Permeabilized mammalian cells as an experimental system for nuclear import of geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions.
The plant-infecting geminiviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two approximately 2.6 kb DNAs or a monopartite genome of approximately 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 peptides containing aa 3-20 of TYLCV CP (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 alpha, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CP and BV1, probably via importin alpha.
Kass
Gideon
G
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Arad
Gabriel
G
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Rosenbluh
Joseph
J
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Gafni
Yedidya
Y
Department of Plant Genetics, ARO, The Volcani Center, Bet-Dagan 50250, Israel.
Graessmann
Adolf
A
Institut für Molekularbiologie und Biochemie, Free University of Berlin, 14195 Berlin, Germany.
Rojas
Maria R
MR
Department of Plant Pathology, University of California, Davis, CA 95616, USA.
Gilbertson
Robert L
RL
Department of Plant Pathology, University of California, Davis, CA 95616, USA.
Loyter
Abraham
A
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D017791:Geminiviridae / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D028884:Karyopherins / Q000378:metabolism; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D010944:Plants / Q000821:virology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D028884:Karyopherins; D019913:Nuclear Localization Signals; D011994:Recombinant Proteins; D014764:Viral Proteins; D012710:Serum Albumin, Bovine