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

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 μ1 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

Rab11 Helps Maintain Apical Crumbs and Adherens Junctions in the Drosophila Embryonic Ectoderm

BackgroundTissue morphogenesis and organogenesis require that cells retain stable cell-cell adhesion while changing shape and moving. One mechanism to accommodate this plasticity in cell adhesion involves regulated trafficking of junctional proteins.Methodology/Principal FindingsHere we explored trafficking of junctional proteins in two well-characterized model epithelia, the Drosophila embryonic ectoderm and amnioserosa. We find that DE-cadherin, the transmembrane protein of adherens junctions, is actively trafficked through putative vesicles, and appears to travel through both Rab5-positive and Rab11-positive structures. We manipulated the functions of Rab11 and Rab5 to examine the effects on junctional stability and morphogenesis. Reducing Rab11 function, either using a dominant negative construct or loss of function alleles, disrupts integrity of the ectoderm and leads to loss of adherens junctions. Strikingly, the apical junctional regulator Crumbs is lost before AJs are destabilized, while the basolateral protein Dlg remains cortical. Altering Rab5 function had less dramatic effects, not disrupting adherens junction integrity but affecting dorsal closure.Conclusions/SignificanceWe contrast our results with what others saw when disrupting other trafficking regulators, and when disrupting Rab function in other tissues; together these data suggest distinct mechanisms regulate junctional stability and plasticity in different tissues

HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

To facilitate viral infection and spread, HIV-1 Nef disrupts the surface expression of the viral receptor (CD4) and molecules capable of presenting HIV antigens to the immune system (MHC-I). To accomplish this, Nef binds to the cytoplasmic tails of both molecules and then, by mechanisms that are not well understood, disrupts the trafficking of each molecule in different ways. Specifically, Nef promotes CD4 internalization after it has been transported to the cell surface, whereas Nef uses the clathrin adaptor, AP-1, to disrupt normal transport of MHC-I from the TGN to the cell surface. Despite these differences in initial intracellular trafficking, we demonstrate that MHC-I and CD4 are ultimately found in the same Rab7+ vesicles and are both targeted for degradation via the activity of the Nef-interacting protein, β-COP. Moreover, we demonstrate that Nef contains two separable β-COP binding sites. One site, an arginine (RXR) motif in the N-terminal α helical domain of Nef, is necessary for maximal MHC-I degradation. The second site, composed of a di-acidic motif located in the C-terminal loop domain of Nef, is needed for efficient CD4 degradation. The requirement for redundant motifs with distinct roles supports a model in which Nef exists in multiple conformational states that allow access to different motifs, depending upon which cellular target is bound by Nef

Modulation of protein trafficking pathways by HIV-1 Nef.

HIV-1 Nef is a viral pathogenic determinant that manipulates the host cell biology to promote the viral life cycle. HIV-1 Nef has been reported to alter the intracellular trafficking of important molecules that participate in the anti-HIV immune response, such as class I and II major histocompatibility complex molecules (MHC-I and MHC-II), CD4, CD28, and DC-SIGN. The molecular details of MHC-I and CD4 disruption are poorly understood; however, it has been proposed that Nef affects intracellular trafficking pathways by acting as a molecular adaptor protein. In support of this theory, we determined that Nef interacted with the cytoplasmic domain of MHC-I both in vivo and in vitro, and required a highly conserved tyrosine-based sequence in MHC-I. Interestingly, MHC-I allotypes that harbored substitutions in this region were not susceptible to Nef. This differential regulation of MHC-I would allow the virus to simultaneously escape immune surveillance by both cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. To further define the exact mechanism of MHC-I disruption, we carefully examined MHC-I biology in T cells that expressed HIV-1 Nef. We found that MHC-I assembly, maturation, and transit to the trans-Golgi network (TGN) were not affected by HIV-1 Nef. However, instead of being exported to the cell surface, MHC-I was redirected to acidic compartments for degradation. The ability of Nef to alter MHC-I trafficking was dependent on expression of AP-1A, a cellular protein complex known to mediate TGN-to-endolysosomal vesicle transport. In HIV-infected primary T cells, Nef promoted a physical interaction between endogenous AP-1 and MHC-I, and we determined the recruitment of AP-1 required amino acids in both Nef and the MHC-I cytoplasmic tail. Finally, the Nef-dependent degradation of both MHC-I and CD4 was ultimately mediated through a late endosomal compartment, and required the expression of the small GTPase, Rab7. These molecular details reveal important clues about the biological activities of HIV-1 Nef and its role in the pathogenicity of HIV.Ph.D.Biological SciencesCellular biologyMicrobiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125211/2/3186749.pd

Rab11 helps maintain apical crumbs and adherens junctions in the Drosophila embryonic ectoderm.

BACKGROUND:Tissue morphogenesis and organogenesis require that cells retain stable cell-cell adhesion while changing shape and moving. One mechanism to accommodate this plasticity in cell adhesion involves regulated trafficking of junctional proteins. METHODOLOGY/PRINCIPAL FINDINGS:Here we explored trafficking of junctional proteins in two well-characterized model epithelia, the Drosophila embryonic ectoderm and amnioserosa. We find that DE-cadherin, the transmembrane protein of adherens junctions, is actively trafficked through putative vesicles, and appears to travel through both Rab5-positive and Rab11-positive structures. We manipulated the functions of Rab11 and Rab5 to examine the effects on junctional stability and morphogenesis. Reducing Rab11 function, either using a dominant negative construct or loss of function alleles, disrupts integrity of the ectoderm and leads to loss of adherens junctions. Strikingly, the apical junctional regulator Crumbs is lost before AJs are destabilized, while the basolateral protein Dlg remains cortical. Altering Rab5 function had less dramatic effects, not disrupting adherens junction integrity but affecting dorsal closure. CONCLUSIONS/SIGNIFICANCE:We contrast our results with what others saw when disrupting other trafficking regulators, and when disrupting Rab function in other tissues; together these data suggest distinct mechanisms regulate junctional stability and plasticity in different tissues

Direct Binding of Human Immunodeficiency Virus Type 1 Nef to the Major Histocompatibility Complex Class I (MHC-I) Cytoplasmic Tail Disrupts MHC-I Trafficking

Nef, an essential pathogenic determinant for human immunodeficiency virus type 1, has multiple functions that include disruption of major histocompatibility complex class I molecules (MHC-I) and CD4 and CD28 cell surface expression. The effects of Nef on MHC-I have been shown to protect infected cells from cytotoxic T-lymphocyte recognition by downmodulation of a subset of MHC-I (HLA-A and -B). The remaining HLA-C and -E molecules prevent recognition by natural killer (NK) cells, which would otherwise lyse cells expressing small amounts of MHC-I. Specific amino acid residues in the MHC-I cytoplasmic tail confer sensitivity to Nef, but their function is unknown. Here we show that purified Nef binds directly to the HLA-A2 cytoplasmic tail in vitro and that Nef forms complexes with MHC-I that can be isolated from human cells. The interaction between Nef and MHC-I appears to be weak, indicating that it may be transient or stabilized by other factors. Supporting the fact that these molecules interact in vivo, we found that Nef colocalizes with HLA-A2 molecules in a perinuclear distribution inside cells. In addition, we demonstrated that Nef fails to bind the HLA-E tail and also fails to bind HLA-A2 tails with deletions of amino acids necessary for MHC-I downmodulation. These data provide an explanation for differential downmodulation of MHC-I allotypes by Nef. In addition, they provide the first direct evidence indicating that Nef functions as an adaptor molecule able to link MHC-I to cellular trafficking proteins