Permeabilized mammalian cells as an experimental system for nuclear import geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions

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Permeabilized mammalian cells as an experimental system for nuclear import geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions

Year: 

2006

Source of publication :

Journal of General Virology

Authors :

Gafni, Yedidya

.

Volume :

Co-Authors: 

Kass, G., Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Arad, G., Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Rosenbluh, J., Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Gafni, Y., Department of Plant Genetics, ARO, The Volcani Center, Bet-Dagan 50250, Israel
Graessmann, A., Institut für Molekularbiologie und Biochemie, Free University of Berlin, 14195 Berlin, Germany
Rojas, M.R., Department of Plant Pathology, University of California, Davis, CA 95616, United States
Gilbertson, R.L., Department of Plant Pathology, University of California, Davis, CA 95616, United States
Loyter, A., Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

From page: 

2709

To page: 

2720

(

Total pages: 

12

)

Link :

Permeabilized mammalian cells as an experimental system for nuclear import geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions

Abstract: 

The plant-infecting germiniviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two ∼2.6 kb DNAs or a monopartite genome of ∼3.0 kb DNA. The bipartite genome of Bean dwarf mosaic virus (BDMV) encodes several karyophilic proteins, among them the capsid protein (CP) and BV1 (nuclear shuttle protein). A CP is also encoded by the monopartite genome of Tomato yellow leaf curl virus (TYLCV). Here, an in vitro assay system was used for direct demonstration of nuclear import of BDMV BV1 and TYLCV CP, as well as synthetic peptides containing their putative nuclear localization signals (NLSs). Full-length recombinant BDMV BV1 and TYLCV CP mediated import of conjugated fluorescently labelled BSA molecules into nuclei of permeabilized mammalian cells. Fluorescently labelled and biotinylated BSA conjugates bearing the synthetic pepticles containing aa 3-20 of TYLCV CIP (CP-NLS) or aa 84-106 of BDMV BV1 (BV1-NLS) were also imported into the nuclei of permeabilized cells. This import was blocked by the addition of unlabelled BSA-NLS peptide conjugates or excess unlabelled free NLS peptides. The CP- and BV1-NLS peptides also mediated nuclear import of fluorescently labelled BSA molecules into the nuclei of microinjected mesophyll cells of Nicotiana benthamiana leaves, demonstrating their biological function in intact plant tissue. BV1 -NLS and CP-NLS were shown to mediate specific binding to importin α, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CIP and BV1, probably via importin α. © 2006 SGM.