Mutational Analysis of HIV-1 Nef: Identification of Two Mutants That Are Temperature-Sensitive for CD4 Downregulation

Aiken, Christopher; Krause, Lisa; Chen, Yen-Liang; Trono, Didier

doi:10.1006/viro.1996.0116

Cited by 73 publications

(69 citation statements)

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“…Indeed, none of the five mutants having lost PAK2 binding capability among those studied (Nef G2A , Nef P72A/P75A , Nef P69A , Nef PR105/106AA , and Nef ⌬57-66 ) induced kidney and lung diseases. But the problem with this interpretation is the evidence that some of these mutations are known to affect other functions of Nef, in addition to its binding to PAK: namely abrogating its binding to Hck through indirect structural changes for the P72A/P75A mutation (41,90,91) and disrupting CD4 down-regulation and CD3 signaling for the R105A/R106A mutation (56,81,92,93). It therefore appears that PAK2 binding to Nef may be required for induction of kidney and lung diseases in Tg mice but is clearly not sufficient.…”

Section: Discussionmentioning

confidence: 99%

Activation of p21-activated Kinase 2 and Its Association with Nef Are Conserved in Murine Cells but Are Not Sufficient to Induce an AIDS-like Disease in CD4C/HIV Transgenic Mice

Vincent

Priceputu

Kay

et al. 2006

Journal of Biological Chemistry

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A well conserved feature of human immunodeficiency virus, type 1 (HIV-1) and simian immunodeficiency virus (SIV) Nef is the interaction with and activation of the human p21-activated kinase 2 (PAK2). The conservation of this interaction in other species and its significance for Nef pathogenesis in vivo are poorly documented. In the present study, we measured these parameters in Nef-expressing thymocytes, macrophages, and dendritic cells of a transgenic (Tg) mouse model of AIDS (CD4C/HIV). We found that Nef binds to and activates PAK2, but not PAK1 and -3, in these three cell subsets. Nef associates with only a small fraction of PAK2. The Nef-PAK2 complex also comprises ␤-PIX-COOL. The impact of the Nef-PAK2 association on disease development was also analyzed in Tg mice expressing 10 different Nef mutant alleles. CD4C/HIV Tg mice expressing Nef alleles defective in Nef-PAK2 association (P69A, P72A/P75A, R105A/R106A, ⌬56 -66, or G2A (myristoylation site)) failed to develop disease of the non-lymphoid organs (kidneys and lungs). Among these, only Tg mice expressing Nef P69A and Nef G2Ashowed some depletion of CD4 ؉ T cells, although a down-regulation of the CD4 surface protein was documented in all these Tg lines, except those expressing Nef ⌬56 -66 . Among other Tg mice expressing Nef mutants having conserved the Nef-PAK2 association (RD35AA, D174K, P147A/P150A, ⌬8 -17, and ⌬25-65), only Tg mice expressing Nef ⌬8 -17 develop kidney and lung diseases, but all showed partial CD4 ؉ T cell depletion despite some being defective for CD4 down-regulation (RD35AA and D174K). Therefore, Nef can activate murine PAK2 and associate with a small fraction of it, as in human cells. Such activation and binding of PAK2 is clearly not sufficient but may be required to induce a multiorgan AIDS-like disease in Tg mice.

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Section: Discussionmentioning

confidence: 99%

Activation of p21-activated Kinase 2 and Its Association with Nef Are Conserved in Murine Cells but Are Not Sufficient to Induce an AIDS-like Disease in CD4C/HIV Transgenic Mice

Vincent

Priceputu

Kay

et al. 2006

Journal of Biological Chemistry

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“…Polyclonal antibodies against the Ins(1,4,5)P 3 -receptor type I Rbt04 were kindly provided by Dr. J. Parys (Leuven, Belgium). Polyclonal antibodies against the HIV-1 Nef were as described previously (50). Anti-mouse/rabbit horseradish peroxidase-conjugated IgG were purchased from Amersham Pharmacia Biotech, and anti-mouse/rabbit fluorescein-5-isothiocyanate-conjugated IgG was from DAKO (Copenhagen).…”

Section: Methodsmentioning

confidence: 99%

The HIV Nef Protein Alters Ca2+ Signaling in Myelomonocytic Cells through SH3-mediated Protein-Protein Interactions

Foti¹,

Cartier²,

Piguet³

et al. 1999

Journal of Biological Chemistry

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“…It was previously reported that residue C142 is critical for the correct folding of Nef and that a Nef mutant with C142 to A substitution is unstable (56). Fig.…”

Section: Substitution Of K144 With Arginine Abolished Nef-mediated CDmentioning

confidence: 99%

Lysine 144, a Ubiquitin Attachment Site in HIV-1 Nef, Is Required for Nef-Mediated CD4 Down-Regulation

Jin

Cai

Zhang

et al. 2008

The Journal of Immunology

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Nef is a HIV-1 accessory protein critical for the replication of the virus and the development of AIDS. The major pathological activity of Nef is the down-regulation of CD4, the primary receptor of HIV-1 infection. The mechanism underlying Nef-mediated CD4 endocytosis and degradation remains incompletely understood. Since protein ubiquitination is the predominant sorting signal in receptor endocytosis, we investigated whether Nef is ubiquitinated. The in vivo ubiquitination assay showed that both HIV-1 and SIV Nef proteins expressed in Jurkat T cells and 293T cells were multiple ubiquitinated by ubiquitin-His. The lysine-free HIV-1 Nef mutant (Δ10K) generated by replacing all 10 lysines with arginines was not ubiquitinated and the major ubiquitin-His attachment sites in HIV-1 Nef were determined to be lysine 144 (di-ubiquitinated) and lysine 204 (mono-ubiquitinated). Lysine-free HIV-1 Nef was completely inactive in Nef-mediated CD4 down-regulation, so was the Nef mutant with a single arginine substitution at K144 but not at K204. A mutant HIV-1 provirion NL4–3 with a single arginine substitution in Nef at K144 was also inactive in Nef-mediated CD4 down-regulation. Lysine-free Nef mutant reintroduced with lysine 144 (ΔK10 + K144) was shown active in CD4 down-regulation. These data suggest that ubiquitination of Nef, particularly diubiquitination of the lysine 144, is necessary for Nef-mediated CD4 down-regulation.

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Mutational Analysis of HIV-1 Nef: Identification of Two Mutants That Are Temperature-Sensitive for CD4 Downregulation

Cited by 73 publications

References 0 publications

Activation of p21-activated Kinase 2 and Its Association with Nef Are Conserved in Murine Cells but Are Not Sufficient to Induce an AIDS-like Disease in CD4C/HIV Transgenic Mice

Activation of p21-activated Kinase 2 and Its Association with Nef Are Conserved in Murine Cells but Are Not Sufficient to Induce an AIDS-like Disease in CD4C/HIV Transgenic Mice

The HIV Nef Protein Alters Ca2+ Signaling in Myelomonocytic Cells through SH3-mediated Protein-Protein Interactions

Lysine 144, a Ubiquitin Attachment Site in HIV-1 Nef, Is Required for Nef-Mediated CD4 Down-Regulation

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