The Dileucine-Based Sorting Motif in HIV-1 Nef Is Not Required for Down-Regulation of Class I MHC

Human immunodeficiency virus type 1 (HIV-1) Nef is a critical protein that is necessary for HIV pathogenesis. Its roles include the disruption of major histocompatibility complex class I (MHC-I) and CD4 trafficking to promote immune evasion and viral spread. Mutational analyses have revealed that separate domains of Nef are required to affect these two molecules. To further elucidate how Nef disrupts MHC-I trafficking in T cells, we examined the role of protein domains that are required for this function (N-terminal alpha helix, polyproline, acidic, and oligomerization domains). We found that each of these regions was required for Nef to disrupt the transport of HLA-A2 to the cell surface and for Nef to coprecipitate with HLA-A2.

supporting

confidence: 92%

Human Immunodeficiency Virus Type 1 Nef Domains Required for Disruption of Major Histocompatibility Complex Class I Trafficking Are Also Necessary for Coprecipitation of Nef with HLA-A2

Williams¹,

Roeth²,

Kasper³

et al. 2005

“…It has also been reported that the recognition of tyrosine-based endocytotic signals clearly involves a dimeric 2 chain of the clathrin adapter AP2 complex (49).…”

Section: Discussionmentioning

confidence: 97%

Mutation of a Conserved Residue (D123) Required for Oligomerization of Human Immunodeficiency Virus Type 1 Nef Protein Abolishes Interaction with Human Thioesterase and Results in Impairment of Nef Biological Functions

Liu

Heveker

Fackler

et al. 2000

122

Nef is a myristoylated protein of 27 to 35 kDa that is conserved in primate lentiviruses. In vivo, Nef is required for high viral load and full pathological effects. In vitro, Nef has at least four activities: induction of CD4 and major histocompatibility complex (MHC) class I downregulation, enhancement of viral infectivity, and alteration of T-cell activation pathways. We previously reported that the Nef protein from human immunodeficiency virus type 1 interacts with a novel human thioesterase (hTE). In the present study, by mutational analysis, we identified a region of the Nef core, extending from the residues D108 to W124, that is involved both in Nef-hTE interaction and in Nef-induced CD4 downregulation. This region of Nef is located on the oligomer interface and is in close proximity to the putative CD4 binding site. One of the mutants carrying a mutation in this region, targeted to the conserved residue D123, was also found to be defective in two other functions of Nef, MHC class I downmodulation and enhancement of viral infectivity. Furthermore, mutation of this residue affected the ability of Nef to form dimers, suggesting that the oligomerization of Nef may be critical for its multiple functions.

“…the possibility that the pathways used for downmodulation of CD4 and class I MHC by LAT-Nef and Nef are different. To examine this, we utilized a series of well-characterized mutations in Nef that previously had been shown to differentially affect the two pathways (51,54). The mutations chosen were D123G, previously shown to inhibit both CD4 and class I MHC downmodulation (39); L164, L165/AA (LL/AA), previously shown inhibit only CD4 downmodulation (7,29); and E62, E63, E64, and E65/AAAA (EEEE/AAAA), previously shown to inhibit only class I MHC downmodulation (31).…”

Section: Vol 78 2004 Lipid Raft Targeting and Nef Function 1689mentioning

confidence: 99%

Human Immunodeficiency Virus Type 1 Nef Associates with Lipid Rafts To Downmodulate Cell Surface CD4 and Class I Major Histocompatibility Complex Expression and To Increase Viral Infectivity

Alexander

Bor

Ravichandran

et al. 2004

Lipid rafts are membrane microdomains that are functionally distinct from other membrane regions. We have shown that 10% of human immunodeficiency virus type 1 (HIV-1) Nef expressed in SupT1 cells is present in lipid rafts and that this represents virtually all of the membrane-associated Nef. To determine whether raft targeting, rather than simply membrane localization, has functional significance, we created a Nef fusion protein (LAT-Nef) containing the N-terminal 35 amino acids from LAT, a protein that is exclusively localized to rafts. Greater than 90% of the LAT-Nef protein was found in the raft fraction. In contrast, a mutated form, lacking two cysteine palmitoylation sites, showed less than 5% raft localization. Both proteins were equally expressed and targeted nearly exclusively to membranes. The LAT-Nef protein was more efficient than its nonraft mutant counterpart at downmodulating both cell surface CD4 and class I major histocompatibility complex (MHC) expression, as well as in enhancing first-round infectivity and being incorporated into virus particles. This demonstrates that targeting of Nef to lipid rafts is mechanistically important for all of these functions. Compared to wild-type Nef, LAT-Nef downmodulated class I MHC nearly as effectively as the wild-type Nef protein, but was only about 60% as effective for CD4 downmodulation and 30% as effective for infectivity enhancement. Since the LAT-Nef protein was found entirely in rafts while the wild-type Nef protein was distributed 10% in rafts and 90% in the soluble fraction, our results suggest that class I MHC downmodulation by Nef may be performed exclusively by raft-bound Nef. In contrast, CD4 downmodulation and infectivity enhancement may require a non-membrane-bound Nef component as well as the membrane-bound form.