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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.

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The cytoplasmic domain of HLA-A2 and the NH2-terminal α helix of Nef are both required for AP-1 binding. (A and B) The NH2-terminal α-helix in Nef is necessary for AP-1 recruitment. CEM-SS cells were transduced with the murine retrovirus encoding HLA-A2 or the indicated A2/Nef fusion constructs. Coprecipitating proteins were detected by Western blotting of anti-HLA-A2 immunoprecipitations. (C) AP-1 binding to the A2/Nef fusion protein requires tyrosine 320 (Y320) in the cytoplasmic tail of HLA-A2. The indicated mutations in the A2/Nef fusion protein were tested for involvement in AP-1 binding using the assay described above. A2ΔTail is an A2/Nef fusion protein that lacks the entire HLA-A2 cytoplasmic tail. All results are representative of at least three independent experiments. White lines indicate that intervening lanes have been spliced out.
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fig6: The cytoplasmic domain of HLA-A2 and the NH2-terminal α helix of Nef are both required for AP-1 binding. (A and B) The NH2-terminal α-helix in Nef is necessary for AP-1 recruitment. CEM-SS cells were transduced with the murine retrovirus encoding HLA-A2 or the indicated A2/Nef fusion constructs. Coprecipitating proteins were detected by Western blotting of anti-HLA-A2 immunoprecipitations. (C) AP-1 binding to the A2/Nef fusion protein requires tyrosine 320 (Y320) in the cytoplasmic tail of HLA-A2. The indicated mutations in the A2/Nef fusion protein were tested for involvement in AP-1 binding using the assay described above. A2ΔTail is an A2/Nef fusion protein that lacks the entire HLA-A2 cytoplasmic tail. All results are representative of at least three independent experiments. White lines indicate that intervening lanes have been spliced out.

Mentions: The domains of HIV Nef specifically involved in MHC-I trafficking have been well characterized. It is known that disruption of Nef's NH2-terminal α helix by deletion (V10EΔ17-26) or point mutation (M20A), mutation of an acidic cluster (E62-65A), or mutation of a polyproline repeat (P69/72/75/78A) specifically affects this activity (Greenberg et al., 1998b; Mangasarian et al., 1999). We have recently reported that each of these mutants fails to coprecipitate with MHC-I (Williams et al., 2004) and thus binding to MHC-I may be the primary role of these domains. However, it is also possible that one or more of these domains plays an additional role. To determine whether any of these domains might also be involved in AP-1 recruitment directly, we bypassed the requirement for Nef binding by directly fusing Nef to the COOH terminus of the HLA-A2 cytoplasmic tail (A2/Nef; Fig. 5 A). As expected, Nef was able to reduce the cell surface expression of HLA-A2 in cis (Fig. 5 B, left), and to promote its colocalization with markers of the Golgi apparatus (Fig. 5 C). In addition, the A2/Nef fusion protein was able to efficiently coprecipitate AP-1 from T cell lysates in a manner that was independent of the Nef dileucine motif (Fig. 6 A). However, the fusion protein was not capable of reducing MHC-I surface expression in trans (Fig. 5 B, right). Thus, A2/Nef was a useful reagent to explore which amino acids in Nef and MHC-I were important for AP-1 recruitment to the Nef–MHC-I complex.


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)

The cytoplasmic domain of HLA-A2 and the NH2-terminal α helix of Nef are both required for AP-1 binding. (A and B) The NH2-terminal α-helix in Nef is necessary for AP-1 recruitment. CEM-SS cells were transduced with the murine retrovirus encoding HLA-A2 or the indicated A2/Nef fusion constructs. Coprecipitating proteins were detected by Western blotting of anti-HLA-A2 immunoprecipitations. (C) AP-1 binding to the A2/Nef fusion protein requires tyrosine 320 (Y320) in the cytoplasmic tail of HLA-A2. The indicated mutations in the A2/Nef fusion protein were tested for involvement in AP-1 binding using the assay described above. A2ΔTail is an A2/Nef fusion protein that lacks the entire HLA-A2 cytoplasmic tail. All results are representative of at least three independent experiments. White lines indicate that intervening lanes have been spliced out.
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Related In: Results  -  Collection

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

fig6: The cytoplasmic domain of HLA-A2 and the NH2-terminal α helix of Nef are both required for AP-1 binding. (A and B) The NH2-terminal α-helix in Nef is necessary for AP-1 recruitment. CEM-SS cells were transduced with the murine retrovirus encoding HLA-A2 or the indicated A2/Nef fusion constructs. Coprecipitating proteins were detected by Western blotting of anti-HLA-A2 immunoprecipitations. (C) AP-1 binding to the A2/Nef fusion protein requires tyrosine 320 (Y320) in the cytoplasmic tail of HLA-A2. The indicated mutations in the A2/Nef fusion protein were tested for involvement in AP-1 binding using the assay described above. A2ΔTail is an A2/Nef fusion protein that lacks the entire HLA-A2 cytoplasmic tail. All results are representative of at least three independent experiments. White lines indicate that intervening lanes have been spliced out.
Mentions: The domains of HIV Nef specifically involved in MHC-I trafficking have been well characterized. It is known that disruption of Nef's NH2-terminal α helix by deletion (V10EΔ17-26) or point mutation (M20A), mutation of an acidic cluster (E62-65A), or mutation of a polyproline repeat (P69/72/75/78A) specifically affects this activity (Greenberg et al., 1998b; Mangasarian et al., 1999). We have recently reported that each of these mutants fails to coprecipitate with MHC-I (Williams et al., 2004) and thus binding to MHC-I may be the primary role of these domains. However, it is also possible that one or more of these domains plays an additional role. To determine whether any of these domains might also be involved in AP-1 recruitment directly, we bypassed the requirement for Nef binding by directly fusing Nef to the COOH terminus of the HLA-A2 cytoplasmic tail (A2/Nef; Fig. 5 A). As expected, Nef was able to reduce the cell surface expression of HLA-A2 in cis (Fig. 5 B, left), and to promote its colocalization with markers of the Golgi apparatus (Fig. 5 C). In addition, the A2/Nef fusion protein was able to efficiently coprecipitate AP-1 from T cell lysates in a manner that was independent of the Nef dileucine motif (Fig. 6 A). However, the fusion protein was not capable of reducing MHC-I surface expression in trans (Fig. 5 B, right). Thus, A2/Nef was a useful reagent to explore which amino acids in Nef and MHC-I were important for AP-1 recruitment to the Nef–MHC-I complex.

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