CD28-dependent HIV-1 Transcription Is Associated with Vav, Rac, and NF-κB Activation

Julie A Cook,Lee Albacker,Avery August,Andrew J Henderson

doi:10.1074/jbc.m302878200

Julie A Cook, Lee Albacker + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m302878200

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Abstract

Activation of HIV-1-infected T cells through the T cell receptor and costimulatory molecule CD28 induces proviral transcription; however, the mechanism behind this enhanced virus expression is unknown. Jurkat T cells and primary CD4+ T cells expressing a CD8 alpha/CD28 chimeric receptor containing a mutation at tyrosine 200 in the cytoplasmic tail were unable to fully induce HIV-1 proviral transcription in response to CD8 alpha/28 receptor cross-linking in comparison to CD28 costimulation. The loss of transactivation seen with the mutant chimeric receptor correlated with a decrease in Vav tyrosine phosphorylation. CD28-dependent activation of HIV-1 transcription also required the GTPase activity of Rac1, which was not activated during costimulation with the mutated receptor. Furthermore, the mutated receptor was unable to induce NF-kappa B DNA binding or transactivation, as demonstrated by electromobility shift assays and HIV-1 long terminal repeat and NF-kappa B-dependent reporter constructs. These studies show that signaling events initiated by tyrosine 200 of CD28 are required for efficient expression of HIV-1 transcription in activated T cells.

Highlights

Activation of Human immunodeficiency virus type 1 (HIV-1)-infected T cells through the T cell receptor and costimulatory molecule CD28 induces proviral transcription; the mechanism behind this enhanced virus expression is unknown
Using Jurkat T cell lines and primary CD4ϩ T lymphocytes expressing chimeric CD8␣/28 receptors with mutations in critical tyrosines located in the cytoplasmic tail, we have shown that CD28 signaling is required for efficient HIV-1 transcription [18]
Tyr200 Is Necessary for CD28-induced HIV-1 Transcription—We have previously shown that Tyr173 within the cytoplasmic tail of CD28 initiates a negative signal in CD28-mediated costimulation of HIV-1 transcription [18]

Summary

The abbreviations used are

TcR, T cell receptor; HIV-1, human immunodeficiency virus type 1; LTR, long terminal repeat; CHO, Chinese hamster ovary; PLAP, placental alkaline phosphatase; Grb-2, growth receptor-bound protein 2; PI3K, phosphatidylinositol 3-kinase, NFAT, nuclear factor of activated T cells; AP-1, activator protein 1; SH2, Src-homology 2; SH3, Src homology 3; NF-␬B, nuclear factor ␬B; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; EMSA, electrophoretic mobility shift assay; PBD, P21 binding domain. Tyrosine residues in its cytoplasmic tail, which when phosphorylated, recruit and activate various kinases, phosphatases, and adapter molecules including LCK, ITK, GRID, GRB2, MKP6, and PI3K leading to changes in IL-2 production as well as HIV-1 transcription [11,12,13,14,15,16]. Signaling events initiated by the TcR and CD28 lead to increases in intracellular calcium, changes in cytoskeletal organization, and triggering of several kinase cascades Activation of these signaling pathways targets various transcription factors including NF-␬B, NFAT, AP-1, Sp1, and Ets-1 [25,26,27,28]. We show that Tyr200 is necessary for efficient HIV-1 transcription in Jurkat T cells and primary CD4ϩ T cells through initiation of signaling events that enhance Vav phosphorylation, Rac activation, and NF-␬B binding activity

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DISCUSSION