Intracellular trafficking and interactions of the HIV-1 Tat protein.
Fusions of the human immunodeficiency virus type 1 (HIV-1) transactivator protein Tat to the green fluorescent protein (GFP) were used to study the intracellular localization, trafficking, and interactions of Tat in human cells. 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 the nucleus, whereas overexpression resulted in nucleolar accumulation. A Tat-GFP hybrid protein containing in addition the HIV-1 Rev nuclear export signal (NES) localized predominantly to the cytoplasm. This shuttle protein, Tat-GFP-NES, transactivated the HIV-1 long terminal repeat. Thus a Tat molecule 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 protein [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 between the nucleus and the cytoplasm. Tat therefore has the potential to perform functions in the nucleus as well as in the cytoplasm.
Stauber
R H
RH
Human Retrovirus Section, NCI-FCRDC, Frederick, Maryland 21702-1201, USA.
Pavlakis
G N
GN
D001692:Biological Transport; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015694:Gene Products, rev / Q000378:metabolism; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D015497:HIV-1; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D000090243:Nucleophosmin; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D015696:Gene Products, tat; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; D000090243:Nucleophosmin; D049452:Green Fluorescent Proteins