Human Immunodeficiency Virus Type 1 Nef Protein Targets CD4 to the Multivesicular Body Pathway

Abstract

The Nef protein of human immunodeficiency virus type 1 downregulates the CD4 coreceptor from the surface of host cells by accelerating the rate of CD4 endocytosis through a clathrin/AP-2 pathway. Herein, we report that Nef has the additional function of targeting CD4 to the multivesicular body (MVB) pathway for eventual delivery to lysosomes. This targeting involves the endosomal sorting complex required for transport (ESCRT) machinery. Perturbation of this machinery does not prevent removal of CD4 from the cell surface but precludes its lysosomal degradation, indicating that accelerated endocytosis and targeting to the MVB pathway are separate functions of Nef. We also show that both CD4 and Nef are ubiquitinated on lysine residues, but this modification is dispensable for Nef-induced targeting of CD4 to the MVB pathway.Primate immunodeficiency viruses infect helper T lymphocytes and cells of the macrophage/monocyte lineage by binding of their viral envelope glycoprotein, Env, to a combination of two host cell-specific surface proteins, CD4 and either the CCR5 or CXCR4 chemokine receptors (reviewed in reference 62). Ensuing fusion of the viral envelope with the host cell plasma membrane delivers the viral genetic material into the cytoplasm. Remarkably, the most highly transcribed viral gene in the early phase of infection does not encode an enzyme or structural protein but an accessory protein named Nef. Early expression of Nef is thought to reprogram the host cell for optimal replication of the virus. Indeed, Nef has been shown to enhance virus production (19,24,59,74) and to promote progression to AIDS (23,47,48), making it an attractive candidate for pharmacologic intervention.Nef is an N-terminally myristoylated protein with a molecular mass of 27 kDa for human immunodeficiency virus type 1 (HIV-1) and 35 kDa for HIV-2 and simian immunodeficiency virus (27,29,50,65). Nef has been ascribed many functions, the best characterized of which is the downregulation of the CD4 coreceptor from the surface of infected cells (28,35,57). CD4 downregulation is believed to prevent superinfection (8, 52) and to preclude the cellular retention of newly synthesized Env (8, 49), thus allowing the establishment of a robust infection (30, 71).The molecular mechanism by which Nef downregulates CD4 has been extensively studied. A consensus has emerged that Nef accelerates the endocytosis of cell surface CD4 (2, 64) by linking the cytosolic tail of CD4 to the heterotetrameric (␣-␤2-2-2) adaptor protein-2 (AP-2) complex (17,25,34,45,67). Determinants in the CD4 tail bind to a hydrophobic pocket comprising tryptophan-57 and leucine-58 on the folded core domain of Nef (34). On the other hand, a dileucine motif (i.e., ENTSLL, residues 160 to 165) (14, 22, 32) and a diacidic motif (i.e., DD, residues 174 and 175) (3) (residues correspond to the NL4-3 clone of HIV-1) within a C-terminal, flexible loop of Nef bind to the ␣ and 2 subunits of AP-2 (17, 18, 25, 51). AP-2, in turn, binds to clathrin, leading to the concentration of CD4 with...

CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the γ1 and γ2 subunits of the AP-1 complex in protein trafficking

“…4M; Table S2). It is possible that proteolytic degradation prevents accumulation of CD4 within lysosomes and undermines detection by immunofluorescence microscopy (daSilva et al, 2009). Taken together, these results further support the notion that γ2 is required for efficient downregulation of CD4 by Nef.…”

“…1D) hemi-complexes, and failed to reduce the total cell levels of CD4 ( Fig. 2A,B), as previously shown (daSilva et al, 2009). The KD of γ1 did not significantly alter CD4 depletion by Nef-WT ( Fig.…”

“…We have previously shown that this process involves ESCRT-dependent targeting of CD4 to the MVB pathway (Amorim et al, 2014;daSilva et al, 2009). Signaling receptors such as the EGF receptor (EGFR) are also targeted to the MVB pathway after they undergo signal-mediated endocytosis (Felder et al, 1990).…”

“…Alix (also known as PDCD6IP), an ESCRT-accessory protein, that directly binds Nef (Amorim et al, 2014;Costa et al, 2006; Jesus da Costa et al, 2009), was recently shown to be required for Nef-mediated targeting of CD4 to lysosomes (Amorim et al, 2014). However, depletion of Alix or of Tsg101 (an ESCRT-I component), which inhibits lysosomal targeting of CD4 by Nef, does not impair the Nef-mediated reduction of CD4 surface levels (Amorim et al, 2014;daSilva et al, 2009). This observation suggests that in addition to mediating MVB sorting through Alix, Nef might also promote endosomal retention of CD4 earlier in the pathway to MVBs.…”

“…CD4 molecules that have been internalized by Nef do not efficiently enter a retrieval pathway from early endosomes back to the plasma membrane, but are instead delivered to late endosomes or multivesicular bodies (MVBs) en route to lysosomes (daSilva et al, 2009;Schaefer et al, 2008). Delivery of transmembrane cargo to the intraluminal vesicles (ILVs) of MVBs, in most cases, requires the recognition of sorting signals by the endosomal sorting complexes required for transport (ESCRTs).…”