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Title: Identification of a cytoplasmic interaction partner of the large regulatory proteins Rep78/Rep68 of adeno-associated virus type 2 (AAV-2)

Abstract

Through yeast two-hybrid analysis and coimmunoprecipitation studies, we have identified a novel cellular AAV-2 Rep78/Rep68 interaction partner located predominantly in the cytoplasm. In public databases, it has been assigned as KCTD5, because of a region of high similarity to the cytoplasmic tetramerization domain of voltage-gated potassium channels. Whereas Rep/KCTD5 interaction relied on the region surrounding the Rep nuclear localization signal, nuclear accumulation of Rep was not required. Wildtype Rep78/Rep68 proteins induced the translocation of large portions of KCTD5 into the nucleus pointing to functional interactions both in the cytoplasm and the nucleus. In line with an anticipated functional interference in the cytoplasm, KCTD5 overexpression completely abrogated Rep68-mediated posttranscriptional activation of a HIV-LTR driven luciferase reporter gene. Our study expands the panel of already identified nuclear Rep interaction partners to a cytoplasmic protein, which raises the awareness that important steps in the AAV life cycle may be regulated in this compartment.

Authors:
 [1];  [2];  [2];  [2]
  1. Institut fuer Virologie, Charite Campus Benjamin Franklin, Freie Universitaet Berlin, Hindenburgdamm 27, 12203 Berlin (Germany). E-mail: stefan.weger@charite.de
  2. Institut fuer Virologie, Charite Campus Benjamin Franklin, Freie Universitaet Berlin, Hindenburgdamm 27, 12203 Berlin (Germany)
Publication Date:
OSTI Identifier:
20977028
Resource Type:
Journal Article
Resource Relation:
Journal Name: Virology; Journal Volume: 362; Journal Issue: 1; Other Information: DOI: 10.1016/j.virol.2006.12.010; PII: S0042-6822(06)00892-0; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AIDS VIRUS; CYTOPLASM; ELECTRIC POTENTIAL; GENES; HYBRIDIZATION; LIFE CYCLE; LUCIFERASE; POTASSIUM; TRANSLOCATION; YEASTS

Citation Formats

Weger, Stefan, Hammer, Eva, Goetz, Anne, and Heilbronn, Regine. Identification of a cytoplasmic interaction partner of the large regulatory proteins Rep78/Rep68 of adeno-associated virus type 2 (AAV-2). United States: N. p., 2007. Web. doi:10.1016/j.virol.2006.12.010.
Weger, Stefan, Hammer, Eva, Goetz, Anne, & Heilbronn, Regine. Identification of a cytoplasmic interaction partner of the large regulatory proteins Rep78/Rep68 of adeno-associated virus type 2 (AAV-2). United States. doi:10.1016/j.virol.2006.12.010.
Weger, Stefan, Hammer, Eva, Goetz, Anne, and Heilbronn, Regine. Fri . "Identification of a cytoplasmic interaction partner of the large regulatory proteins Rep78/Rep68 of adeno-associated virus type 2 (AAV-2)". United States. doi:10.1016/j.virol.2006.12.010.
@article{osti_20977028,
title = {Identification of a cytoplasmic interaction partner of the large regulatory proteins Rep78/Rep68 of adeno-associated virus type 2 (AAV-2)},
author = {Weger, Stefan and Hammer, Eva and Goetz, Anne and Heilbronn, Regine},
abstractNote = {Through yeast two-hybrid analysis and coimmunoprecipitation studies, we have identified a novel cellular AAV-2 Rep78/Rep68 interaction partner located predominantly in the cytoplasm. In public databases, it has been assigned as KCTD5, because of a region of high similarity to the cytoplasmic tetramerization domain of voltage-gated potassium channels. Whereas Rep/KCTD5 interaction relied on the region surrounding the Rep nuclear localization signal, nuclear accumulation of Rep was not required. Wildtype Rep78/Rep68 proteins induced the translocation of large portions of KCTD5 into the nucleus pointing to functional interactions both in the cytoplasm and the nucleus. In line with an anticipated functional interference in the cytoplasm, KCTD5 overexpression completely abrogated Rep68-mediated posttranscriptional activation of a HIV-LTR driven luciferase reporter gene. Our study expands the panel of already identified nuclear Rep interaction partners to a cytoplasmic protein, which raises the awareness that important steps in the AAV life cycle may be regulated in this compartment.},
doi = {10.1016/j.virol.2006.12.010},
journal = {Virology},
number = 1,
volume = 362,
place = {United States},
year = {Fri May 25 00:00:00 EDT 2007},
month = {Fri May 25 00:00:00 EDT 2007}
}
  • Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites ofmore » AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.« less
  • Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites ofmore » AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.« less
  • The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, couldmore » be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68.« less
  • No abstract prepared.
  • No abstract prepared.