Regulation of human immunodeficiency virus type 1 gene expression by clade-specific Tat proteins.

The major group of human immunodeficiency virus type 1 (HIV-1) strains that comprise the current global pandemic have diversified during their worldwide spread into at least 10 distinct subtypes, or clades. Subtype C predominates in sub-Saharan Africa and is responsible for the majority of worldwide HIV-1 infections, subtype B predominates in North America and Europe, and subtype E is prevalent in Southeast Asia. Significant amino acid variations have been observed among the clade-specific Tat proteins. For the present study, we examined clade-specific interactions between Tat, transactivation-responsive (TAR) element, and P-TEFb proteins and how these interactions may modulate the efficiency of HIV-1 transcription. Clade-specific Tat proteins significantly modified viral gene expression. Tat proteins derived from HIV-1 clades C and E were strong transactivators of long terminal repeat (LTR) activity; Tat E also had a longer half-life than the other Tat proteins and interacted more efficiently with the stem-loop TAR element. Chimeric Tat proteins harboring the Tat E activation domain were strong transactivators of LTR expression. While Tat B, C, and E were able to rescue a Tat-defective HIV-1 proviral clone, Tat E was significantly more efficient at rescue than Tat C, possibly due to the relative stability of the Tat protein. Swapping the activation domains of Tat B, C, and E identified the cyclin T1 association domain as a critical determinant of the transactivation efficiency and of Tat-defective HIV-1 provirus rescue.

c-Myc potentiates the mitochondrial pathway of apoptosis by acting upstream of apoptosis signal-regulating kinase 1 (Ask1) in the p38 signalling cascade.

Cell transformation by growth-promoting oncoproteins renders cells extremely sensitive to apoptosis through an unknown mechanism affecting the mitochondrial pathway of apoptosis. We have shown previously that sensitization to apoptosis also correlated with the activation of the stress-activated protein kinase p38. In the present study, we investigated the role of p38 in c-Myc-dependent apoptosis induced by the anticancer agent cisplatin. Cisplatin treatment of Rat1 cells with deregulated expression of c-Myc resulted in nuclear fragmentation that was accompanied in all cells by the activation of Bax and the translocation of cytochrome c from the mitochondria to the cytoplasm. None of these features of apoptosis was induced in control Rat-1 cells. p38 was also activated by cisplatin only in cells with deregulated expression of c-Myc, but, in contrast with all features of apoptosis, this activation was not affected by Bcl-2. Remarkably, overexpression of an interfering mutant of the p38alpha isoform, but not p38beta, blocked cisplatin-induced Bax activation or cytochrome c release and nuclear fragmentation. Analysis of the kinase cascade upstream of p38 revealed a c-Myc-dependent activation by cisplatin of mitogen-activated protein kinase kinase (MKK) 3/6 and apoptosis signal-regulating kinase 1 (Ask1). Inhibition of Ask1 blocked p38 activation by cisplatin and all features of apoptosis. Several of these data were confirmed using other DNA-damaging agents. The findings indicated that c-Myc potentiation of the mitochondrial pathway of apoptosis results, at least in part, from a sensitization of Ask1 activation, allowing DNA-damaging agents to induce in cascade Ask1, p38alpha and Bax.