HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

Schaefer, Malinda; Wonderlich, Elizabeth R.; Roeth, Jeremiah F.; Leonard, Jolie A.; Collins, Kathleen L.

doi:10.1371/journal.ppat.1000131

Cited by 125 publications

(174 citation statements)

References 74 publications

(145 reference statements)

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“…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).…”

Section: Introductionmentioning

confidence: 99%

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

Tavares

Silva

Silva-Januário

et al. 2017

Journal of Cell Science

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The HIV accessory protein Nef is a major determinant of viral pathogenesis that facilitates viral particle release, prevents viral antigen presentation and increases infectivity of new virus particles. These functions of Nef involve its ability to remove specific host proteins from the surface of infected cells, including the CD4 receptor. Nef binds to the adaptor protein 2 (AP-2) and CD4 in clathrin-coated pits, forcing CD4 internalization and its subsequent targeting to lysosomes. Herein, we report that this lysosomal targeting requires a variant of AP-1 containing isoform 2 of γ-adaptin (AP1G2, hereafter γ2). Depletion of the γ2 or μ1A (AP1M1) subunits of AP-1, but not of γ1 (AP1G1), precludes Nef-mediated lysosomal degradation of CD4. In γ2-depleted cells, CD4 internalized by Nef accumulates in early endosomes and this alleviates CD4 removal from the cell surface. Depletion of γ2 also hinders EGFR-EGF-complex targeting to lysosomes, an effect that is not observed upon γ1 depletion. Taken together, our data provide evidence that the presence of γ1 or γ2 subunits delineates two distinct variants of AP-1 complexes, with different functions in protein sorting.

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Section: Introductionmentioning

confidence: 99%

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

Tavares

Silva

Silva-Januário

et al. 2017

Journal of Cell Science

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“…Nef acts on CD4 expression early in the viral life cycle, enhancing CD4 internalization from the cell surface by linking it to the AP-2 clathrin adaptor [8,9]. Via its interaction with β-COP, Nef appears to target the internalized receptor to endosomes [10] and subsequently to the Multivesicular Body Pathway (MVB) in an Endosomal Sorting Complex Required for Transport (ESCRT)-dependent manner [11]. Vpu is expressed later in the life cycle of the HIV-1 virus and causes the degradation of newly synthesized CD4 receptor, retained in the ER by the envelope glycoprotein gp160.…”

Section: Introductionmentioning

confidence: 99%

Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-induced CD4 down-regulation

et al. 2014

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Background: Down-modulation of the CD4 receptor is one of the hallmarks of HIV-1 infection and it is believed to confer a selective replicative advantage to the virus in vivo. This process is mainly mediated by three viral proteins: Env, Vpu and Nef. To date, the mechanisms that lead to CD4 depletion from the surface of infected cells during HIV-1 infection are still only partially characterized. In this study, we sought to identify and characterize cellular host factors in HIV-1-induced CD4 down-modulation. Results: To identify host factors involved in CD4 down-regulation, we used a whole genome-targeting shRNA lentiviral library in HeLa CD4+ cells expressing Nef as an inducer of CD4 down-modulation. We identified 55 genes, mainly encoding for proteins involved in various steps of clathrin-mediated endocytosis. For confirmation and further selection of the hits we performed several rounds of validation, using individual shRNA lentiviral vectors with a different target sequence for gene knock-down in HIV-1-infected T cells. By this stringent validation set-up, we could demonstrate that the knock-down of DNM3 (dynamin 3), SNX22 (sorting nexin 22), ATP6AP1 (ATPase, H+ Transporting, Lysosomal Accessory Protein 1), HRBL (HIV-Rev binding protein Like), IDH3G (Isocitrate dehydrogenase), HSP90B1 (Heat shock protein 90 kDa beta member 1) and EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15) significantly increases CD4 levels in HIV-infected SupT1 T cells compared to the non-targeting shRNA control. Moreover, EPS15, DNM3, IDH3G and ATP6AP1 knock-down significantly decreases HIV-1 replication in T cells.

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“…AP-1 then directs MHC-I into an endolysosomal pathway as opposed to the cell surface (77,(89)(90)(91). In contrast, CD4 anterograde traffic to the plasma membrane is not inhibited in the presence of Nef.…”

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confidence: 99%

HIV-1 Nef Disrupts Intracellular Trafficking of Major Histocompatibility Complex Class I, CD4, CD8, and CD28 by Distinct Pathways That Share Common Elements

Leonard

Filzen²,

Carter

et al. 2011

J Virol

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The Nef protein is an important HIV virulence factor that promotes the degradation of host proteins to augment virus production and facilitate immune evasion. The best-characterized targets of Nef are major histocompatibility complex class I (MHC-I) and CD4, but Nef also has been reported to target several other proteins, including CD8␤, CD28, CD80, CD86, and CD1d. To compare and contrast the effects of Nef on each protein, we constructed a panel of chimeric proteins in which the extracellular and transmembrane regions of the MHC-I allele HLA-A2 were fused to the cytoplasmic tails of CD4, CD28, CD8␤, CD80, CD86, and CD1d. We found that Nef coprecipitated with and disrupted the expression of molecules with cytoplasmic tails from MHC-I HLA-A2, CD4, CD8␤, and CD28, but Nef did not bind to or alter the expression of molecules with cytoplasmic tails from CD80, CD86, and CD1d. In addition, we used short interfering RNA (siRNA) knockdown and coprecipitation experiments to implicate AP-1 as a cellular cofactor for Nef in the downmodulation of both CD28 and CD8␤. The interaction with AP-1 required for CD28 and CD8␤ differed from the AP-1 interaction required for MHC-I downmodulation in that it was mediated through the dileucine motif within Nef (LL 164,165 AA) and did not require the tyrosine binding pocket of the AP-1 subunit. In addition, we demonstrate a requirement for ␤-COP as a cellular cofactor for Nef that was necessary for the degradation of targeted molecules HLA-A2, CD4, and CD8. These studies provide important new information on the similarities and differences with which Nef affects intracellular trafficking and help focus future research on the best potential pharmaceutical targets.

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HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

Cited by 125 publications

References 74 publications

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

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

Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-induced CD4 down-regulation

HIV-1 Nef Disrupts Intracellular Trafficking of Major Histocompatibility Complex Class I, CD4, CD8, and CD28 by Distinct Pathways That Share Common Elements

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