An MHC-I Cytoplasmic Domain/HIV-1 Nef Fusion Protein Binds Directly to the μ Subunit of the AP-1 Endosomal Coat Complex

The down-regulation of the major histocompatibility complex class I (MHC-I) from the surface of infected cells by the Nef proteins of primate immunodeficiency viruses likely contributes to pathogenesis by providing evasion of cell-mediated immunity. HIV-1 Nef-induced down-regulation involves endosomal trafficking and a cooperative interaction between the cytoplasmic domain (CD) of MHC-I, Nef, and the clathrin adaptor protein complex-1 (AP-1). The CD of MHC-I contains a key tyrosine within the sequence YSQA that is required for down-regulation by Nef, but this sequence does not conform to the canonical AP-binding tyrosine-based motif Yxxphi, which mediates binding to the medium (micro) subunits of AP complexes. We previously proposed that Nef allows the MHC-I CD to bind the mu subunit of AP-1 (micro1) as if it contained a Yxxphimotif.Here, we show that a direct interaction between the MHC-I CD/Nef and micro1 plays a primary role in the down-regulation of MHC-I: GST pulldown assays using recombinant proteins indicated that most of the MHC-I CD and Nef residues that are required for the down-regulation in human cells contribute to direct interactions with a truncated version of micro1. Specifically, the tyrosine residue of the YSQA sequence in the MHC-I CD as well as Nef residues E62-65 and P78 each contributed to the interaction between MHC-I CD/Nef and micro1 in vitro, whereas Nef M20 had little to no role. Conversely, residues F172/D174 and V392/L395 of the binding pocket on micro1 for Yxxphi motifs were required for a robust interaction.These data indicate that the MHC-I cytoplasmic domain, Nef, and the C-terminal two thirds of the mu subunit of AP-1 are sufficient to constitute a biologically relevant interaction. The data also reveal an unexpected role for a hydrophobic pocket in micro1 for interaction with MHC-I CD/Nef

Downregulation of MHC-I by HIV-1 Nef : evolution after sexual transmission and mechanism of action

The Human Immunodeficiency Virus (HIV) Nef protein has evolved multiple methods to evade the host immune system. Reduction in cell-surface major histocompatibility complex I (MHC-I) is one such method; this enables virally- infected cells to evade lysis by cytotoxic T lymphocytes (CTL). In addition, Nef also performs functions related to HIV infectivity and replication. The main objective of this dissertation is to delineate the mechanism by which Nef downregulates MHC-I, and how this function may be sacrificed in order to optimize infectivity and replication. I first focused on the optimization of Nef functions after sexual transmission by studying the modulation of MHC-I and CD4 by Nef clones from phylogenetically- and epidemiologically-linked transmission partners. Flow cytometry of T cells transfected with these Nef clones showed that both CD4 and MHC-I downregulation are important to the establishment of a successful infection in a new host and neither function is expendible. Next I addressed how Nef modulates MHC-I trafficking in the cell by examining the interactions of Nef, MHC-I, and the non-clathrin endocytic pathway. I studied the role of the GTP cycle of the ADP-ribosylation factor 6 (ARF6) in the Nef-mediated downregulation of MHC- I. Immunofluorescence microscopy of cells transfected with both Nef and epitope-labeled ARF6 were examined for colocalization. Additionally, T cells transfected with Nef, and mutants of ARF6 that interfered with the ARF6-GTP cycle, were examined for cell-surface MHC-I by flow cytometry. While disruptions of the ARF6-GTP cycle did appear to interfere with MHC-I cell-surface expression, this result was not related to MHC-I downregulation by Nef. Finally, I changed the focus of the mechanistic studies to the intracellular endosomal system, specifically to adaptor protein-1(AP-1)-containing vesicles. Studying protein-protein interactions, I found that Nef and MHC-I can synergize to create a novel AP-1 interaction surface, and I determined residues in Nef, MHC -I, and the subunit of AP-1 involved in this interaction. In summary, my dissertation research has contributed to a greater understanding of not only the importance of MHC-I downregulation by HIV-1 Nef, but also to its mechanism in the cel

Cooperative Binding of the Class I Major Histocompatibility Complex Cytoplasmic Domain and Human Immunodeficiency Virus Type 1 Nef to the Endosomal AP-1 Complex via Its μ Subunit▿

Human immunodeficiency virus type 1 Nef provides immune evasion by decreasing the expression of major histocompatibility complex class I (MHC-I) at the surfaces of infected cells. The endosomal clathrin adaptor protein complex AP-1 is a key cellular cofactor for this activity, and it is recruited to the MHC-I cytoplasmic domain (CD) in the presence of Nef by an uncharacterized mechanism. To determine the molecular basis of this recruitment, we used an MHC-I CD-Nef fusion protein to represent the MHC-I CD/Nef complex during protein interaction assays. The MHC-I CD had no intrinsic ability to bind AP-1, but it conferred binding activity when fused to Nef. This activity was independent of the canonical leucine-based AP-binding motif in Nef; it required residue Y320 in the MHC-I CD and residues E62-65 and P78 in Nef, and it involved the μ but not the γ/σ subunits of AP-1. The impaired binding of mutants encoding substitutions of E62-65 or P78 in Nef was rescued by replacing the Y320SQA sequence in the MHC-I CD with YSQL, suggesting that Nef allows the YSQA sequence to act as if it were a canonical μ-binding motif. These data identify the μ subunit of AP-1 (μ1) as the key target of the MHC-I CD/Nef complex, and they indicate that both Y320 in the MHC-I CD and E62-65 in Nef interact directly with μ1. The data support a cooperative binding model in which Nef functions as a clathrin-associated sorting protein that allows recognition of an incomplete, tyrosine-based μ-binding signal in the MHC-I CD by AP-1

Surface expression in CEM T cells of CD8 chimeras containing the MHC-I CD, Nef, or the MHC-I CD-Nef chimeric sequence as cytoplasmic domains was measured by flow cytometry.

<p>Cells were transfected to express GFP as well as the CD8-chimeras, and the cells were gated for low and mid-intensity GFP expression. (A) Relative cell number vs. relative fluorescence intensity of CD8 [phycoerythrin (PE)]. Mean fluorescence intensities for CD8 (PE) are shown. The chimeric MHC-I CD-NefLL/AA sequence directs decreased expression at the cell surface relative to the MHC-I CD alone or NefLL/AA alone. This activity of the MHC-I CD when fused to Nef depends on Y320. (B) The total cellular expression of the chimeric molecules containing Nef was evaluated by western blot. Equal volumes of the transfected cell suspensions were collected before the FACS staining and lysed for western blot analysis using anti-Nef antibody. All chimeric molecules were expressed at a similar level. Actin was probed as a loading control.</p

Structural insights into opposing actions of neurosteroids on GABAA receptors

Abstract γ-Aminobutyric acid type A (GABAA) receptors mediate fast inhibitory signaling in the brain and are targets of numerous drugs and endogenous neurosteroids. A subset of neurosteroids are GABAA receptor positive allosteric modulators; one of these, allopregnanolone, is the only drug approved specifically for treating postpartum depression. There is a consensus emerging from structural, physiological and photolabeling studies as to where positive modulators bind, but how they potentiate GABA activation remains unclear. Other neurosteroids are negative modulators of GABAA receptors, but their binding sites remain debated. Here we present structures of a synaptic GABAA receptor bound to allopregnanolone and two inhibitory sulfated neurosteroids. Allopregnanolone binds at the receptor-bilayer interface, in the consensus potentiator site. In contrast, inhibitory neurosteroids bind in the pore. MD simulations and electrophysiology support a mechanism by which allopregnanolone potentiates channel activity and suggest the dominant mechanism for sulfated neurosteroid inhibition is through pore block

Direct interaction between the MHC-I CD-Nef and μ1: partially purified recombinant proteins were used in GST pulldown assays and analyzed on SDS-PAGE gels by staining with Coomassie blue.

<p>(A) From left to right: protein ladder, recombinant μ121 and μ158 proteins as markers, then six lanes of negative controls as indicated. (B) Specific binding between μ1 and MHC-I CD-Nef. Recombinant proteins (“Input”) were run as markers. Right four lanes show binding of μ121 or μ158 with either MHC-I CD-Nef or MHC-I CD-Nef LL/AA. Nef LL/AA indicates alanine substitution of Nef residues L164 and L165.</p

GST pulldown of recombinant μ1 <i>in vitro</i> using chimeric proteins in which the MHC-I CD is fused to the N-terminus of Nef and by MHC-I CD-Nef chimera mutants.

<p>Various residues within the CD of MHC-I and Nef were mutated to alanine as shown. (A) A western blot of the pulldown was probed with anti-His antibody to detect the recombinant μ1 (μ121). Band quantitation using Image J and setting the pulldown of μ1 by GST-CD-Nef to 100% yielded the following efficiencies for the constructs: GST-CD-NefLL/AA: 77%; GST-CD(Y320A)-NefLL/AA: 16%; GST-CD-Nef M20A,LL/AA: 53%; GST-CD-Nef E62-65A,LL/AA: 30%; GST-CD-Nef P78A,LL/AA: 19%. The results shown are representative of three independent experiments. (B) The amounts of GST-proteins used for binding setup are shown by Coomassie blue staining.</p

Purified recombinant truncated μ1 and μ1 mutants (F172A/D174S and V392A/L395A) were subjected to GST pulldown assays using MHC-I CD-Nef and MHC-I CD-Nef LL/AA chimeras:

<p>(A) Ponceau red staining of PVDF membrane. (B) A western blot of the pulldown was probed with anti-His6 antibody to detect the wild-type μ1 and μ1 mutants. Band quantitation using Image J and setting the pulldown of wild-type μ1 by GST-CD-Nef to 100% yielded the following efficiencies for the μ1 mutants: μ1_FD/AS: 57%; μ1_VL/AA: 24%. The results shown are representative of three independent experiments. (C) The amounts of GST-proteins used for binding setup are shown by Coomassie blue staining.</p

Schematic representation of the GST-MHC-I CD-Nef fusion protein.

<p>Key residues in MHC-I CD (Y320) and HIV1-Nef (M20, E62-65, P78) used in mutational and binding studies are shown.</p