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

Gideon Kass, Gabriel Arad, Joseph Rosenbluh, Yedidya Gafni, Adolf Graessmann, Maria R. Rojas, Robert L. Gilbertson, Abraham Loyter, Abraham Loyter

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6 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2709-2720
Number of pages12
JournalJournal of General Virology
Volume87
Issue number9
DOIs
Publication statusPublished - 1 Sep 2006
Externally publishedYes

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