CD4 Down-regulation by HIV-1 and Simian Immunodeficiency Virus (SIV) Nef Proteins Involves Both Internalization and Intracellular Retention Mechanisms

Jeremy J Rose,Katy Janvier,Soundararajulu Chandrasekhar,Rafick P Sekaly,Juan S Bonifacino,Sundararajan Venkatesan

doi:10.1074/jbc.m409420200

Abstract

Among the pleiotropic effects of Nef proteins of HIV and simian immunodeficiency virus (SIV), down-modulation of cell surface expression of CD4 is a prominent phenotype. It has been presumed that Nef proteins accelerate endocytosis of CD4 by linking the receptor to the AP-2 clathrin adaptor. However, the related AP-1 and AP-3 adaptors have also been shown to interact with Nef, hinting at role(s) for these complexes in the intracellular retention of CD4. By using genetic inhibitors of endocytosis and small interfering RNA-induced knockdown of AP-2, we show that accelerated CD4 endocytosis is not a dominant mechanism of HIV-1 (NL4-3 strain) Nef in epithelial cells, T lymphocyte cell lines, or peripheral blood lymphocytes. Furthermore, we show that both the CD4 recycling from the plasma membrane and the nascent CD4 in transit to the plasma membrane are susceptible to intracellular retention in HIV-1 Nef-expressing cells. In contrast, AP-2-mediated enhanced endocytosis constitutes the predominant mechanism for SIV (MAC-239 strain) Nef-induced down-regulation of human CD4 in human cells.

Highlights

The cytoplasmic domains of plasma membrane receptors contain sequence determinants that regulate orderly protein sorting during anterograde transport, specify organelle targeting, and determine the intracellular fate of internalized receptors after agonist binding or cell activation [1,2,3]
Using a yeast three-hybrid assay, we demonstrated recently that (D/E)XXXL(L/I)-type signals from HIV and SIV Nefs interacted in a bipartite manner with the adaptor hemicomplexes, ␥1⁄7␴1 of AP-1 and ␦1⁄7␴3 of AP-3
HIV-1 Nef Delayed the Cell Surface Expression and Reduced the Cell Surface Density of de novo Synthesized CD4 —Initially, we examined the kinetics of transport to the plasma membrane of de novo synthesized CD4 in the context of Nef expression

Summary

Introduction

The cytoplasmic domains of plasma membrane receptors contain sequence determinants that regulate orderly protein sorting during anterograde transport, specify organelle targeting, and determine the intracellular fate of internalized receptors after agonist binding or cell activation [1,2,3]. SIV Nef has an additional tyrosine-based sorting signal of the type YXXØ, which presumably binds to the AP-2 complex and is implicated in enhancing endocytosis of CD4 [17, 18]. From these findings, Nef is presumed to serve as a “connector” between CD4 and the endocytic machinery [6, 16]. We have extended the above studies to show that the loss of CD4 at the cell surface of HIV-1 Nef-expressing cells did not result exclusively from accelerated endocytosis of CD4 Both the recycling CD4 and the nascent receptor in transit to the plasma membrane were susceptible to intracellular retention and degradation in HIV-1 Nef-expressing cells. CD4 Down-regulation by HIV-1 and SIV Nef Proteins inhibitors of endocytosis or small interfering RNA (siRNA)driven knockdown of AP-2 complexes

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