ANALYSIS OF HIV-1 TAT EFFECTS IN XENOPUS-LAEVIS EMBRYOS

Tat is one of the regulatory proteins of the HIV-1 virus. To date, bes ides the transactivation activity, a myriad of effects exerted by HN-I 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 reg ulatory factors in the embryo has been widely used as a quick and effe ctive first screen for protein function. Xenopus' early development is well characterized by stage-specific phenotypes, therefore, an in viv o HIV-1 Tat-mediated aberrant phenotype can easily be detected and ana lyzed. HIV-1 Tat protein expression through injection of synthetic mRN A into zygotes produced a marked delay in gastrulation leading to alte red specification of the anterior-posterior axis and to partial or tot al loss of anterior structures. HIV-1 Tat effects resulted in a genera l 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 fu nction' mutant (Tat-C37S), lacking a single cysteine residue, which di d not yield any effect. Both Tat and Tat-C37S were found to be localiz ed 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 seq uence. The Tat-BDM protein localized in the cytoplasm and accumulated at the cell membrane. Embryos injected with Tat-BDM mRNA did not devel op beyond gastrulation. The importance of proper protein conformation and subcellular localization in determining Tat effects is discussed.