REGULATED EXPRESSION VECTORS DEMONSTRATE CELL-TYPE-SPECIFIC SENSITIVITY TO HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NEF-INDUCED CYTOSTASIS

The nef gene product of both human and simian immunodeficiency viruses is critically important for virus replication and disease progression in vivo. However, the precise biological function of Nef remains poor ly characterized in vitro, with previous reports suggesting that Nef m ight be either cytotoxic or cytostatic, As a result of difficulties en countered by several groups in establishing cell lines constitutively expressing Nef, we have developed two inducible systems resulting in s table Nef expression in various mammalian cell lines. Tetracycline-reg ulated Nef expression was achieved in HeLa cells but could not be esta blished in human T cell lines, Jurkat E6-1 T cell and RAW264.7 murine macrophage cell lines expressing a regulated nef gene were generated u sing a system in which Nef expression was controlled by a mutated vers ion of the heavy metal-inducible human metallothionein IIA promoter. I nduction of high levels of Nef expression in HeLa-Nef and Jurkat-Nef c ells resulted in a moderate (2-fold) and a dramatic (10-fold) retardat ion of cell growth respectively, supporting the contention that Nef ma y be a cytotoxic or cytostatic factor, This property was also observed at low basal levels of Nef expression in RAW264.7-Nef macrophage clon es (5-fold reduction in growth) and was associated with an altered mor phological phenotype suggesting that different cell types may be more susceptible to the cytostatic activity of Nef. The regulated Nef-expre ssion systems provide tools for investigating the molecular basis of N ef function, including Nef-mediated cytopathogenicity, CD4 down-regula tion and enhancement of virus infectivity.