Design and use of an inducibly activated human immunodeficiency virus type1 Nef to study immune modulation

The Nef protein of the human immunodeficiency virus type 1 (HIV-1) has been shown to enhance the infectivity of virus particles, downmodulate cell sur face proteins, and associate with many intracellular proteins that are thou ght to facilitate HIV infection. One of the challenges in defining the mole cular events regulated hy Nef has been obtaining good expression of Nef pro tein in T cells. This has been attributed to effects of Nef on cell prolife ration and apoptosis. We have designed a Nef protein that is readily expres sed in T cell lines and whose function is inducibly activated. It is compos ed of a fusion between full-length Nef and the estrogen receptor hormone-bi nding domain (Nef-ER). The Nef-ER is kept in an inactive state due to steri c hindrance, and addition of the membrane-permeable drug 4-hydroxytamoxifen (4-HT), which binds to the ER domain, leads to inducible activation of Nef -ER within cells. We demonstrate that Nef-ER inducibly associates with the 62-kDa Ser/Thr kinase and is localized to specific membrane microdomains (l ipid rafts) only after activation. Using this inducible Nef, we also compar ed the specific requirements for CD4 and HLA-A2 downmodulation in a SupT1 T -cell line. Half-maximal downmodulation of cell surface CD4 required very l ittle active Nef-ER and occurred as early as 4 h after addition of 4-HT. In contrast, 50% downmodulation of HLA-A2 by Nef required 16 to 24 h and abou t 50- to 100-fold-greater concentrations of 4-HT. These data suggest that H LA-A2 downmodulation may require certain threshold levels of active Nef. Th e differential timing of CD4 and HLA-A2 downmodulation may have implication s for HIV pathogenesis and immune evasion.