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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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HIV-1 Nef redirects MHC-I from the TGN to lysosomes. (A and B) Nef does not affect the maturation of HLA-A2 through the TGN. CEM HA-HLA-A2 cells were transduced with a control adenovirus (nef−) or an adenovirus expressing HIV-1 Nef (nef+) and subjected to a pulse-chase metabolic labeling assay to follow proteins through the biosynthetic pathway. HA-HLA-A2 species with different gel mobilities were identified based on enzymatic digestion profiles: (a) N-glycosylated and sialylated HA-HLA-A2; (b) N-glycosylated HA-HLA-A2; and (c) core HA-HLA-A2 protein. (B) Quantitation of the relative percentage of endo H resistant and sialylated HA-HLA-A2. The mean percentage ± SD from two independent experiments is plotted over time. (C) Nef-induced degradation of mature MHC-I is blocked by inhibitors of acidic degradation. CEM HA-HLA-A2 cells were treated with adenovirus, pulsed with radioactive amino acids, and were either collected immediately (lanes 1 and 2) or chased for 4 h in media containing the indicated chemical inhibitor (lanes 3–12). Cellular lysates were divided equally, and HA-HLA-A2 or transferrin receptor was recovered by immunoprecipitation. All samples were digested with endo H before SDS-PAGE. The results are representative of three independent experiments. (D) Bafilomycin A1 treatment increases the degree of colocalization between HLA-A2 and LAMP-1. Cells transduced with the indicated adenovirus were treated with either solvent alone (DMSO) or bafilomycin A1 (Baf A1) for 4 h before staining. HLA-A2 and LAMP-1 were detected by indirect immunofluorescence using mAbs as described in Materials and methods. Arrows indicate colocalization between HLA-A2 and LAMP-1. Images were collected using a confocal microscope. Individual z-sections are shown. Bar, 5 μm.
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fig1: HIV-1 Nef redirects MHC-I from the TGN to lysosomes. (A and B) Nef does not affect the maturation of HLA-A2 through the TGN. CEM HA-HLA-A2 cells were transduced with a control adenovirus (nef−) or an adenovirus expressing HIV-1 Nef (nef+) and subjected to a pulse-chase metabolic labeling assay to follow proteins through the biosynthetic pathway. HA-HLA-A2 species with different gel mobilities were identified based on enzymatic digestion profiles: (a) N-glycosylated and sialylated HA-HLA-A2; (b) N-glycosylated HA-HLA-A2; and (c) core HA-HLA-A2 protein. (B) Quantitation of the relative percentage of endo H resistant and sialylated HA-HLA-A2. The mean percentage ± SD from two independent experiments is plotted over time. (C) Nef-induced degradation of mature MHC-I is blocked by inhibitors of acidic degradation. CEM HA-HLA-A2 cells were treated with adenovirus, pulsed with radioactive amino acids, and were either collected immediately (lanes 1 and 2) or chased for 4 h in media containing the indicated chemical inhibitor (lanes 3–12). Cellular lysates were divided equally, and HA-HLA-A2 or transferrin receptor was recovered by immunoprecipitation. All samples were digested with endo H before SDS-PAGE. The results are representative of three independent experiments. (D) Bafilomycin A1 treatment increases the degree of colocalization between HLA-A2 and LAMP-1. Cells transduced with the indicated adenovirus were treated with either solvent alone (DMSO) or bafilomycin A1 (Baf A1) for 4 h before staining. HLA-A2 and LAMP-1 were detected by indirect immunofluorescence using mAbs as described in Materials and methods. Arrows indicate colocalization between HLA-A2 and LAMP-1. Images were collected using a confocal microscope. Individual z-sections are shown. Bar, 5 μm.

Mentions: It has been previously shown that Nef does not affect the transport of MHC-I into the medial Golgi apparatus (Schwartz et al., 1996), but does inhibit its transport to the cell surface (Kasper and Collins, 2003). To further define where in the secretory pathway Nef exerts its effects, we asked whether MHC-I was transported normally into the TGN. This was accomplished by monitoring the rate at which HLA-A2 acquired sialic acid residues in this compartment, which can be measured by testing sensitivity to neuraminidase digestion. We detected sialylation of HLA-A2 as a slight increase in HLA-A2 molecular weight (Fig. 1 A, lanes 4 and 7) that was eliminated by neuraminidase treatment (Fig. 1 A, lanes 5 and 8). Based on these data (quantified in Fig. 1 B), Nef does not delay the transport of HLA-A2 into the TGN. As a control, we also confirmed the previously published observation that Nef does not affect transit into the medial Golgi (Schwartz et al., 1996), as assessed by sensitivity to endoglycosidase H (endo H) digestion (Fig. 1, A and B).


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)

HIV-1 Nef redirects MHC-I from the TGN to lysosomes. (A and B) Nef does not affect the maturation of HLA-A2 through the TGN. CEM HA-HLA-A2 cells were transduced with a control adenovirus (nef−) or an adenovirus expressing HIV-1 Nef (nef+) and subjected to a pulse-chase metabolic labeling assay to follow proteins through the biosynthetic pathway. HA-HLA-A2 species with different gel mobilities were identified based on enzymatic digestion profiles: (a) N-glycosylated and sialylated HA-HLA-A2; (b) N-glycosylated HA-HLA-A2; and (c) core HA-HLA-A2 protein. (B) Quantitation of the relative percentage of endo H resistant and sialylated HA-HLA-A2. The mean percentage ± SD from two independent experiments is plotted over time. (C) Nef-induced degradation of mature MHC-I is blocked by inhibitors of acidic degradation. CEM HA-HLA-A2 cells were treated with adenovirus, pulsed with radioactive amino acids, and were either collected immediately (lanes 1 and 2) or chased for 4 h in media containing the indicated chemical inhibitor (lanes 3–12). Cellular lysates were divided equally, and HA-HLA-A2 or transferrin receptor was recovered by immunoprecipitation. All samples were digested with endo H before SDS-PAGE. The results are representative of three independent experiments. (D) Bafilomycin A1 treatment increases the degree of colocalization between HLA-A2 and LAMP-1. Cells transduced with the indicated adenovirus were treated with either solvent alone (DMSO) or bafilomycin A1 (Baf A1) for 4 h before staining. HLA-A2 and LAMP-1 were detected by indirect immunofluorescence using mAbs as described in Materials and methods. Arrows indicate colocalization between HLA-A2 and LAMP-1. Images were collected using a confocal microscope. Individual z-sections are shown. Bar, 5 μm.
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Related In: Results  -  Collection

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

fig1: HIV-1 Nef redirects MHC-I from the TGN to lysosomes. (A and B) Nef does not affect the maturation of HLA-A2 through the TGN. CEM HA-HLA-A2 cells were transduced with a control adenovirus (nef−) or an adenovirus expressing HIV-1 Nef (nef+) and subjected to a pulse-chase metabolic labeling assay to follow proteins through the biosynthetic pathway. HA-HLA-A2 species with different gel mobilities were identified based on enzymatic digestion profiles: (a) N-glycosylated and sialylated HA-HLA-A2; (b) N-glycosylated HA-HLA-A2; and (c) core HA-HLA-A2 protein. (B) Quantitation of the relative percentage of endo H resistant and sialylated HA-HLA-A2. The mean percentage ± SD from two independent experiments is plotted over time. (C) Nef-induced degradation of mature MHC-I is blocked by inhibitors of acidic degradation. CEM HA-HLA-A2 cells were treated with adenovirus, pulsed with radioactive amino acids, and were either collected immediately (lanes 1 and 2) or chased for 4 h in media containing the indicated chemical inhibitor (lanes 3–12). Cellular lysates were divided equally, and HA-HLA-A2 or transferrin receptor was recovered by immunoprecipitation. All samples were digested with endo H before SDS-PAGE. The results are representative of three independent experiments. (D) Bafilomycin A1 treatment increases the degree of colocalization between HLA-A2 and LAMP-1. Cells transduced with the indicated adenovirus were treated with either solvent alone (DMSO) or bafilomycin A1 (Baf A1) for 4 h before staining. HLA-A2 and LAMP-1 were detected by indirect immunofluorescence using mAbs as described in Materials and methods. Arrows indicate colocalization between HLA-A2 and LAMP-1. Images were collected using a confocal microscope. Individual z-sections are shown. Bar, 5 μm.
Mentions: It has been previously shown that Nef does not affect the transport of MHC-I into the medial Golgi apparatus (Schwartz et al., 1996), but does inhibit its transport to the cell surface (Kasper and Collins, 2003). To further define where in the secretory pathway Nef exerts its effects, we asked whether MHC-I was transported normally into the TGN. This was accomplished by monitoring the rate at which HLA-A2 acquired sialic acid residues in this compartment, which can be measured by testing sensitivity to neuraminidase digestion. We detected sialylation of HLA-A2 as a slight increase in HLA-A2 molecular weight (Fig. 1 A, lanes 4 and 7) that was eliminated by neuraminidase treatment (Fig. 1 A, lanes 5 and 8). Based on these data (quantified in Fig. 1 B), Nef does not delay the transport of HLA-A2 into the TGN. As a control, we also confirmed the previously published observation that Nef does not affect transit into the medial Golgi (Schwartz et al., 1996), as assessed by sensitivity to endoglycosidase H (endo H) digestion (Fig. 1, A and B).

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