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.2005 Sep;79(17):11422-33.
doi: 10.1128/JVI.79.17.11422-11433.2005.

Human immunodeficiency virus Nef induces rapid internalization of the T-cell coreceptor CD8alphabeta

Affiliations

Human immunodeficiency virus Nef induces rapid internalization of the T-cell coreceptor CD8alphabeta

Veronique Stove et al. J Virol.2005 Sep.

Erratum in

  • J Virol. 2005 Oct;79(20):13195

Abstract

Human immunodeficiency virus (HIV) Nef is a membrane-associated protein decreasing surface expression of CD4, CD28, and major histocompatibility complex class I on infected cells. We report that Nef strongly down-modulates surface expression of the beta-chain of the CD8alphabeta receptor by accelerated endocytosis, while CD8 alpha-chain expression is less affected. By mutational analysis of the cytoplasmic tail of the CD8 beta-chain, an FMK amino acid motif was shown to be critical for Nef-induced endocytosis. Although independent of CD4, endocytosis of the CD8 beta-chain was abrogated by the same mutations in Nef that affect CD4 down-regulation, suggesting common molecular interactions. The ability to down-regulate the human CD8 beta-chain was conserved in HIV-1, HIV-2, and simian immunodeficiency virus SIVmac239 Nef and required an intact AP-2 complex. The Nef-mediated internalization of receptors, such as CD4, major histocompatibility complex class I, CD28, and CD8alphabeta, may contribute to the subversion of the host immune system and progression towards AIDS.

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Figures

FIG. 1.
FIG. 1.
Flow cytometric evaluation of Nef-mediated CD8 down-regulation in retrovirally transduced cells. (A) Bivariate dot plots (CD8α-allophycocyanin, CD8αβ-phycoerythrin versus EGFP) of flow cytometric measurement of Nef− (control) and Nef+ (NA-7 allele) transduced peripheral blood mononuclear cells, gated on CD8+ cells, at day 3 after transduction. (B) Bivariate dot plots of flow cytometric measurement (CD8α-allophycocyanin, CD8αβ-phycoerythrin versus EGFP) of Nef NA-7 wild-type and NA-7 LLAA transduced SupT1 cells (left) and SupT1 cells overexpressing CD8α (right), at day 2 after transduction. (C) The solid and open histograms show the CD8αβ expression profile of SupT1 CD8α+ cells and SupT1 CD8α+ cells (CD8α-transduced population), respectively, gated as shown in the inset. (D) Daudi CD8αα cells and Daudi CD8αβ cells were transduced with control, HIV-1 (NL4-3, LAI and NA-7), SIV (mac239), and HIV-2 (Rod) Nef. Percent down-regulation is shown with white bars for CD8α in Daudi CD8αα, with gray bars for CD8α in Daudi CD8αβ, and with black bars for CD8αβ in Daudi CD8αβ. All percentages were calculated, as described in Materials and Methods, using the ranges E− and E+, as indicated in A.
FIG.2.
FIG.2.
Flow cytometric mutational analysis of Nef-mediated down- or up-regulation of cell surface molecules in retrovirally transduced SupT1 and Daudi cells. SupT1 cells and Daudi cells were transduced with control virus or wild-type or mutant HIV-1 Nef (alleles NL4-3, LAI, and NA-7). Percent down-regulation was calculated, as described in the Materials and Methods, using the ranges indicated in Fig. 1A. (A) Alignment of the amino acid sequences of the HIV-1 Nef alleles LAI, NL4-3, and NA-7. Underlined amino acids represent the positions that are changed within the indicated mutant Nef protein, and shaded amino acids in NL4-3 and NA-7 are different from the LAI sequence. (B) Comparison of the down-regulating activity of control and Nef constructs in SupT1 cells by using monoclonal antibodies against CD4-allophycocyanin, CD28-phycoerythrin, CD8α-allophycocyanin, and CD8αβ-phycoerythrin. (C) Comparison of the modulating activity of control and Nef constructs in Daudi cells by using monoclonal antibodies CD74/goat anti-mouse-phycoerythrin and HLA-DR-allophycocyanin. The values in B and C are means and standard deviations calculated from the data generated from three independent experiments. Mutations that abrogate the modulating capacity of Nef for more than 50%, compared to the respective wild-type Nef, are underlined. (D) Western blot analysis of Nef expression in transduced SupT1 cells. Lysates of sorted SupT1 cells, transduced with virus as indicated, were stained for Nef. The marker indicates the position of 31 kDa.
FIG. 3.
FIG. 3.
Flow cytometric analysis of Nef-mediated receptor down-regulation and internalization in retrovirally transduced SupT1 cells. SupT1 cells were transduced with an inducible NA-7.ER construct. At time zero, 4-hydroxytamoxifen (1 μM) was added to the culture medium. (A) Percent down-regulation was calculated, as described in the Materials and Methods. CD4-allophycocyanin (solid line) and CD8β-phycoerythrin (dashed line) were measured as a function of time. (B) The figure shows the percentage of CD4, CD8αβ, and CD28 molecules internalized by HIV-1 NA-7.ER, calculated as described in Materials and Methods. In each graph, Nef-positive (EGFP expressing Nef, solid line) and Nef-negative (EGFP not expressing Nef, dashed line) cells are depicted. The EGFP ranges used for calculation are indicated in Fig. 1A.
FIG. 4.
FIG. 4.
Mutations in the CD8 β-chain and their effect on endocytosis and down-regulation. Daudi cells were cotransduced with CD8α, wild-type or mutant CD8β, and control or wild-type Nef. (A and C) Alignment of amino acid sequences of wild-type (210*) and mutant CD8 β-chain cytoplasmic tails. An asterisk indicates a stop codon. (A) The bar chart shows the percent down-regulation of (mutant) CD8αβ by HIV-1 Nef alleles NA-7, LAI, and NL4-3. In both B and D the percentage of (mutant) CD8αβ molecules internalized by wild-type HIV-1 NA-7 is shown, including in both the same data for a Nef-negative construct as a control (wild-type Nef−). (C) The bar chart represents the percentage of (mutant) CD8αβ down-regulation after transduction with either control virus or wild-type Nef NL4-3. Each bar represents a (mutant) CD8 β-chain, as indicated by the changed amino acid sequence compared with CD8αβ. Percent down-regulation and internalization were calculated as described in Materials and Methods, using the ranges indicated in Fig. 1A. In A, B, and D, mean values are shown and standard deviations are calculated from the data generated from three independent experiments. In B the results for NL4-3 are representative of the results with HIV-1 alleles NA-7 and LAI.
FIG. 5.
FIG. 5.
Chimeric constructs. Daudi cells were retrovirally transduced with the CD8α(EC-TM)-CD8α(IC) chimera (cyt tail) or a CD8α(EC-TM)-CD8β(IC) chimera (cyt tail), using bicistronic constructs with ΔNGFR as the reporter. Bivariate dot plots are gated on ΔNGFR-positive cells, at day 2 after transduction of these cells with control virus, HIV-1 Nef (NA-7 allele), and SIV Nef (mac239), using bicistronic constructs with EGFP as the reporter. CD8α-phycoerythrin versus EGFP expression is shown.
FIG. 6.
FIG. 6.
Confocal images of 293T cells. Nef.EGFP was detected by direct fluorescence (green, left panels) and CD8β.HA or CD8β by monoclonal antibodies as indicated in Materials and Methods (red, middle panels). Nuclei were visualized by DAPI staining (blue). Right panels show the merged images from Nef.EGFP and CD8β. Areas of colocalization of Nef.EGFP/CD8β are shown in yellow. As indicated, the upper panels show cells expressing wild-type LAI, the middle panels show the LLAA mutant, and alower panels show the PPAA mutant. Scale bars represent 5 μm.
FIG. 7.
FIG. 7.
Blocking Nef-mediated internalization and down-regulation by ikarugamycin and RNA interference. (A) The figures show the percentage of CD8αβ molecules internalized in Daudi CD8αβ cells by HIV-1 NA-7.ER. Cells were incubated for 2 h with (IKA+) or without (IKA−) ikarugamycin (2 μM) prior to the internalization experiment. At time zero, 4-hydroxytamoxifen (1 μM) was added to NA-7.ER-transduced Daudi cells. (B) Western blot, performed as indicated in Materials and Methods, shows protein expression levels of AP-2 μ2 subunit, clathrin heavy chain (Chc), and dynamin 2 (Dyn-2, arrowhead) in control and RNAi-transduced SupT1 cells, with equal amounts of protein loaded. (C) Bivariate dot plots of flow cytometric measurement of SupT1 cells transduced with AP-2i and HIV-1 Nef (LAI). CD8αβ (phycoerythrin) versus ΔNGFR (allophycocyanin) expression is shown, gated on EGFP-negative and EGFP-positive cells. (D) The bar charts represent the effect of AP-2, clathrin heavy chain (Chc), and dynamin 2 RNAi on HIV-1 Nef (LAI) and SIV (mac239) Nef-induced CD4 (left panel) and CD8αβ (right panel) down-regulation in transduced SupT1 cells, gated on EGFP-negative and EGFP-positive cells. Percent down-regulation was calculated, as described in Materials and Methods, using the ranges N− and N+, as indicated in C. Mean values and standard deviations are shown, calculated from data generated from three independent experiments.
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