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.