Plasma membrane-targeted Raf kinase activates NF-κB and human immunodeficiency virus type 1 replication in T lymphocytes

Increasing evidence points to a role of the mitogenic Ras/Raf/MEK/ERK signaling cascade in regulation of human immunodeficiency virus type 1 (HIV-1) gene expression. Stimulation of elements of this pathway leads to transactivation of the HIV-1 promoter. In particular, the NF-kappa B motif in the HIV long terminal repeat (LTR) represents a Raf-responsive element in fibroblasts. Regulation of the Raf kinase in T cells differs from findings with a variety of cell lines that the catalytic domain of Raf (Raf(Delta 26-303)) shows no activity, In this study, we restored the activity of the kinase in T cells by fusing its catalytic domain to the CAAX motif (-Cx) of Ras,thus targeting the enzyme to the plasma membrane, Constitutive activity of Raf was demonstrated by phosphorylation of mitogen-activated protein kinase kinase (MEK) and endogenous mitogen-activated protein kinase 1/2 (ERK1/2) in A3.01 T cells transfected with Raf(Delta 26-303)-Cx. Membrane-targeted Raf also stimulates NF-kappa B, as judged by kappa B-dependent reporter assays and enhanced NF-kappa B p65 binding on band shift analysis. Moreover, we found that active Raf transactivates the HIVNL4-3 LTR in A3.01 T lymphocytes and that dominant negative Raf (C4) blocked 12-O-tetradecanoylphorbol-13-acetate induced transactivation. When cotransfected with infectious HIVNL4-3 DNA, membrane-targeted Raf induces viral replication up to 10-fold over basal levels, as determined by the release of newly synthesized p24(gag) protein, Our study clearly demonstrates that the activity of the catalytic domain of Raf in A3.01 T cells is dependent on its cellular localization, The functional consequences of active Raf in T lymphocytes include not only NF-kappa B activation and transactivation of the HIVNL4-3 LTR but also synthesis and release of HIV particles.