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

AIDS. 1995 Sep;9(9):995-1000. doi: 10.1097/00002030-199509000-00003.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Nucleus / virology*
  • Gene Expression Regulation, Viral / physiology
  • Genes, Reporter / genetics
  • Genes, tat / genetics*
  • HIV-1 / genetics*
  • HeLa Cells
  • Humans
  • Signal Transduction / genetics
  • Tetrahydrofolate Dehydrogenase / genetics
  • Transcriptional Activation / genetics
  • Translocation, Genetic / genetics*
  • Viral Fusion Proteins / genetics*
  • Virus Replication / genetics

Substances

  • Viral Fusion Proteins
  • Tetrahydrofolate Dehydrogenase