Limits...
HIV-1 Nef disrupts MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail.

Roeth JF, Williams M, Kasper MR, Filzen TM, Collins KL - J. Cell Biol. (2004)

Bottom Line: We demonstrate that in HIV-infected primary T cells, Nef promotes a physical interaction between endogenous AP-1 and MHC-I.Moreover, we present data that this interaction uses a novel AP-1 binding site that requires amino acids in the MHC-I cytoplasmic tail.In sum, our evidence suggests that binding of AP-1 to the Nef-MHC-I complex is an important step required for inhibition of antigen presentation by HIV.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
To avoid immune recognition by cytotoxic T lymphocytes (CTLs), human immunodeficiency virus (HIV)-1 Nef disrupts the transport of major histocompatibility complex class I molecules (MHC-I) to the cell surface in HIV-infected T cells. However, the mechanism by which Nef does this is unknown. We report that Nef disrupts MHC-I trafficking by rerouting newly synthesized MHC-I from the trans-Golgi network (TGN) to lysosomal compartments for degradation. The ability of Nef to target MHC-I from the TGN to lysosomes is dependent on expression of the mu1 subunit of adaptor protein (AP) AP-1A, a cellular protein complex implicated in TGN to endolysosomal pathways. We demonstrate that in HIV-infected primary T cells, Nef promotes a physical interaction between endogenous AP-1 and MHC-I. Moreover, we present data that this interaction uses a novel AP-1 binding site that requires amino acids in the MHC-I cytoplasmic tail. In sum, our evidence suggests that binding of AP-1 to the Nef-MHC-I complex is an important step required for inhibition of antigen presentation by HIV.

Show MeSH

Related in: MedlinePlus

Nef expression results in coprecipitation of AP-1 with HLA-A2. (A) HLA-A2+ primary human T cells or control CEM-SS cells that did not express HLA-A2 (Ctrl) were transduced with an HIV molecular clone expressing Nef (nef+), or a matched control HIV that did not express Nef (nef−). Cells were harvested, and HLA-A2 was immunoprecipitated as described in Materials and methods. Proteins that coprecipitated with HLA-A2 were detected by Western blotting as indicated. Western blots of protein inputs are also shown as a control for relative protein levels in each sample before immunoprecipitation (Input Controls). Results are typical of two independent experiments. (B) AP-3 does not coprecipitate with HLA-A2. CEM-SS cells (lanes 1 and 4) or CEM HA-HLA-A2 cells (lanes 2, 3, 5, and 6) were infected with HIV, and HLA-A2 was recovered by immunoprecipitation as described in Materials and methods. Coprecipitating proteins were detected by Western blotting (lanes 1–3). A fraction of the protein input before immunoprecipitation was also analyzed to ensure similar protein expression levels (lanes 4–6). Western blots of δ-adaptin (AP-3) and γ-adaptin (AP-1) were performed simultaneously using the same antibody concentrations. Apparent molecular masses of protein standards are denoted in kilodaltons on the left.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172469&req=5

fig3: Nef expression results in coprecipitation of AP-1 with HLA-A2. (A) HLA-A2+ primary human T cells or control CEM-SS cells that did not express HLA-A2 (Ctrl) were transduced with an HIV molecular clone expressing Nef (nef+), or a matched control HIV that did not express Nef (nef−). Cells were harvested, and HLA-A2 was immunoprecipitated as described in Materials and methods. Proteins that coprecipitated with HLA-A2 were detected by Western blotting as indicated. Western blots of protein inputs are also shown as a control for relative protein levels in each sample before immunoprecipitation (Input Controls). Results are typical of two independent experiments. (B) AP-3 does not coprecipitate with HLA-A2. CEM-SS cells (lanes 1 and 4) or CEM HA-HLA-A2 cells (lanes 2, 3, 5, and 6) were infected with HIV, and HLA-A2 was recovered by immunoprecipitation as described in Materials and methods. Coprecipitating proteins were detected by Western blotting (lanes 1–3). A fraction of the protein input before immunoprecipitation was also analyzed to ensure similar protein expression levels (lanes 4–6). Western blots of δ-adaptin (AP-3) and γ-adaptin (AP-1) were performed simultaneously using the same antibody concentrations. Apparent molecular masses of protein standards are denoted in kilodaltons on the left.

Mentions: To determine whether Nef functions as an AP linking MHC-I to AP-1, we immunoprecipitated endogenous HLA-A2 from HIV-infected primary T cells and then immunoblotted for Nef and for endogenous subunits of AP-1. As expected, we detected Nef protein coprecipitating with HLA-A2 (Fig. 3 A). In addition, we also detected the μ and γ subunits of AP-1 coprecipitating with HLA-A2. This result is particularly striking because only ∼25% of the primary T cells were infected in this experiment. The interaction was highly specific based on the fact that the related AP, AP-3, did not coprecipitate with HLA-A2 (Fig. 3 B). Thus, these studies strongly indicate that Nef promotes the formation of a complex containing MHC-I and AP-1.


HIV-1 Nef disrupts MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail.

Roeth JF, Williams M, Kasper MR, Filzen TM, Collins KL - J. Cell Biol. (2004)

Nef expression results in coprecipitation of AP-1 with HLA-A2. (A) HLA-A2+ primary human T cells or control CEM-SS cells that did not express HLA-A2 (Ctrl) were transduced with an HIV molecular clone expressing Nef (nef+), or a matched control HIV that did not express Nef (nef−). Cells were harvested, and HLA-A2 was immunoprecipitated as described in Materials and methods. Proteins that coprecipitated with HLA-A2 were detected by Western blotting as indicated. Western blots of protein inputs are also shown as a control for relative protein levels in each sample before immunoprecipitation (Input Controls). Results are typical of two independent experiments. (B) AP-3 does not coprecipitate with HLA-A2. CEM-SS cells (lanes 1 and 4) or CEM HA-HLA-A2 cells (lanes 2, 3, 5, and 6) were infected with HIV, and HLA-A2 was recovered by immunoprecipitation as described in Materials and methods. Coprecipitating proteins were detected by Western blotting (lanes 1–3). A fraction of the protein input before immunoprecipitation was also analyzed to ensure similar protein expression levels (lanes 4–6). Western blots of δ-adaptin (AP-3) and γ-adaptin (AP-1) were performed simultaneously using the same antibody concentrations. Apparent molecular masses of protein standards are denoted in kilodaltons on the left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172469&req=5

fig3: Nef expression results in coprecipitation of AP-1 with HLA-A2. (A) HLA-A2+ primary human T cells or control CEM-SS cells that did not express HLA-A2 (Ctrl) were transduced with an HIV molecular clone expressing Nef (nef+), or a matched control HIV that did not express Nef (nef−). Cells were harvested, and HLA-A2 was immunoprecipitated as described in Materials and methods. Proteins that coprecipitated with HLA-A2 were detected by Western blotting as indicated. Western blots of protein inputs are also shown as a control for relative protein levels in each sample before immunoprecipitation (Input Controls). Results are typical of two independent experiments. (B) AP-3 does not coprecipitate with HLA-A2. CEM-SS cells (lanes 1 and 4) or CEM HA-HLA-A2 cells (lanes 2, 3, 5, and 6) were infected with HIV, and HLA-A2 was recovered by immunoprecipitation as described in Materials and methods. Coprecipitating proteins were detected by Western blotting (lanes 1–3). A fraction of the protein input before immunoprecipitation was also analyzed to ensure similar protein expression levels (lanes 4–6). Western blots of δ-adaptin (AP-3) and γ-adaptin (AP-1) were performed simultaneously using the same antibody concentrations. Apparent molecular masses of protein standards are denoted in kilodaltons on the left.
Mentions: To determine whether Nef functions as an AP linking MHC-I to AP-1, we immunoprecipitated endogenous HLA-A2 from HIV-infected primary T cells and then immunoblotted for Nef and for endogenous subunits of AP-1. As expected, we detected Nef protein coprecipitating with HLA-A2 (Fig. 3 A). In addition, we also detected the μ and γ subunits of AP-1 coprecipitating with HLA-A2. This result is particularly striking because only ∼25% of the primary T cells were infected in this experiment. The interaction was highly specific based on the fact that the related AP, AP-3, did not coprecipitate with HLA-A2 (Fig. 3 B). Thus, these studies strongly indicate that Nef promotes the formation of a complex containing MHC-I and AP-1.

Bottom Line: We demonstrate that in HIV-infected primary T cells, Nef promotes a physical interaction between endogenous AP-1 and MHC-I.Moreover, we present data that this interaction uses a novel AP-1 binding site that requires amino acids in the MHC-I cytoplasmic tail.In sum, our evidence suggests that binding of AP-1 to the Nef-MHC-I complex is an important step required for inhibition of antigen presentation by HIV.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
To avoid immune recognition by cytotoxic T lymphocytes (CTLs), human immunodeficiency virus (HIV)-1 Nef disrupts the transport of major histocompatibility complex class I molecules (MHC-I) to the cell surface in HIV-infected T cells. However, the mechanism by which Nef does this is unknown. We report that Nef disrupts MHC-I trafficking by rerouting newly synthesized MHC-I from the trans-Golgi network (TGN) to lysosomal compartments for degradation. The ability of Nef to target MHC-I from the TGN to lysosomes is dependent on expression of the mu1 subunit of adaptor protein (AP) AP-1A, a cellular protein complex implicated in TGN to endolysosomal pathways. We demonstrate that in HIV-infected primary T cells, Nef promotes a physical interaction between endogenous AP-1 and MHC-I. Moreover, we present data that this interaction uses a novel AP-1 binding site that requires amino acids in the MHC-I cytoplasmic tail. In sum, our evidence suggests that binding of AP-1 to the Nef-MHC-I complex is an important step required for inhibition of antigen presentation by HIV.

Show MeSH
Related in: MedlinePlus