Two independent regions of HIV-1 Nef are required for connection with the endocytic pathway through binding to the mu 1 chain of AP1 complex.

The Nef protein from the human immunodeficiency virus (HIV) induces down-regulation of the CD4 and major histocompatibility complex class I molecules from the cell surface by interfering with the endocytic machinery. This work focuses on the interaction of HIV-1 Nef with the mu 1 chain of adaptor protein type 1 (AP1) complex and its contribution to the Nef-induced alterations of membrane trafficking. Two independent regions surrounding a disordered loop located in the C-terminal part of Nef are involved in mu 1 binding. Each region can separately interact with mu 1, and simultaneous point mutations within both regions are needed to abolish binding. We used CD8 chimeras in which the cytoplasmic tail was replaced by Nef mutants to show that these mu 1-binding sites contain determinants required to induce CD4 down-regulation and to target the chimera to the endocytic pathway by promoting AP1 complex recruitment. Ultrastructural analysis revealed that the CD8-Nef chimera provokes morphological alterations of the endosomal compartments and co-localizes with AP1 complexes. These data indicate that the recruitment by Nef of AP1 via binding to mu 1 participates in the connection of Nef with the endocytic pathway.

Interactions of the cytoplasmic domains of human and simian retroviral transmembrane proteins with components of the clathrin adaptor complexes modulate intracellular and cell surface expression of envelope glycoproteins.

The cytoplasmic domains of the transmembrane (TM) envelope proteins (TM-CDs) of most retroviruses have a Tyr-based motif, YXXO, in their membrane-proximal regions. This signal is involved in the trafficking and endocytosis of membrane receptors via clathrin-associated AP-1 and AP-2 adaptor complexes. We have used CD8-TM-CD chimeras to investigate the role of the Tyr-based motif of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and human T-leukemia virus type 1 (HTLV-1) TM-CDs in the cell surface expression of the envelope glycoprotein. Flow cytometry and confocal microscopy studies showed that this motif is a major determinant of the cell surface expression of the CD8-HTLV chimera. The YXXO motif also plays a key role in subcellular distribution of the envelope of lentiviruses HIV-1 and SIV. However, these viruses, which encode TM proteins with a long cytoplasmic domain, have additional determinants distal to the YXXO motif that participate in regulating cell surface expression. We have also used the yeast two-hybrid system and in vitro binding assays to demonstrate that all three retroviral YXXO motifs interact with the micro1 and micro2 subunits of AP complexes and that the C-terminal regions of HIV-1 and SIV TM proteins interact with the beta2 adaptin subunit. The TM-CDs of HTLV-1, HIV-1, and SIV also interact with the whole AP complexes. These results clearly demonstrate that the cell surface expression of retroviral envelope glycoproteins is governed by interactions with adaptor complexes. The YXXO-based signal is the major determinant of this interaction for the HTLV-1 TM, which contains a short cytoplasmic domain, whereas the lentiviruses HIV-1 and SIV have additional determinants distal to this signal that are also involved.

Interaction with the p6 domain of the gag precursor mediates incorporation into virions of Vpr and Vpx proteins from primate lentiviruses.

Vpr and Vpx proteins from human and simian immunodeficiency viruses (HIV and SIV) are incorporated into virions in quantities equivalent to those of the viral Gag proteins. We demonstrate here that Vpr and Vpx proteins from distinct lineages of primate lentiviruses were able to bind to their respective Gag precursors. The capacity of HIV type 1 (HIV-1) Vpr mutants to bind to Pr55(Gag) was correlated with their incorporation into virions. Molecular analysis of these interactions revealed that they required the C-terminal p6 domain of the Gag precursors. While the signal for HIV-1 Vpr binding lies in the leucine triplet repeat region of the p6 domain reported to be essential for incorporation, SIVsm Gag lacking the equivalent region still bound to SIVsm Vpr and Vpx, indicating that the determinants for Gag binding are located upstream of this region of the p6 domain. Binding to Gag cleavage products showed that HIV-1 Vpr interacted directly with the nucleocapsid protein (NC), whereas SIVsm Vpr and Vpx did not interact with NC but with the p6 protein. These results (i) reveal differences between HIV-1 and SIVsm for the p6 determinants required for Vpr and Vpx binding to Gag and (ii) suggest that HIV-1 Vpr and SIVsm Vpr and Vpx interact with distinct cleavage products of the precursor following proteolytic processing in the virions.

Nef interacts with the mu subunit of clathrin adaptor complexes and reveals a cryptic sorting signal in MHC I molecules.

The surface expression of MHC I is reduced in HIV-infected cells. We show that the Nef protein affects the intracellular sorting of HLA-A and -B molecules. In the presence of Nef, these proteins accumulate in the Golgi and colocalize with clathrin-coated vesicles. MHC I modulation relies on a tyrosine-based sorting signal located in the cytoplasmic domain of HLA-A and -B heavy chains. This cryptic sorting signal becomes operative only in the presence of Nef. Nef interacts with the medium (mu) subunit of AP adaptor complexes involved in the recognition of tyrosine-based sorting signals, likely facilitating the connection between MHC I and the clathrin-dependent sorting machinery.