Nuclear translocation of an exogenous fusion protein containing HIV Tat requires unfolding.
OBJECTIVE
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.


METHODS
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.


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-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.


CONCLUSIONS
HIV-1 Tat can act as a vector to drive polypeptides into the nucleoplasm of living cells. The inhibitor effects of MTX on the nuclear localization of Tat-DHFR suggest that an unfolding step is required for the internalization of exogenous Tat.
Bonifaci
N
N
Laboratory of Clinical Pathology, National Institute of Cancer Research, Genoa, Italy.
Sitia
R
R
Rubartelli
A
A
D002467:Cell Nucleus / Q000821:virology*; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D017930:Genes, Reporter / Q000235:genetics; D015756:Genes, tat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D015398:Signal Transduction / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics; D014178:Translocation, Genetic / Q000235:genetics*; D014760:Viral Fusion Proteins / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics
D014760:Viral Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase