Intracellular trafficking and interactions of the HIV-1 Tat protein

Fusions of the human immunodeficiency virus type 1 (HIV-I) transactivator p rotein Tat to the green fluorescent protein (GFP) were used to study the in tracellular localization, trafficking, and interactions of Tat in human cel ls. Tagging Tat with GFP did not change its nuclear localization or ability to act as a transactivator. Tat-GFP expressed at low levels was found in t he nucleus, whereas overexpression resulted in nucleolar accumulation. A Ta t-GFP hybrid protein containing in addition the HIV-I Rev nuclear export si gnal (NES) localized predominantly to the cytoplasm. This shuttle protein, Tat-GFP-NES, transactivated the HIV-1 long terminal repeat. Thus a Tat mole cule being only transiently present in the nucleus is active and nucleolar accumulation of Tat is not prerequisite for function. A coexpression assay previously used to define protein interaction domains in the HIV-1 Rev prot ein [R. H. Stauber, E. Afonina, S. Gulnik, J. Erickson, and G. N. Pavlakis (1998a). Virology 251, 38-48.] indicated that Tat exists predominantly as a monomer and does not form stable multimers with B23 in living cells. Using a heterokaryon fusion assay, we found that Tat-GFP was able to shuttle bet ween the nucleus and the cytoplasm. Tat therefore has the potential to perf orm functions in the nucleus as well as in the cytoplasm.