Analysis of HIV-1 Tat effects in Xenopus laevis embryos.
Tat is one of the regulatory proteins of the HIV-1 virus. To date, besides the transactivation activity, a myriad of effects exerted by HIV-1 Tat on cellular and viral genes have been observed. The present study investigated the in vivo effects of HIV-1 Tat protein in the Xenopus embryo. We adopted the Xenopus system since expression of putative regulatory factors in the embryo has been widely used as a quick and effective first screen for protein function. Xenopus' early development is well characterized by stage-specific phenotypes, therefore, an in vivo HIV-1 Tat-mediated aberrant phenotype can easily be detected and analyzed. HIV-1 Tat protein expression through injection of synthetic mRNA into zygotes produced a marked delay in gastrulation leading to altered specification of the anterior-posterior axis and to partial or total loss of anterior structures. HIV-1 Tat effects resulted in a general suppression of gene expression, including that of Xbra and gsc, two early genes whose expression is required for proper gastrulation. The specificity of Tat effects was demonstrated by injecting a 'loss of function' mutant (Tat-C37S), lacking a single cysteine residue, which did not yield any effect. Both Tat and Tat-C37S were found to be localized mainly in the nucleus. The importance of subcellular targeting for the effects caused by HIV-1 Tat was demonstrated by injecting a second mutant (Tat-BDM), carrying an altered nuclear localization signal sequence. The Tat-BDM protein localized in the cytoplasm and accumulated at the cell membrane. Embryos injected with Tat-BDM mRNA did not develop beyond gastrulation. The importance of proper protein conformation and subcellular localization in determining Tat effects is discussed.
Reparto di Genetica Molecolare, Laboratorio di Biologia Cellulare, Istituto Superiore di Sanità, Rome, Italy. s.venanz@iss.it
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D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism / Q000821:virology; D003593:Cytoplasm / Q000378:metabolism / Q000821:virology; D015536:Down-Regulation; D004625:Embryo, Nonmammalian / Q000502:physiology; D018507:Gene Expression Regulation, Developmental; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D012333:RNA, Messenger; D014162:Transfection; D014982:Xenopus laevis / Q000196:embryology*; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D010446:Peptide Fragments; D012333:RNA, Messenger; C111139:Tat peptide (37-72), Human immunodeficiency virus 1; D054322:tat Gene Products, Human Immunodeficiency Virus