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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 targets MHC-I into an AP-1–dependent pathway. (A and B) Western blot analysis of adaptin and Nef expression in siRNA-treated cells. Astrocytic cells (A) or CEM T cells (B) were transfected with the indicated siRNA and transduced with control or Nef-expressing adenoviruses as described in Materials and methods. Lysates were harvested and immunoblotted for the indicated protein. (C and D) Flow cytometric analysis of siRNA-treated cells. Astrocytic cells (C) or CEM T cells (D) were stained for surface HLA-A2 expression (left column) or surface CD4 expression (right column); shaded curve, control adenovirus; black line, Nef-expressing adenovirus. (E) Depletion of AP-1 inhibits Nef-dependent HLA-A2 degradation. CEM T cells were transduced and treated with siRNAs as described above except that an additional siRNA transfection was included before transduction. Lysates were prepared and analyzed by immunoblot to examine the expression levels of the indicated proteins.
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fig2: HIV-1 Nef targets MHC-I into an AP-1–dependent pathway. (A and B) Western blot analysis of adaptin and Nef expression in siRNA-treated cells. Astrocytic cells (A) or CEM T cells (B) were transfected with the indicated siRNA and transduced with control or Nef-expressing adenoviruses as described in Materials and methods. Lysates were harvested and immunoblotted for the indicated protein. (C and D) Flow cytometric analysis of siRNA-treated cells. Astrocytic cells (C) or CEM T cells (D) were stained for surface HLA-A2 expression (left column) or surface CD4 expression (right column); shaded curve, control adenovirus; black line, Nef-expressing adenovirus. (E) Depletion of AP-1 inhibits Nef-dependent HLA-A2 degradation. CEM T cells were transduced and treated with siRNAs as described above except that an additional siRNA transfection was included before transduction. Lysates were prepared and analyzed by immunoblot to examine the expression levels of the indicated proteins.

Mentions: Transport of lysosomal hydrolases and LAMP-1 to the lysosomes is known to require the APs AP-1 and AP-3, respectively. Interestingly, both of these adaptors are known to interact with HIV-1 Nef. Therefore, to explore a possible requirement for these complexes, we transfected cells with siRNAs directed at the μ subunit of AP-1A and AP-3. As shown in Fig. 2 (A and B), we were able to reduce expression of these molecules in both astrocytic cells and T cells. The effect was more dramatic in astrocytic cells, probably because we were able to transfect them more efficiently than T cells. In both cell types, we found that inhibiting the expression of AP-1A, but not that of AP-3, reduced the effect of Nef on MHC-I cell surface expression (Fig. 2, C and D). In addition, AP-1A expression was required for HLA-A2 degradation (Fig. 2 E). However, reducing the expression of μ1A and μ3 did not affect Nef's ability to downmodulate CD4 (Fig. 2, C and D), nor did it affect Nef expression (Fig. 2, A, B, and E). These results suggest that AP-1A expression is specifically required for Nef to disrupt MHC-I cell surface expression and to target it for degradation.


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 targets MHC-I into an AP-1–dependent pathway. (A and B) Western blot analysis of adaptin and Nef expression in siRNA-treated cells. Astrocytic cells (A) or CEM T cells (B) were transfected with the indicated siRNA and transduced with control or Nef-expressing adenoviruses as described in Materials and methods. Lysates were harvested and immunoblotted for the indicated protein. (C and D) Flow cytometric analysis of siRNA-treated cells. Astrocytic cells (C) or CEM T cells (D) were stained for surface HLA-A2 expression (left column) or surface CD4 expression (right column); shaded curve, control adenovirus; black line, Nef-expressing adenovirus. (E) Depletion of AP-1 inhibits Nef-dependent HLA-A2 degradation. CEM T cells were transduced and treated with siRNAs as described above except that an additional siRNA transfection was included before transduction. Lysates were prepared and analyzed by immunoblot to examine the expression levels of the indicated proteins.
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Related In: Results  -  Collection

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

fig2: HIV-1 Nef targets MHC-I into an AP-1–dependent pathway. (A and B) Western blot analysis of adaptin and Nef expression in siRNA-treated cells. Astrocytic cells (A) or CEM T cells (B) were transfected with the indicated siRNA and transduced with control or Nef-expressing adenoviruses as described in Materials and methods. Lysates were harvested and immunoblotted for the indicated protein. (C and D) Flow cytometric analysis of siRNA-treated cells. Astrocytic cells (C) or CEM T cells (D) were stained for surface HLA-A2 expression (left column) or surface CD4 expression (right column); shaded curve, control adenovirus; black line, Nef-expressing adenovirus. (E) Depletion of AP-1 inhibits Nef-dependent HLA-A2 degradation. CEM T cells were transduced and treated with siRNAs as described above except that an additional siRNA transfection was included before transduction. Lysates were prepared and analyzed by immunoblot to examine the expression levels of the indicated proteins.
Mentions: Transport of lysosomal hydrolases and LAMP-1 to the lysosomes is known to require the APs AP-1 and AP-3, respectively. Interestingly, both of these adaptors are known to interact with HIV-1 Nef. Therefore, to explore a possible requirement for these complexes, we transfected cells with siRNAs directed at the μ subunit of AP-1A and AP-3. As shown in Fig. 2 (A and B), we were able to reduce expression of these molecules in both astrocytic cells and T cells. The effect was more dramatic in astrocytic cells, probably because we were able to transfect them more efficiently than T cells. In both cell types, we found that inhibiting the expression of AP-1A, but not that of AP-3, reduced the effect of Nef on MHC-I cell surface expression (Fig. 2, C and D). In addition, AP-1A expression was required for HLA-A2 degradation (Fig. 2 E). However, reducing the expression of μ1A and μ3 did not affect Nef's ability to downmodulate CD4 (Fig. 2, C and D), nor did it affect Nef expression (Fig. 2, A, B, and E). These results suggest that AP-1A expression is specifically required for Nef to disrupt MHC-I cell surface expression and to target it for degradation.

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