Two Functional Variants of AP-1 Complexes Composed of either γ2 or γ1 Subunits Are Independently Required for Major Histocompatibility Complex Class I Downregulation by HIV-1 Nef

Abstract

The HIV-1 accessory protein Nef downregulates the cell surface expression of major histocompatibility complex class I (MHC-I) molecules to facilitate virus spreading. The Nef-induced downregulation of MHC-I molecules such as HLA-A requires the clathrin adaptor protein 1 (AP-1) complex. The cooperative interaction of Nef, AP-1, and the cytosolic tail (CT) of HLA-A leads to a redirection of HLA-A targeting from the trans-Golgi network (TGN) to lysosomes for degradation. Although the γ-adaptin subunit of AP-1 has two distinct isoforms (γ1 and γ2), which may form two AP-1 complex variants, so far, only the importance of AP-1γ1 in MHC-I downregulation by Nef has been investigated. Here, we report that the AP-1γ2 isoform also participates in this process. We found that AP-1γ2 forms a complex with Nef and HLA-A2_CT and that this interaction depends on the Y320 residue in HLA-A2_CT and Nef expression. Moreover, Nef targets AP-1γ1 and AP-1γ2 to different compartments in T cells, and the depletion of either AP-1 variant impairs the Nef-mediated reduction of total endogenous HLA-A levels and rescues HLA-A levels on the cell surface. Finally, immunofluorescence and immunoelectron microscopy analyses reveal that the depletion of γ2 in T cells compromises both the Nef-mediated retention of HLA-A molecules in the TGN and targeting to multivesicular bodies/late endosomes. Altogether, these results show that in addition to AP-1γ1, Nef also requires the AP-1γ2 variant for efficient MHC-I downregulation. IMPORTANCE HIV-1 Nef mediates evasion of the host immune system by inhibiting MHC-I surface presentation of viral antigens. To achieve this goal, Nef modifies the intracellular trafficking of MHC-I molecules in several ways. Despite being the subject of intense study, the molecular details underlying these modifications are not yet fully understood. Adaptor protein 1 (AP-1) plays an essential role in the Nef-mediated downregulation of MHC-I molecules such as HLA-A in different cell types. However, AP-1 has two functionally distinct variants composed of either γ1 or γ2 subunit isoforms. Because previous studies on the role of AP-1 in MHC-I downregulation by Nef focused on AP-1γ1, an important open question is the participation of AP-1γ2 in this process. Here, we show that AP-1γ2 is also essential for Nef-mediated depletion of surface HLA-A molecules in T cells. Our results indicate that Nef hijacks AP-1γ2 to modify HLA-A intracellular transport, redirecting these proteins to lysosomes for degradation.

Host Retromer Protein Sorting Nexin 2 Interacts with Human Respiratory Syncytial Virus Structural Proteins and is Required for Efficient Viral Production

“…This protocol was based on a previous paper by Perez-Victoria ( 22 , 46 ). HEp-2 cells were infected with HRSV (MOI = 1).…”

Clathrin’s life beyond 40: Connecting biochemistry with physiology and disease

“…The relatively weak interaction between nef and tetherin reduces AP1's ability to recruit clathrin, leading to retention of tetherin at the TGN, while the strong nef-MHCI interaction promotes AP1 clathrin binding that subsequently targets MHCI for lysosomal degradation [48]. Supporting this model, Tavares et al reported that HIV nef interacts with both 1 and 2 forms of AP1 to deplete MHCI from the PM [50].…”

HIV-1 Hijacking of Host ATPases and GTPases That Control Protein Trafficking

“…As previously mentioned, the clathrin adaptor AP-1 is recruited by ARF-1 to TGN membranes to form clathrin-coated vesicles (CCVs) that are destined for endosomes. In fact, MHC-I downregulation by Nef was shown to require AP-1 ( Williams et al, 2002 ; Roeth et al, 2004 ; Lubben et al, 2007 ; Schaefer et al, 2008 ; Leonard et al, 2011 ; Jia et al, 2012 ; Tavares et al, 2020 ) and ARF-1 activity ( Wonderlich et al, 2011 ). Structural analysis revealed that ARF-1-GTP binding to AP-1 triggers conformational changes in AP-1, which acquires an ‘unlocked’ state that is compatible with vesicle-cargo binding ( Ren et al, 2013 ).…”

“…In fact, the expression of an ARF-1 dominant-negative form inhibited Nef-CD4 complex migration toward the lysosome ( Fauré et al, 2004 ), highlighting the importance of ARF-1 in other receptor downregulation strategies utilized by Nef. Interestingly, it has been shown that Nef uses a variant of AP-1 (AP-1-γ2) to retain CD4 ( Tavares et al, 2017 ) and MHC-I ( Tavares et al, 2020 ) in endosomes for lysosomal delivery.…”