right). (C and D) Down-regulation of human LSP1 in human DCs and Raji DC-SIGN cell line causes an increase in HIV-1 transfer to T cells. Mature human DCs were transiently transfected by GeneSilencer with siRNA for LSP1 (siA and siC) or scrambled siRNA (control). To label transfected cells, unrelated Cy5 siRNA was mixed with both control siRNA and LSP1 siRNAs at a ratio of 4:1. 8 h after the siRNA transfection, the cells were sorted for the Cy5 label and plated onto 96-well plates. 24 h after transfection, cells were pulsed with luciferase expressing 780 ng/ml HIV-1ADA for 2 h at 37°C, washed five times, and incubated with A3R5 T cells. Cells were lysed 72 h after transfection and analyzed for luciferase activity. These experiments were performed in three independent HIV-1− donors in triplicate samples, and two such experiments are represented in the figure.

Mentions: To determine whether LSP1 was detectable in immature and mature mDCs, mDCs from healthy individuals were examined before and after maturation with poly:IC. Both immature and mature mDCs expressed LSP1, and the levels did not change with maturation (Fig. 5 A), even though HIV-1 transfers more efficiently in mature mDCs (22). The physiological consequences of LSP1–DC-SIGN interactions were further studied by LSP1-specific siRNAs. Four LSP1-specific siRNAs (Fig. 5 B) were synthesized, and their effectiveness in down-regulating LSP1 was determined initially in Raji cells. Two siRNAs effectively decreased endogenous LSP1 in Raji B cells (Fig. 5 B, siRNAs in A and C), and this knockdown was most efficient at 24 h, with LSP1 levels returning to normal in 48 h. Down-regulation of LSP1 did not change expression of DC-SIGN, CD80, or MHC class II; however, it enhanced the transfer of HIV-1 to T cells (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20061604/DC1).


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 down-regulation causes increased HIV-1 transfer in human DCs. (A) LSP1 expression in human DCs. Cell lysates prepared from immature or poly:IC-matured mDCs isolated from human elutriated monocytes were assayed for LSP1 expression by a polyclonal anti-LSP1 antibody. Expression was measured against control cell lysates from 293T transfected with the LSP1 gene. (B) Specific siRNAs for human LSP1 cause a down-regulation of LSP1 expression. Raji–DC-SIGN cells were transfected with the indicated LSP1, and control siRNA duplexes were introduced by nucleofection. 24 h after transfection, cell lysates (20 μg of total protein) were assayed for LSP1 expression by immunoblot (left). The film images were digitized using an Epson scanner and quantified using Bio-Rad Quantity One software. The numbers obtained for LSP1 were corrected using the numbers obtained from actin and plotted relative to control in a Microsoft Excel graph as indicated (right). (C and D) Down-regulation of human LSP1 in human DCs and Raji DC-SIGN cell line causes an increase in HIV-1 transfer to T cells. Mature human DCs were transiently transfected by GeneSilencer with siRNA for LSP1 (siA and siC) or scrambled siRNA (control). To label transfected cells, unrelated Cy5 siRNA was mixed with both control siRNA and LSP1 siRNAs at a ratio of 4:1. 8 h after the siRNA transfection, the cells were sorted for the Cy5 label and plated onto 96-well plates. 24 h after transfection, cells were pulsed with luciferase expressing 780 ng/ml HIV-1ADA for 2 h at 37°C, washed five times, and incubated with A3R5 T cells. Cells were lysed 72 h after transfection and analyzed for luciferase activity. These experiments were performed in three independent HIV-1− donors in triplicate samples, and two such experiments are represented in the figure.
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Related In: Results  -  Collection

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fig5: LSP1 down-regulation causes increased HIV-1 transfer in human DCs. (A) LSP1 expression in human DCs. Cell lysates prepared from immature or poly:IC-matured mDCs isolated from human elutriated monocytes were assayed for LSP1 expression by a polyclonal anti-LSP1 antibody. Expression was measured against control cell lysates from 293T transfected with the LSP1 gene. (B) Specific siRNAs for human LSP1 cause a down-regulation of LSP1 expression. Raji–DC-SIGN cells were transfected with the indicated LSP1, and control siRNA duplexes were introduced by nucleofection. 24 h after transfection, cell lysates (20 μg of total protein) were assayed for LSP1 expression by immunoblot (left). The film images were digitized using an Epson scanner and quantified using Bio-Rad Quantity One software. The numbers obtained for LSP1 were corrected using the numbers obtained from actin and plotted relative to control in a Microsoft Excel graph as indicated (right). (C and D) Down-regulation of human LSP1 in human DCs and Raji DC-SIGN cell line causes an increase in HIV-1 transfer to T cells. Mature human DCs were transiently transfected by GeneSilencer with siRNA for LSP1 (siA and siC) or scrambled siRNA (control). To label transfected cells, unrelated Cy5 siRNA was mixed with both control siRNA and LSP1 siRNAs at a ratio of 4:1. 8 h after the siRNA transfection, the cells were sorted for the Cy5 label and plated onto 96-well plates. 24 h after transfection, cells were pulsed with luciferase expressing 780 ng/ml HIV-1ADA for 2 h at 37°C, washed five times, and incubated with A3R5 T cells. Cells were lysed 72 h after transfection and analyzed for luciferase activity. These experiments were performed in three independent HIV-1− donors in triplicate samples, and two such experiments are represented in the figure.
Mentions: To determine whether LSP1 was detectable in immature and mature mDCs, mDCs from healthy individuals were examined before and after maturation with poly:IC. Both immature and mature mDCs expressed LSP1, and the levels did not change with maturation (Fig. 5 A), even though HIV-1 transfers more efficiently in mature mDCs (22). The physiological consequences of LSP1–DC-SIGN interactions were further studied by LSP1-specific siRNAs. Four LSP1-specific siRNAs (Fig. 5 B) were synthesized, and their effectiveness in down-regulating LSP1 was determined initially in Raji cells. Two siRNAs effectively decreased endogenous LSP1 in Raji B cells (Fig. 5 B, siRNAs in A and C), and this knockdown was most efficient at 24 h, with LSP1 levels returning to normal in 48 h. Down-regulation of LSP1 did not change expression of DC-SIGN, CD80, or MHC class II; however, it enhanced the transfer of HIV-1 to T cells (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20061604/DC1).

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