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.
Venanzi
S
S
Reparto di Genetica Molecolare, Laboratorio di Biologia Cellulare, Istituto Superiore di Sanità, Rome, Italy. s.venanz@iss.it
Destrée
O H
OH
Gigliani
F
F
Battaglia
P A
PA
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