       Document 0501
 DOCN  M9640501
 TI    Nuclear translocation of an exogenous fusion protein containing HIV Tat
       requires unfolding.
 DT    9604
 AU    Bonifaci N; Sitia R; Rubartelli A; Laboratory of Clinical Pathology,
       National Institute of Cancer; Research, Genoa, Italy.
 SO    AIDS. 1995 Sep;9(9):995-1000. Unique Identifier : AIDSLINE MED/96085713
 AB    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.
 DE    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

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

