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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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Requirement of the TCR ζ chain for binding of NAK/p62 to Nef. (A) In vitro kinase assay after immunoprecipitation (IP) of CD8-Nef  chimeras using the CD8 tag from stably transfected wild-type and mutant Jurkat cell lines lacking Lck, TCR, or CD45. Lane 1, control (Cont.) Jurkat  transfected with the CD8 tag. (B) Control immunoprecipitation showing expression of 35S-labeled CD8-Nef. (C) In vitro kinase assay after immunoprecipitation of CD8-Nef to study NAK/p62 association/phosphorylation in wild-type (Cont., lane 1) and TCR− Jurkat cell lines (lane 2), and after coexpression (stable transfection) of CD8-Nef with either CD16ε, CD16ζ2 (ITAM 2), CD16ζ3 (ITAM 3), or CD16ζmu (mutation of all three ITAMs) in  the TCR− cell line (lanes 3–6). (D) Control immunoprecipitation of 35S-labeled CD8-Nef from cell lines shown in C. (E) In vitro kinase assay after immunoprecipitation of AU-1–tagged Nef from transiently transfected 293T cells. Lane 1, transfection with Nef alone; lanes 2–7, cotransfection with increasing amounts (0.5 and 1 μg) of CD16ε, CD16ζ, or CD16ζmu. (F) The nitrocellulose filter shown in E was blotted (WB) with an anti-Nef (AU-1)  antibody to verify comparable Nef expression.
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Figure 1: Requirement of the TCR ζ chain for binding of NAK/p62 to Nef. (A) In vitro kinase assay after immunoprecipitation (IP) of CD8-Nef chimeras using the CD8 tag from stably transfected wild-type and mutant Jurkat cell lines lacking Lck, TCR, or CD45. Lane 1, control (Cont.) Jurkat transfected with the CD8 tag. (B) Control immunoprecipitation showing expression of 35S-labeled CD8-Nef. (C) In vitro kinase assay after immunoprecipitation of CD8-Nef to study NAK/p62 association/phosphorylation in wild-type (Cont., lane 1) and TCR− Jurkat cell lines (lane 2), and after coexpression (stable transfection) of CD8-Nef with either CD16ε, CD16ζ2 (ITAM 2), CD16ζ3 (ITAM 3), or CD16ζmu (mutation of all three ITAMs) in the TCR− cell line (lanes 3–6). (D) Control immunoprecipitation of 35S-labeled CD8-Nef from cell lines shown in C. (E) In vitro kinase assay after immunoprecipitation of AU-1–tagged Nef from transiently transfected 293T cells. Lane 1, transfection with Nef alone; lanes 2–7, cotransfection with increasing amounts (0.5 and 1 μg) of CD16ε, CD16ζ, or CD16ζmu. (F) The nitrocellulose filter shown in E was blotted (WB) with an anti-Nef (AU-1) antibody to verify comparable Nef expression.

Mentions: As shown previously, Nef associates with a serine kinase, termed p62 or Nef-associated kinase (NAK [31]). The Nef–NAK interaction is complex: Nef stimulates the phosphorylation/activation of NAK, and it is only in this activated form that NAK can bind Nef (32). This suggests that Nef must act upstream of NAK to promote NAK activation. Our previous results showing that Nef interfered with early signals emanating from the TCR suggested it may interact with a component of the TCR signaling complex. This prompted us to study Nef-mediated NAK/p62 activation in cell lines with TCR signaling defects. CD8-Nef chimeras (CD8-Nef), containing the extracellular domain of CD8α fused to Nef, were stably transfected into wild-type Jurkat and a variety of Jurkat mutant cell lines lacking either Lck (J.CaM.1), CD45 (CD45−), or the entire TCR signaling complex (RT3.T3.5). Expression of CD8-Nef in these cell lines was verified by metabolic labeling and immunoprecipitation (Fig. 1 B). The Nef chimeras from these transfectants were immunoprecipitated and subjected to an in vitro kinase assay. NAK/p62 association was observed in all cell lines except the TCR− cells (Fig. 1 A). The latter result was confirmed in a second, independently transfected cell clone (data not shown).


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)

Requirement of the TCR ζ chain for binding of NAK/p62 to Nef. (A) In vitro kinase assay after immunoprecipitation (IP) of CD8-Nef  chimeras using the CD8 tag from stably transfected wild-type and mutant Jurkat cell lines lacking Lck, TCR, or CD45. Lane 1, control (Cont.) Jurkat  transfected with the CD8 tag. (B) Control immunoprecipitation showing expression of 35S-labeled CD8-Nef. (C) In vitro kinase assay after immunoprecipitation of CD8-Nef to study NAK/p62 association/phosphorylation in wild-type (Cont., lane 1) and TCR− Jurkat cell lines (lane 2), and after coexpression (stable transfection) of CD8-Nef with either CD16ε, CD16ζ2 (ITAM 2), CD16ζ3 (ITAM 3), or CD16ζmu (mutation of all three ITAMs) in  the TCR− cell line (lanes 3–6). (D) Control immunoprecipitation of 35S-labeled CD8-Nef from cell lines shown in C. (E) In vitro kinase assay after immunoprecipitation of AU-1–tagged Nef from transiently transfected 293T cells. Lane 1, transfection with Nef alone; lanes 2–7, cotransfection with increasing amounts (0.5 and 1 μg) of CD16ε, CD16ζ, or CD16ζmu. (F) The nitrocellulose filter shown in E was blotted (WB) with an anti-Nef (AU-1)  antibody to verify comparable Nef expression.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193060&req=5

Figure 1: Requirement of the TCR ζ chain for binding of NAK/p62 to Nef. (A) In vitro kinase assay after immunoprecipitation (IP) of CD8-Nef chimeras using the CD8 tag from stably transfected wild-type and mutant Jurkat cell lines lacking Lck, TCR, or CD45. Lane 1, control (Cont.) Jurkat transfected with the CD8 tag. (B) Control immunoprecipitation showing expression of 35S-labeled CD8-Nef. (C) In vitro kinase assay after immunoprecipitation of CD8-Nef to study NAK/p62 association/phosphorylation in wild-type (Cont., lane 1) and TCR− Jurkat cell lines (lane 2), and after coexpression (stable transfection) of CD8-Nef with either CD16ε, CD16ζ2 (ITAM 2), CD16ζ3 (ITAM 3), or CD16ζmu (mutation of all three ITAMs) in the TCR− cell line (lanes 3–6). (D) Control immunoprecipitation of 35S-labeled CD8-Nef from cell lines shown in C. (E) In vitro kinase assay after immunoprecipitation of AU-1–tagged Nef from transiently transfected 293T cells. Lane 1, transfection with Nef alone; lanes 2–7, cotransfection with increasing amounts (0.5 and 1 μg) of CD16ε, CD16ζ, or CD16ζmu. (F) The nitrocellulose filter shown in E was blotted (WB) with an anti-Nef (AU-1) antibody to verify comparable Nef expression.
Mentions: As shown previously, Nef associates with a serine kinase, termed p62 or Nef-associated kinase (NAK [31]). The Nef–NAK interaction is complex: Nef stimulates the phosphorylation/activation of NAK, and it is only in this activated form that NAK can bind Nef (32). This suggests that Nef must act upstream of NAK to promote NAK activation. Our previous results showing that Nef interfered with early signals emanating from the TCR suggested it may interact with a component of the TCR signaling complex. This prompted us to study Nef-mediated NAK/p62 activation in cell lines with TCR signaling defects. CD8-Nef chimeras (CD8-Nef), containing the extracellular domain of CD8α fused to Nef, were stably transfected into wild-type Jurkat and a variety of Jurkat mutant cell lines lacking either Lck (J.CaM.1), CD45 (CD45−), or the entire TCR signaling complex (RT3.T3.5). Expression of CD8-Nef in these cell lines was verified by metabolic labeling and immunoprecipitation (Fig. 1 B). The Nef chimeras from these transfectants were immunoprecipitated and subjected to an in vitro kinase assay. NAK/p62 association was observed in all cell lines except the TCR− cells (Fig. 1 A). The latter result was confirmed in a second, independently transfected cell clone (data not shown).

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