NUCLEAR TRANSLOCATION OF AN EXOGENOUS FUSION PROTEIN CONTAINING HIV TAT REQUIRES UNFOLDING

Objective: To characterize the transcellular transport of HIV-1 Tat. H IV-1 Tat contains a putative localization signal and no leader peptide ; however, it can be released from virus-infected cells and taken up b y uninfected cells. Design and methods: We constructed a chimeric prot ein between Tat and dihydrofolate reductase (DHFR), a cytosolic enzyme that binds tightly to the folate analogue methotrexate (MTX). As conf irmed by protease sensitivity assays, binding to MTX results in stabil ization of the three-dimensional structure of the DHFR domain. The nuc lear translocation of recombinant proteins was monitored by both funct ional [transcellular transactivation of a long terminal repeat-chloram phenicol acetyl transferase (LTR-CAT) reporter gene] and biochemical ( subcellular localization in HeLa cells of exogenous radiolabelled prot eins) assays and the effects of MTX-induced stabilization were evaluat ed. Results: 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-ty pe 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 polypep tides. Pre-treatment of Tat-DHFR with MTX blocks the nuclear transloca tion of the chimeric protein. MTX has no effect on wild-type Tat. Conc lusion: HIV-1 Tat can act as a vector to drive polypeptides into the n ucleoplasm of living cells. The inhibitory effects of MTX on the nucle ar localization of Tat-DHFR suggest that an unfolding step is required for the internalization of exogenous Tat.