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Induction of Fas ligand expression by HIV involves the interaction of Nef with the T cell receptor zeta chain.

Xu XN, Laffert B, Screaton GR, Kraft M, Wolf D, Kolanus W, Mongkolsapay J, McMichael AJ, Baur AS - J. Exp. Med. (1999)

Bottom Line: A single ITAM motif of zeta but not CD3epsilon was both required and sufficient to promote activation and binding of the Nef-associated kinase (NAK/p62).Our data imply that Nef can form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T cell activation and subsequently upregulation of FasL expression.Thus, our study may provide critical insights into the molecular mechanism whereby the HIV-1 accessory protein Nef contributes to the pathogenesis of HIV.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom.

ABSTRACT
During HIV/SIV infection, there is widespread programmed cell death in infected and, perhaps more importantly, uninfected cells. Much of this apoptosis is mediated by Fas-Fas ligand (FasL) interactions. Previously we demonstrated in macaques that induction of FasL expression and apoptotic cell death of both CD4(+) and CD8(+) T cells by SIV is dependent on a functional nef gene. However, the molecular mechanism whereby HIV-1 induces the expression of FasL remained poorly understood. Here we report a direct association of HIV-1 Nef with the zeta chain of the T cell receptor (TCR) complex and the requirement of both proteins for HIV-mediated upregulation of FasL. Expression of FasL through Nef depended upon the integrity of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain. Conformation for the importance of zeta for Nef-mediated signaling in T cells came from an independent finding. A single ITAM motif of zeta but not CD3epsilon was both required and sufficient to promote activation and binding of the Nef-associated kinase (NAK/p62). Our data imply that Nef can form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T cell activation and subsequently upregulation of FasL expression. Thus, our study may provide critical insights into the molecular mechanism whereby the HIV-1 accessory protein Nef contributes to the pathogenesis of HIV.

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FasL upregulation by  HIV requires the TCR ζ chain.  FasL expression was assessed 48 h  after infection with HIV (IIIB  strain) by immunoprecipitation  using an anti-FasL mAb (A) or  by flow cytometry (B). Wild-type (WT) and TCR− Jurkat are  compared with the TCR− cells  stably transfected with CD16ζ or  CD16ζmu (signaling-defective  CD16ζ). The level of viral replication in Jurkat cells was comparable as determined by p24 assay  (wild-type, 3.5 ± 0.5; TCR−,  3.8 ± 1.0; CD16ζ, 3.8 ± 0.5;  CD16ζmu, 3.1 ± 0.25 ng/ml).
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Figure 4: FasL upregulation by HIV requires the TCR ζ chain. FasL expression was assessed 48 h after infection with HIV (IIIB strain) by immunoprecipitation using an anti-FasL mAb (A) or by flow cytometry (B). Wild-type (WT) and TCR− Jurkat are compared with the TCR− cells stably transfected with CD16ζ or CD16ζmu (signaling-defective CD16ζ). The level of viral replication in Jurkat cells was comparable as determined by p24 assay (wild-type, 3.5 ± 0.5; TCR−, 3.8 ± 1.0; CD16ζ, 3.8 ± 0.5; CD16ζmu, 3.1 ± 0.25 ng/ml).

Mentions: We have previously shown that the upregulation of FasL in SIV infection requires an intact nef gene (22). In general, the level of cell surface FasL expression is quite low when analyzed by FACS® even when metalloproteinase inhibitors are used which block cleavage of FasL from the cell surface. In view of this difficulty, we used additional experimental approaches to analyze Nef-mediated FasL expression (see below). Since stimulation of TCR-ζ effectively upregulates FasL expression (34, 36), we speculated that the interaction of Nef with TCR-ζ would lead to a similar effect. First, to show that HIV-Nef is required for FasL upregulation, we infected Jurkat with wild-type HIV (NL4-3.SF2Nef) or a mutant lacking the nef gene (NL4-3ΔNef) (Fig. 3). Little if any FasL is seen on cells infected with Nef-deleted HIV, thus confirming our previous results with SIV. The level of viral replication in Jurkat cells was comparable, as determined by RT activity (NL4-3.SF2Nef, 4.6 × 103 cpm; NL4-3ΔNef, 5.8 × 103 cpm). Upregulation of FasL by HIV is also lost in mutant Jurkat cells lacking the TCR complex, whereas cells reconstituted with ζ but not with the ζ mutant restored the FasL expression upon HIV infection as determined by both immunoprecipitation (Fig. 4 A) and FACS® analysis (Fig. 4 B). Viral replication assessed by p24 assay indicated that these cell lines were comparably infected (wild-type, 3.5 ± 0.5; TCR−, 3.8 ± 1.0; CD16ζ, 3.8 ± 0.5; CD16ζmu, 3.1 ± 0.25 ng/ml).


Induction of Fas ligand expression by HIV involves the interaction of Nef with the T cell receptor zeta chain.

Xu XN, Laffert B, Screaton GR, Kraft M, Wolf D, Kolanus W, Mongkolsapay J, McMichael AJ, Baur AS - J. Exp. Med. (1999)

FasL upregulation by  HIV requires the TCR ζ chain.  FasL expression was assessed 48 h  after infection with HIV (IIIB  strain) by immunoprecipitation  using an anti-FasL mAb (A) or  by flow cytometry (B). Wild-type (WT) and TCR− Jurkat are  compared with the TCR− cells  stably transfected with CD16ζ or  CD16ζmu (signaling-defective  CD16ζ). The level of viral replication in Jurkat cells was comparable as determined by p24 assay  (wild-type, 3.5 ± 0.5; TCR−,  3.8 ± 1.0; CD16ζ, 3.8 ± 0.5;  CD16ζmu, 3.1 ± 0.25 ng/ml).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2193060&req=5

Figure 4: FasL upregulation by HIV requires the TCR ζ chain. FasL expression was assessed 48 h after infection with HIV (IIIB strain) by immunoprecipitation using an anti-FasL mAb (A) or by flow cytometry (B). Wild-type (WT) and TCR− Jurkat are compared with the TCR− cells stably transfected with CD16ζ or CD16ζmu (signaling-defective CD16ζ). The level of viral replication in Jurkat cells was comparable as determined by p24 assay (wild-type, 3.5 ± 0.5; TCR−, 3.8 ± 1.0; CD16ζ, 3.8 ± 0.5; CD16ζmu, 3.1 ± 0.25 ng/ml).
Mentions: We have previously shown that the upregulation of FasL in SIV infection requires an intact nef gene (22). In general, the level of cell surface FasL expression is quite low when analyzed by FACS® even when metalloproteinase inhibitors are used which block cleavage of FasL from the cell surface. In view of this difficulty, we used additional experimental approaches to analyze Nef-mediated FasL expression (see below). Since stimulation of TCR-ζ effectively upregulates FasL expression (34, 36), we speculated that the interaction of Nef with TCR-ζ would lead to a similar effect. First, to show that HIV-Nef is required for FasL upregulation, we infected Jurkat with wild-type HIV (NL4-3.SF2Nef) or a mutant lacking the nef gene (NL4-3ΔNef) (Fig. 3). Little if any FasL is seen on cells infected with Nef-deleted HIV, thus confirming our previous results with SIV. The level of viral replication in Jurkat cells was comparable, as determined by RT activity (NL4-3.SF2Nef, 4.6 × 103 cpm; NL4-3ΔNef, 5.8 × 103 cpm). Upregulation of FasL by HIV is also lost in mutant Jurkat cells lacking the TCR complex, whereas cells reconstituted with ζ but not with the ζ mutant restored the FasL expression upon HIV infection as determined by both immunoprecipitation (Fig. 4 A) and FACS® analysis (Fig. 4 B). Viral replication assessed by p24 assay indicated that these cell lines were comparably infected (wild-type, 3.5 ± 0.5; TCR−, 3.8 ± 1.0; CD16ζ, 3.8 ± 0.5; CD16ζmu, 3.1 ± 0.25 ng/ml).

Bottom Line: A single ITAM motif of zeta but not CD3epsilon was both required and sufficient to promote activation and binding of the Nef-associated kinase (NAK/p62).Our data imply that Nef can form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T cell activation and subsequently upregulation of FasL expression.Thus, our study may provide critical insights into the molecular mechanism whereby the HIV-1 accessory protein Nef contributes to the pathogenesis of HIV.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom.

ABSTRACT
During HIV/SIV infection, there is widespread programmed cell death in infected and, perhaps more importantly, uninfected cells. Much of this apoptosis is mediated by Fas-Fas ligand (FasL) interactions. Previously we demonstrated in macaques that induction of FasL expression and apoptotic cell death of both CD4(+) and CD8(+) T cells by SIV is dependent on a functional nef gene. However, the molecular mechanism whereby HIV-1 induces the expression of FasL remained poorly understood. Here we report a direct association of HIV-1 Nef with the zeta chain of the T cell receptor (TCR) complex and the requirement of both proteins for HIV-mediated upregulation of FasL. Expression of FasL through Nef depended upon the integrity of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain. Conformation for the importance of zeta for Nef-mediated signaling in T cells came from an independent finding. A single ITAM motif of zeta but not CD3epsilon was both required and sufficient to promote activation and binding of the Nef-associated kinase (NAK/p62). Our data imply that Nef can form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T cell activation and subsequently upregulation of FasL expression. Thus, our study may provide critical insights into the molecular mechanism whereby the HIV-1 accessory protein Nef contributes to the pathogenesis of HIV.

Show MeSH
Related in: MedlinePlus