Characterization of E3 ligases involved in lysosomal sorting of the HIV-1 restriction factor BST2.

Nicolas Roy,Grégory Pacini,Katy Janvier,Clarisse Berlioz-Torrent

doi:10.1242/jcs.195412

Abstract

ABSTRACTThe cellular protein BST2 (also known as tetherin) acts as a major intrinsic antiviral protein that prevents the release of enveloped viruses by trapping nascent viral particles at the surface of infected cells. Viruses have evolved specific strategies to displace BST2 from viral budding sites in order to promote virus egress. In HIV-1, the accessory protein Vpu counters BST2 antiviral activity and promotes sorting of BST2 for lysosomal degradation. Vpu increases polyubiquitylation of BST2, a post-translation modification required for Vpu-induced BST2 downregulation, through recruitment of the E3 ligase complex SCF adaptors β-TrCP1 and β-TrCP2 (two isoforms encoded by BTRC and FBXW11, respectively). Herein, we further investigate the role of the ubiquitylation machinery in the lysosomal sorting of BST2. Using a small siRNA screen, we highlighted two additional regulators of BST2 constitutive ubiquitylation and sorting to the lysosomes: the E3 ubiquitin ligases NEDD4 and MARCH8. Interestingly, Vpu does not hijack the cellular machinery that is constitutively involved in BST2 ubiquitylation to sort BST2 for degradation in the lysosomes but instead promotes the recognition of BST2 by β-TrCP proteins. Altogether, our results provide further understanding of the mechanisms underlying BST2 turnover in cells.

Highlights

IntroductionBST2 contains a short N-terminal cytoplasmic tail linked to a transmembrane domain and a large extracellular coiled-coil domain anchored to the membrane through a C-terminal glycosylphosphatidylinositol (GPI) moiety (Evans et al, 2010; Kupzig et al, 2003; Masuyama et al, 2009)
membrane-associated RING-CH 8 (MARCH8), NEDD4 or β-TrCP depletion leads to increased levels of BST2 To characterize the E3 ligase(s) involved in the constitutive ubiquitylation of BST2 and its sorting for lysosomal degradation, we performed a small siRNA screen targeting a subset of wellcharacterized E3 ligases involved in the regulation of protein trafficking in the endo-lysosomal pathway: the HECT-E3 ligases NEDD4, NEDD4-L, ITCH, WWP1 (Boase and Kumar, 2015; Ingham et al, 2004); the RING domain-containing E3 ligases c-Cbl and the transmembrane protein MARCH8 (Goh and Sorkin, 2013; Ohmura-Hoshino et al, 2006a)
Quantification of BST2 transcripts in cells depleted of NEDD4, MARCH8 or β-TrCP was further assessed by performing RT-qPCR (Fig. 1E) and showed no significant difference compared to control cells

Summary

Introduction

BST2 contains a short N-terminal cytoplasmic tail linked to a transmembrane domain and a large extracellular coiled-coil domain anchored to the membrane through a C-terminal glycosylphosphatidylinositol (GPI) moiety (Evans et al, 2010; Kupzig et al, 2003; Masuyama et al, 2009) This atypical topology is responsible for its restrictive function towards viral release; BST2 assembles as parallel disulfide-bonded homo-dimers and acts as a bridge between virions and cellular membranes via its GPI anchors and its transmembrane domain, respectively, leading to the retention of the nascent viral particles at the surface of infected cells (Iwabu et al, 2009; Neil et al, 2008; Perez-Caballero et al, 2009; Van Damme et al, 2008; Venkatesh and Bieniasz, 2013). Several virus-encoded proteins devoted to this function have been characterized, notably Vpu of human immunodeficiency virus type 1 (HIV-1) (Neil et al, 2008; Van Damme et al, 2008), Nef of most strains of simian immunodeficiency virus (SIV) and of HIV-1 Group O (Jia et al, 2009; Kluge et al, 2014; Sauter et al, 2009; Zhang et al, 2009), envelope glycoproteins (Env) of HIV-2 and SIVtan, (Gupta et al, 2009; Le Tortorec and Neil, 2009) and K5 of Kaposi’s sarcomaassociated herpes virus (KSHV) (Mansouri et al, 2009)

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Results

Conclusion