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Blocking of HIV-1 infection by targeting CD4 to nonraft membrane domains.

Del Real G, Jiménez-Baranda S, Lacalle RA, Mira E, Lucas P, Gómez-Moutón C, Carrera AC, Martínez-A C, Mañes S - J. Exp. Med. (2002)

Bottom Line: We generated CD4 partitioning mutants by substituting or deleting CD4 transmembrane and cytoplasmic domains and the CD4 ectodomain was unaltered.Conversely, CD4 ectodomain targeting to a nonraft membrane fraction results in a CD4 receptor with severely diminished capacity to mediate Lck activation or HIV-1 entry, although this mutant binds gp120 as well as CD4wt.In addition, the nonraft CD4 mutant inhibits HIV-1 X4 and R5 entry in a CD4(+) cell line.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología/Spanish Council for Scientific Research (CSIC), E-28049 Madrid, Spain.

ABSTRACT
Human immunodeficiency virus (HIV)-1 infection depends on multiple lateral interactions between the viral envelope and host cell receptors. Previous studies have suggested that these interactions are possible because HIV-1 receptors CD4, CXCR4, and CCR5 partition in cholesterol-enriched membrane raft domains. We generated CD4 partitioning mutants by substituting or deleting CD4 transmembrane and cytoplasmic domains and the CD4 ectodomain was unaltered. We report that all CD4 mutants that retain raft partitioning mediate HIV-1 entry and CD4-induced Lck activation independently of their transmembrane and cytoplasmic domains. Conversely, CD4 ectodomain targeting to a nonraft membrane fraction results in a CD4 receptor with severely diminished capacity to mediate Lck activation or HIV-1 entry, although this mutant binds gp120 as well as CD4wt. In addition, the nonraft CD4 mutant inhibits HIV-1 X4 and R5 entry in a CD4(+) cell line. These results not only indicate that HIV-1 exploits host membrane raft domains as cell entry sites, but also suggest new strategies for preventing HIV-1 infection.

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Partitioning of CD4 mutants into distinct membrane domains. (A) The scheme shows the amino acid sequence of the CD4 mutants generated. Mutations or foreign sequences added to the CD4 extracellular domain are indicated in bold. (B) HEK-293 cells expressing CD4 mutants were fractionated in flotation gradients and CD4 partitioning was analyzed by Western blot. Fraction 1 represents the top and fraction 5 represents the bottom of the gradient. Filters were hybridized with anti-TfR and anti-VIP21 (caveolin-1) as controls for nonraft- and raft-associated proteins, respectively. (C–G) Confocal microscopy of CD4 mutant–expressing cells stained with cholera toxin β subunit (green) and anti-CD4 antibody (red). Yellow staining indicates colocalization of the molecules. The two-color overlay shows the representative cells for (C) CD4wt, (D) CD4–GPI, (E) CD4–LDL, (F) CD4–LDL–CD4, and (G) CD4–C394/397A (n = 50/mutant). Bar, 5 μm.
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fig1: Partitioning of CD4 mutants into distinct membrane domains. (A) The scheme shows the amino acid sequence of the CD4 mutants generated. Mutations or foreign sequences added to the CD4 extracellular domain are indicated in bold. (B) HEK-293 cells expressing CD4 mutants were fractionated in flotation gradients and CD4 partitioning was analyzed by Western blot. Fraction 1 represents the top and fraction 5 represents the bottom of the gradient. Filters were hybridized with anti-TfR and anti-VIP21 (caveolin-1) as controls for nonraft- and raft-associated proteins, respectively. (C–G) Confocal microscopy of CD4 mutant–expressing cells stained with cholera toxin β subunit (green) and anti-CD4 antibody (red). Yellow staining indicates colocalization of the molecules. The two-color overlay shows the representative cells for (C) CD4wt, (D) CD4–GPI, (E) CD4–LDL, (F) CD4–LDL–CD4, and (G) CD4–C394/397A (n = 50/mutant). Bar, 5 μm.

Mentions: Double acylation and GPI modification are major signals for protein partitioning in rafts by anchoring proteins to the inner or outer leaflet of the membrane raft, respectively. Nonetheless, integral membrane proteins have no clear consensus signal that indicates preferential raft association. The best studied raft-associated transmembrane protein is the influenza hemagglutinin, whose raft targeting is determined by three acylation acceptor cysteines and specific amino acids in its transmembrane domain (31, 32). CD4 has a 26–amino acid transmembrane region with two putative palmitoylation acceptor cysteines in the juxtamembrane domain (33). We generated a panel of CD4 chimeras and mutants that affect both transmembrane and cytoplasmic domains (Fig. 1 A). The CD4 extracellular domain was fused to the LDL-R transmembrane and juxtamembrane region (CD4–LDL). As a control for this construct, a CD4 mutant was generated by replacing the CD4 transmembrane domain with that of the LDL-R (CD4–LDL–CD4). This mutant retains the palmitoylated cysteines. The CD4 ectodomain was also fused to a GPI consensus sequence (CD4–GPI) to target CD4 luminal domain to rafts. Finally, we generated CD4 mutants, including three in which palmitoylated Cys394 and/or Cys397 are eliminated by alanine scanning of the transmembrane and juxtamembrane CD4 domains.


Blocking of HIV-1 infection by targeting CD4 to nonraft membrane domains.

Del Real G, Jiménez-Baranda S, Lacalle RA, Mira E, Lucas P, Gómez-Moutón C, Carrera AC, Martínez-A C, Mañes S - J. Exp. Med. (2002)

Partitioning of CD4 mutants into distinct membrane domains. (A) The scheme shows the amino acid sequence of the CD4 mutants generated. Mutations or foreign sequences added to the CD4 extracellular domain are indicated in bold. (B) HEK-293 cells expressing CD4 mutants were fractionated in flotation gradients and CD4 partitioning was analyzed by Western blot. Fraction 1 represents the top and fraction 5 represents the bottom of the gradient. Filters were hybridized with anti-TfR and anti-VIP21 (caveolin-1) as controls for nonraft- and raft-associated proteins, respectively. (C–G) Confocal microscopy of CD4 mutant–expressing cells stained with cholera toxin β subunit (green) and anti-CD4 antibody (red). Yellow staining indicates colocalization of the molecules. The two-color overlay shows the representative cells for (C) CD4wt, (D) CD4–GPI, (E) CD4–LDL, (F) CD4–LDL–CD4, and (G) CD4–C394/397A (n = 50/mutant). Bar, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2193941&req=5

fig1: Partitioning of CD4 mutants into distinct membrane domains. (A) The scheme shows the amino acid sequence of the CD4 mutants generated. Mutations or foreign sequences added to the CD4 extracellular domain are indicated in bold. (B) HEK-293 cells expressing CD4 mutants were fractionated in flotation gradients and CD4 partitioning was analyzed by Western blot. Fraction 1 represents the top and fraction 5 represents the bottom of the gradient. Filters were hybridized with anti-TfR and anti-VIP21 (caveolin-1) as controls for nonraft- and raft-associated proteins, respectively. (C–G) Confocal microscopy of CD4 mutant–expressing cells stained with cholera toxin β subunit (green) and anti-CD4 antibody (red). Yellow staining indicates colocalization of the molecules. The two-color overlay shows the representative cells for (C) CD4wt, (D) CD4–GPI, (E) CD4–LDL, (F) CD4–LDL–CD4, and (G) CD4–C394/397A (n = 50/mutant). Bar, 5 μm.
Mentions: Double acylation and GPI modification are major signals for protein partitioning in rafts by anchoring proteins to the inner or outer leaflet of the membrane raft, respectively. Nonetheless, integral membrane proteins have no clear consensus signal that indicates preferential raft association. The best studied raft-associated transmembrane protein is the influenza hemagglutinin, whose raft targeting is determined by three acylation acceptor cysteines and specific amino acids in its transmembrane domain (31, 32). CD4 has a 26–amino acid transmembrane region with two putative palmitoylation acceptor cysteines in the juxtamembrane domain (33). We generated a panel of CD4 chimeras and mutants that affect both transmembrane and cytoplasmic domains (Fig. 1 A). The CD4 extracellular domain was fused to the LDL-R transmembrane and juxtamembrane region (CD4–LDL). As a control for this construct, a CD4 mutant was generated by replacing the CD4 transmembrane domain with that of the LDL-R (CD4–LDL–CD4). This mutant retains the palmitoylated cysteines. The CD4 ectodomain was also fused to a GPI consensus sequence (CD4–GPI) to target CD4 luminal domain to rafts. Finally, we generated CD4 mutants, including three in which palmitoylated Cys394 and/or Cys397 are eliminated by alanine scanning of the transmembrane and juxtamembrane CD4 domains.

Bottom Line: We generated CD4 partitioning mutants by substituting or deleting CD4 transmembrane and cytoplasmic domains and the CD4 ectodomain was unaltered.Conversely, CD4 ectodomain targeting to a nonraft membrane fraction results in a CD4 receptor with severely diminished capacity to mediate Lck activation or HIV-1 entry, although this mutant binds gp120 as well as CD4wt.In addition, the nonraft CD4 mutant inhibits HIV-1 X4 and R5 entry in a CD4(+) cell line.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología/Spanish Council for Scientific Research (CSIC), E-28049 Madrid, Spain.

ABSTRACT
Human immunodeficiency virus (HIV)-1 infection depends on multiple lateral interactions between the viral envelope and host cell receptors. Previous studies have suggested that these interactions are possible because HIV-1 receptors CD4, CXCR4, and CCR5 partition in cholesterol-enriched membrane raft domains. We generated CD4 partitioning mutants by substituting or deleting CD4 transmembrane and cytoplasmic domains and the CD4 ectodomain was unaltered. We report that all CD4 mutants that retain raft partitioning mediate HIV-1 entry and CD4-induced Lck activation independently of their transmembrane and cytoplasmic domains. Conversely, CD4 ectodomain targeting to a nonraft membrane fraction results in a CD4 receptor with severely diminished capacity to mediate Lck activation or HIV-1 entry, although this mutant binds gp120 as well as CD4wt. In addition, the nonraft CD4 mutant inhibits HIV-1 X4 and R5 entry in a CD4(+) cell line. These results not only indicate that HIV-1 exploits host membrane raft domains as cell entry sites, but also suggest new strategies for preventing HIV-1 infection.

Show MeSH
Related in: MedlinePlus