Inhibition of human immunodeficiency virus type 1 and type 2 Tat function by transdominant Tat protein localized to both the nucleus and cytoplasm.
We introduced various mutations into the activation and RNA binding domains of human immunodeficiency virus type 1 (HIV-1) Tat in order to develop a novel and potent transdominant Tat protein and to characterize its mechanism of action. The different mutant Tat proteins were characterized for their abilities to activate the HIV LTR and inhibit the function of wild-type Tat in trans. A Tat protein containing a deletion of the basic domain (Tat(delta)49-57) localized exclusively to the cytoplasm of transfected human cells was nonfunctional and inhibited both HIV-1 and HIV-2 Tat function in a transdominant manner. Tat proteins containing mutations in the cysteine-rich and core domains were nonfunctional but failed to inhibit Tat function in trans. When Tat nuclear or nucleolar localization signals were fused to the carboxy terminus of Tat(delta)49-57, the chimeric proteins localized to the nucleus or nucleolus, respectively, and remained capable of acting in a transdominant manner. Introduction of secondary mutations in the cysteine-rich and core domains of the various transdominant Tat proteins completely eliminated their abilities to act in a transdominant fashion. Our data best support a mechanism in which these transdominant Tat proteins squelch a cellular factor or factors that interact with the Tat activation domain and are required for Tat to function.
Orsini
M J
MJ
Department of Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
Debouck
C M
CM
D000595:Amino Acid Sequence; D002460:Cell Line; D002467:Cell Nucleus; D003001:Cloning, Molecular; D003593:Cytoplasm; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D015498:HIV-2 / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus