Interaction of HIV-1 Nef Protein with the Host Protein Alix Promotes Lysosomal Targeting of CD4 Receptor

Nathaly A Amorim,Eulália M.L Da Silva,Rodrigo O De Castro,Mara E Da Silva-Januário,Luiza M Mendonça,Juan S Bonifacino,Luciana J Da Costa,Luis L.P Dasilva

doi:10.1074/jbc.m114.560193

Abstract

Nef is an accessory protein of human immunodeficiency viruses that promotes viral replication and progression to AIDS through interference with various host trafficking and signaling pathways. A key function of Nef is the down-regulation of the coreceptor CD4 from the surface of the host cells. Nef-induced CD4 down-regulation involves at least two independent steps as follows: acceleration of CD4 endocytosis by a clathrin/AP-2-dependent pathway and targeting of internalized CD4 to multivesicular bodies (MVBs) for eventual degradation in lysosomes. In a previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. Here, we report that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We show that Nef interacts with both the Bro1 and V domains of Alix. Depletion of Alix or overexpression of the Alix V domain impairs lysosomal degradation of CD4 induced by Nef. In contrast, the V domain overexpression does not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical ubiquitination-dependent MVB pathway. We also show that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, our results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef.

Highlights

human immunodeficiency virus (HIV)-1 Nef targets the coreceptor CD4 to the multivesicular body (MVB) pathway for degradation via an unknown mechanism
We report that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs
Bro1 and V Domain of Alix Mediate Interaction with Nef— Alix is composed of three distinct structural elements that individually interact with a number of cellular proteins and with retroviral Gag proteins (Fig. 1A)

Summary

Background

HIV-1 Nef targets the coreceptor CD4 to the multivesicular body (MVB) pathway for degradation via an unknown mechanism. Our results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef. The human immunodeficiency virus (HIV) interferes with the host cell protein sorting machinery to promote viral replication and evade defense mechanisms [1, 2]. Unlike typical endocytic recycling receptors, CD4 molecules that are internalized by Nef do not return to the cell surface but are instead delivered to lysosomes for degradation [13, 15, 24] This pathway is similar to that followed by signaling receptors, transporters, and other transmembrane proteins that undergo ubiquitination-mediated incorporation into nascent intraluminal vesicles (ILVs) of multivesicular bodies (MVBs) for eventual transport to lysosomes [25]. These observations indicate that Nef uses Alix as an adaptor for ubiquitination-independent targeting of transmembrane cargo to the MVB pathway

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION