Association of simian immunodeficiency virus Nef with cellular serine/threonine kinases is dispensable for the development of AIDS in rhesus macaques

Lang, Simone; Aj, Iafrate; Stahl–Hennig∥, Christiane; Em, Kuhn; Nisslein, Thomas; Fj, Kaup; Haupt, Marianne; Hunsmann, Gerhard; Skowroński, Jacek; Kirchhoff, Frank

doi:10.1038/nm0897-860

Cited by 85 publications

(139 citation statements)

References 39 publications

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“…Of interest, the SIV Nef WTQ mutant appears to co-precipitate more efficiently with fulllength Hck than HIV-1 Nef in vivo, suggesting some additional mechanisms of binding such as through its SH2 domain. Indeed, although a strong selective pressure was noted in vivo to restore an experimentally disrupted proline motif in SIV Nef (11), this motif appears dispensable for Src binding in intact cells (10). This is in strike contrast to HIV-Nef binding to Hck which requires an intact proline motif (30).…”

Section: Resultsmentioning

confidence: 99%

“…Elucidation of incongruences in functional aspects between SIV and HIV-1 Nef molecules is indispensable for the use of SIV Nef as a model for drug design. As an alternative to mutational analysis in the proline motif (4,10,11,26), we now propose to design mutations that selectively inactivate specific Nef functions to assess their effect on viral replication and pathogenesis in vivo.…”

Section: Resultsmentioning

confidence: 99%

“…Controversial data has further been collected for the requirement of the proline-rich motif for AIDS disease progression in infected macaques (10,11). Especially, it was reported that a PXXP to AXXA mutated SIV mac239 protein could still bind to Src kinase (10), suggesting that SIV might have different strategies of interacting with the Src family of protein tyrosine kinases.…”

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confidence: 99%

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HIV-2 and SIV Nef Proteins Target Different Src Family SH3 Domains than Does HIV-1 Nef because of a Triple Amino Acid Substitution

Collette¹,

Arold²,

Picard³

et al. 2000

Journal of Biological Chemistry

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The nef gene is required for optimal viral spread of human and simian immunodeficiency viruses. However, the molecular mechanisms underlying the action of the Nef proteins may not be identical for all viral families. Here we investigate the interaction between the Nef protein of human and simian immunodeficiency viruses and SH3 domains from Src family kinases. Using the yeast two-hybrid system and immunoblotting we show that, in contrast to HIV-1 Nef, SIV and HIV-2 Nef poorly interact with Hck SH3 but bind to Src and Fyn SH3 domains. The molecular basis of these differences in SH3 targeting was revealed by sequence analysis and homology modeling of the putative SH3-Nef structures. Three amino acids (Trp-113, Thr-117, and Gln-118) that localize in a "hydrophobic pocket" implicated in SH3 binding of HIV-1 Nef, are systematically substituted in SIV/HIV-2 alleles (by Tyr, Glu, and Glu, respectively). We demonstrate that site-directed mutagenesis of these residues in SIV mac239 Nef suffices to restore Hck SH3 binding and co-immunoprecipitation with full-length Hck from transfected cells. Our findings identify fundamental mechanistic differences in targeting of Src family kinases by HIV and SIV Nef. The herein described mechanism of SH3 selection by Nef via a "pocket" proximal to the canonical proline-rich motif may be a common feature for SH3 recognition by their natural ligands.The nef gene is conserved among all primate lentiviruses and has been implicated in optimal viral replication and disease induction by HIV-1 1 and SIV (1, 2). Substantial sequence polymorphism has been detected among nef alleles from various cloned isolates of HIV and SIV, suggesting that Nef proteins may have subtype-specific functions or possess different strategies to perform the same task. However, four stretches of residues have been identified that were highly conserved among sequences derived from AIDS patients but also from among sequences of HIV-2 and SIV (3). One of these stretches includes a Pro-Xaa-Xaa-Pro motif, the canonical Src homology (SH) 3 domain binding consensus, indicating that SH3 binding is a common feature of all types of Nef alleles.An intact PXXP motif is required for the potential of HIV-1 Nef to enhance viral growth and infectivity in cell cultures (4 -6). It has subsequently been shown that HIV-1 Nef binds with high affinity to the SH3 domains of the Src family kinases Hck and Lyn, whereas only with a modest affinity to SH3 domains of other members of the Src family (Lck, Fyn, and Src) (7,8). However, despite the PXXP motif being strictly conserved in all Nef proteins, an HIV-2 Nef allele has been reported to have lost the high affinity for Hck SH3 (9). Controversial data has further been collected for the requirement of the proline-rich motif for AIDS disease progression in infected macaques (10, 11). Especially, it was reported that a PXXP to AXXA mutated SIV mac239 protein could still bind to Src kinase (10), suggesting that SIV might have different strategies of interacting with the Src family of protein tyro...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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HIV-2 and SIV Nef Proteins Target Different Src Family SH3 Domains than Does HIV-1 Nef because of a Triple Amino Acid Substitution

Collette¹,

Arold²,

Picard³

et al. 2000

Journal of Biological Chemistry

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“…Phospho-amino acids analysis demonstrated that the majority of the phosphorylation of the p62/p95/p85 species occurred on serine residues (Fig. 1C), indicating that murine NAK, like its human and simian homologues (47,48,56,59,64,65,74), is a serine-threonine kinase. These results indicated that Nef binds to a murine serine kinase in primary immune cells of Tg mice.…”

Section: Nak Activity Is Present In Thymocytes Macrophages and Dcs mentioning

confidence: 94%

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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“…Observations from naturally HIV-1-infected individuals indicated that Nef functions on downmodulation of CD4 and MHC-I could be related to the pathogenesis of AIDS (Carl et al, 2001;Tobiume et al, 2002), however the real contribution of these functions still needs to be demonstrated (Crotti et al, 2006). The motifs in Nef that mediate CD4 downregulation were considered critical for SIVmac replication in rhesus macaques, however MHC-I downregulation by Nef is not sufficient for optimal virulence of SIVmac early in infection Lang et al, 1997;Schindler et al, 2004). Moreover, the solely importance of the CD4 downmodulation for SIVmac pathogenesis in experimental models has been challenged (Jesus da Costa et al, 2009).…”

Section: Downmodulation Of Cd4 and Mhc-imentioning

confidence: 99%

Functions of the Lentiviral Accessory Protein Nef During the Distinct Steps of HIV and SIV Replication Cycle

Costa¹,

Mendonça²,

Sampaio³

2011

HIV and AIDS - Updates on Biology, Immunology, Epidemiology and Treatment Strategies

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Association of simian immunodeficiency virus Nef with cellular serine/threonine kinases is dispensable for the development of AIDS in rhesus macaques

Cited by 85 publications

References 39 publications

HIV-2 and SIV Nef Proteins Target Different Src Family SH3 Domains than Does HIV-1 Nef because of a Triple Amino Acid Substitution

HIV-2 and SIV Nef Proteins Target Different Src Family SH3 Domains than Does HIV-1 Nef because of a Triple Amino Acid Substitution

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

Functions of the Lentiviral Accessory Protein Nef During the Distinct Steps of HIV and SIV Replication Cycle

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