The transforming activity of c-Raf-1 can be activated by deletions or insertions in the N-terminal half of the protein. Using a cotransfection assay, we have demonstrated that c-Raf-1 kinase activates transcription from the HIV-LTR. Mutations that activate the transforming activity (e.g., c-Raf-BXB) also increase the transactivation potential of the Raf-I protein. Activation of latent provirus occurs when cells are stimulated with physiological inducers or tumor promoters such as 12-0-tetradecanoyl-phorbol-13-acetate (TPA). We have begun experiments aimed at determining if c-Raf-I is involved in TPA induction of the HIV-LTR. We have recently observed, in agreement with published data, that transcription from the HIV-LTR is activated by TPA. We explored the c-Raf-I dependence of this induction utilizing a dominant negative mutant, c-Raf-301, which carries a single amino acid substitution (LYS-TRP) in the putative ATP binding site of Raf-1 kinase and found that it blocks TPA induction. The ability of c-Raf-301 to block TPA induction strongly suggests that c-Raf-I is mediating TPA induction of the HIV-LTR. Clearly, c-Raf-I is an important mediator of signal transduction from the membrane to the nucleus, and we have demonstrated that the HIV-LTR is a target for activated c-Raf. In many receptor systems where cell proliferation is the end result, triggering of the receptor is followed by hyper-phosphorylation of 100 percent of the c-Raf-1 molecules in the cell. Association of the HIV envelope with the CD4 receptor is an essential step in HIV infection. In the CD4 system, antibody cross-linking results in a transient phosphorylation of 1 to 5 percent of the c-Raf-I molecules in the cell, and the mitogenic signalling is abortive. Thus, the role of c-Raf-I in the initial stages of HIV infection may be different from its role in cellular proliferation.
