['To characterize the transcellular transport of HIV-1 Tat. HIV-1 Tat contains a putative localization signal and no leader peptide; however, it can be released from virus-infected cells and taken up by uninfected cells.', 'We constructed a chimeric protein between Tat and dihydrofolate reductase (DHFR), a cytosolic enzyme that binds tightly to the folate analogue methotrexate (MTX). As confirmed by protease sensitivity assays, binding to MTX results in stabilization of the three-dimensional structure of the DHFR domain. The nuclear translocation of recombinant proteins was monitored by both functional [transcellular transactivation of a long terminal repeat-chloramphenicol acetyl transferase (LTR-CAT) reporter gene] and biochemical (subcellular localization in HeLa cells of exogenous radiolabelled proteins) assays and the effects of MTX-induced stabilization were evaluated.', 'When in vitro translated proteins are added to HeLa cells in culture, both wild-type Tat and the chimeric protein Tat-DHFR are taken up by target cells and accumulate in the nucleus, unlike wild-type DHFR. Cells transfected with Tat-DHFR, when co-cultured with cells harbouring a LTR-CAT gene, induce transactivation of the reporter gene to the same extent as cells expressing wild-type Tat. These findings indicate that Tat can mediate the internalization of unrelated polypeptides. Pre-treatment of Tat-DHFR with MTX blocks the nuclear translocation of the chimeric protein. MTX has no effect on wild-type Tat.'] Does nuclear translocation of an exogenous fusion protein containing HIV Tat require unfolding?