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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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Analysis of MHC-I and Nef domains that contribute to AP-1 recruitment. (A) Recruitment of AP-1 requires an intact Nef–MHC-I complex and is independent of the dileucine motif in Nef. CEM-SS cells (lane 1) or CEM HA-HLA-A2 cells (lanes 2–5) were transduced with the indicated adenoviral vector. The cells were harvested at 72 h and subjected to the coimmunoprecipitation assay. Cells were transduced with each adenovirus to achieve equivalent Nef expression levels (Input Controls): wild-type NL4-3 Nef (MOI = 25); NL4-3 Nef with a NH2-terminal deletion (V10EΔ17-26, MOI = 300); HXB Nef with a mutation in the dileucine motif (LL164,165AA, MOI = 25). (As shown, HXB Nef migrates more slowly on SDS PAGE than NL4-3 Nef. Wild-type HXB Nef and HXB NefLL164,165AA are directly compared in Fig. S3.) All results are typical of at least two independent experiments. (B) HLA-A2 tyrosine 320 (Y320) is required for efficient Nef binding, AP-1 recruitment, and targeting for degradation. CEM-SS (lanes 1 and 2), CEM HA-HLA-A2 (lanes 3 and 4), or CEM cells expressing the MHC-I mutant, HA-HLA-A2 Y320A (lanes 5 and 6), were transduced with a control (−) or Nef-expressing adenovirus (+). At 72 h after transduction, HLA-A2 immunoprecipitations were recovered and analyzed by Western blotting as described in Fig. 3.
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fig4: Analysis of MHC-I and Nef domains that contribute to AP-1 recruitment. (A) Recruitment of AP-1 requires an intact Nef–MHC-I complex and is independent of the dileucine motif in Nef. CEM-SS cells (lane 1) or CEM HA-HLA-A2 cells (lanes 2–5) were transduced with the indicated adenoviral vector. The cells were harvested at 72 h and subjected to the coimmunoprecipitation assay. Cells were transduced with each adenovirus to achieve equivalent Nef expression levels (Input Controls): wild-type NL4-3 Nef (MOI = 25); NL4-3 Nef with a NH2-terminal deletion (V10EΔ17-26, MOI = 300); HXB Nef with a mutation in the dileucine motif (LL164,165AA, MOI = 25). (As shown, HXB Nef migrates more slowly on SDS PAGE than NL4-3 Nef. Wild-type HXB Nef and HXB NefLL164,165AA are directly compared in Fig. S3.) All results are typical of at least two independent experiments. (B) HLA-A2 tyrosine 320 (Y320) is required for efficient Nef binding, AP-1 recruitment, and targeting for degradation. CEM-SS (lanes 1 and 2), CEM HA-HLA-A2 (lanes 3 and 4), or CEM cells expressing the MHC-I mutant, HA-HLA-A2 Y320A (lanes 5 and 6), were transduced with a control (−) or Nef-expressing adenovirus (+). At 72 h after transduction, HLA-A2 immunoprecipitations were recovered and analyzed by Western blotting as described in Fig. 3.

Mentions: Previous studies that have examined the direct interaction of Nef with AP-1 have found that it depends on the Nef dileucine motif (Bresnahan et al., 1998; Craig et al., 2000; Janvier et al., 2003a,b). However, we found that the dileucine motif was not needed for coprecipitation of AP-1 with MHC-I in CEM T cells treated with adeno-Nef (Fig. 4 A) or HIV (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200407031/DC1). These data are consistent with the fact that the dileucine motif is not necessary for disruption of MHC-I trafficking (Greenberg et al., 1998a; Mangasarian et al., 1999), and suggests that there is an alternative AP-1 binding site in 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)

Analysis of MHC-I and Nef domains that contribute to AP-1 recruitment. (A) Recruitment of AP-1 requires an intact Nef–MHC-I complex and is independent of the dileucine motif in Nef. CEM-SS cells (lane 1) or CEM HA-HLA-A2 cells (lanes 2–5) were transduced with the indicated adenoviral vector. The cells were harvested at 72 h and subjected to the coimmunoprecipitation assay. Cells were transduced with each adenovirus to achieve equivalent Nef expression levels (Input Controls): wild-type NL4-3 Nef (MOI = 25); NL4-3 Nef with a NH2-terminal deletion (V10EΔ17-26, MOI = 300); HXB Nef with a mutation in the dileucine motif (LL164,165AA, MOI = 25). (As shown, HXB Nef migrates more slowly on SDS PAGE than NL4-3 Nef. Wild-type HXB Nef and HXB NefLL164,165AA are directly compared in Fig. S3.) All results are typical of at least two independent experiments. (B) HLA-A2 tyrosine 320 (Y320) is required for efficient Nef binding, AP-1 recruitment, and targeting for degradation. CEM-SS (lanes 1 and 2), CEM HA-HLA-A2 (lanes 3 and 4), or CEM cells expressing the MHC-I mutant, HA-HLA-A2 Y320A (lanes 5 and 6), were transduced with a control (−) or Nef-expressing adenovirus (+). At 72 h after transduction, HLA-A2 immunoprecipitations were recovered and analyzed by Western blotting as described in Fig. 3.
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fig4: Analysis of MHC-I and Nef domains that contribute to AP-1 recruitment. (A) Recruitment of AP-1 requires an intact Nef–MHC-I complex and is independent of the dileucine motif in Nef. CEM-SS cells (lane 1) or CEM HA-HLA-A2 cells (lanes 2–5) were transduced with the indicated adenoviral vector. The cells were harvested at 72 h and subjected to the coimmunoprecipitation assay. Cells were transduced with each adenovirus to achieve equivalent Nef expression levels (Input Controls): wild-type NL4-3 Nef (MOI = 25); NL4-3 Nef with a NH2-terminal deletion (V10EΔ17-26, MOI = 300); HXB Nef with a mutation in the dileucine motif (LL164,165AA, MOI = 25). (As shown, HXB Nef migrates more slowly on SDS PAGE than NL4-3 Nef. Wild-type HXB Nef and HXB NefLL164,165AA are directly compared in Fig. S3.) All results are typical of at least two independent experiments. (B) HLA-A2 tyrosine 320 (Y320) is required for efficient Nef binding, AP-1 recruitment, and targeting for degradation. CEM-SS (lanes 1 and 2), CEM HA-HLA-A2 (lanes 3 and 4), or CEM cells expressing the MHC-I mutant, HA-HLA-A2 Y320A (lanes 5 and 6), were transduced with a control (−) or Nef-expressing adenovirus (+). At 72 h after transduction, HLA-A2 immunoprecipitations were recovered and analyzed by Western blotting as described in Fig. 3.
Mentions: Previous studies that have examined the direct interaction of Nef with AP-1 have found that it depends on the Nef dileucine motif (Bresnahan et al., 1998; Craig et al., 2000; Janvier et al., 2003a,b). However, we found that the dileucine motif was not needed for coprecipitation of AP-1 with MHC-I in CEM T cells treated with adeno-Nef (Fig. 4 A) or HIV (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200407031/DC1). These data are consistent with the fact that the dileucine motif is not necessary for disruption of MHC-I trafficking (Greenberg et al., 1998a; Mangasarian et al., 1999), and suggests that there is an alternative AP-1 binding site in 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