Extracellular Nef Protein Activates Signal Transduction Pathway from Ras to Mitogen-Activated Protein Kinase Cascades That Leads to Activation of Human Immunodeficiency Virus from Latency

We previously reported that viral antigen expression was markedly up-regulated by stimulation with extracellular Nef, similar to the effects of tumor necrosis factor (TNF)-α and phorbol myristate acetate, in model cells for HIV-1 latency. In this study, we examined the molecular mechanism of this novel Nef function. Flow cytometry revealed specific binding of Nef on the surface of latently infected cells. Furthermore, activation of Ras in the cells was detected after treatment with Nef, indicating the involvement of Ras in Nef-mediated activation of HIV-1 from latency. This was also confirmed by the observations that HIV-1 long-terminal repeat-luciferase (LTR-Luc) activity was significantly up-regulated by introduction of the active Ras into uninfected cells, and that LTR-Luc activity observed in Nef-treated cells was specifically inhibited by introduction of a dominant negative Ras. In addition, PD98059 inhibited the activation of HIV-1 by Nef, but not by TNF-α. Thus, Nef-mediated reactivation of HIV-1 in latent model cells occurs by signal transduction from Ras to mitogen-activated protein kinase cascades.