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

Important note: Information in this article was accurate in 1996. The state of the art may have changed since the publication date.

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

AIDS. 1995 Sep;9(9):995-1000. Unique Identifier : AIDSLINE MED/96085713
Bonifaci N; Sitia R; Rubartelli A; Laboratory of Clinical Pathology, National Institute of Cancer; Research, Genoa, Italy.

Abstract: 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. DESIGN AND 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. CONCLUSION: 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.

Keywords: Cell Nucleus/*VIROLOGY Gene Expression Regulation, Viral/PHYSIOLOGY Genes, tat/*GENETICS Genes, Reporter/GENETICS Hela Cells Human HIV-1/*GENETICS Signal Transduction/GENETICS Support, Non-U.S. Gov't Tetrahydrofolate Dehydrogenase/GENETICS Trans-Activation (Genetics)/GENETICS Translocation (Genetics)/*GENETICS Viral Fusion Proteins/*GENETICS Virus Replication/GENETICS JOURNAL ARTICLE
960430
M9640850

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