A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor.

Charlotte A Stoneham,Simon Langer,Paul D De Jesus,John Lapek,Andrea Thor,Jacob M Wozniak,David J Gonzalez,Sumit K Chanda,John Guatelli,Thomas Deerinck,Lars Pache,Mark Ellisman

doi:10.1371/journal.ppat.1009409

Abstract

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu’s itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.

Highlights

HIV-1 encodes the accessory proteins Vif, Vpr, Nef, and Vpu to overcome cell-intrinsic, innate, and adaptive host defenses
Vpu acts as an adaptor, linking cellular protein targets to the ubiquitination and membrane trafficking machinery
ascorbate peroxidase 2 (APEX2)-tagged Vpu retained biological activity against BST-2 and CD4, activity was reduced compared to Vpu tagged only with a C-terminal FLAG-epitope

Summary

Introduction

HIV-1 encodes the accessory proteins Vif, Vpr, Nef, and Vpu to overcome cell-intrinsic, innate, and adaptive host defenses. Non-enzymatic, integral membrane protein that functions as an adaptor, linking targeted cellular proteins to the protein quality control and membrane trafficking machinery to induce their re-localization or degradation. Cellular targets of Vpu interfere directly with viral replication or support immune surveillance; these targets include CD4 (the virus’s primary receptor), BST-2 (an interferon-induced protein that traps newly assembled virions on the infected-cell surface), natural killer (NK) cell receptors (NTB-A), class I MHC (HLA-C), CCR7, and tetraspanins [1,2,3,4,5,6,7]. Several Vpu cofactors and co-opted pathways have been identified. These include a Skp1/ cullin1/F-box (SCF) multi-subunit E3 ubiquitin ligase containing β-TrCP [8]. Other Vpu targets, such as BST-2, are instead degraded within the endo-lysosomal system [10,11]

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Discussion

Conclusion