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A role for transportin in the nuclear import of adenovirus core proteins and DNA.

Hindley CE, Lawrence FJ, Matthews DA - Traffic (2007)

Bottom Line: We show that inhibition of transportin results in aberrant localization of protein V and that transportin is necessary for protein V to accumulate in the nucleolus.Furthermore, inhibition of transportin results in inhibition of protein VII and DNA import, whereas disruption of the classical importin alpha-importin beta import pathway has little effect.This is significant because while preVII is important during viral replication and assembly only mature VII is available during viral DNA import to a newly infected cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, Bristol, UK.

ABSTRACT
Adenoviruses target their double-stranded DNA genome and its associated core proteins to the interphase nucleus; this core structure then enters through the nuclear pore complex. We have used digitonin permeabilized cell import assays to study the cellular import factors involved in nuclear entry of virus DNA and the core proteins, protein V and protein VII. We show that inhibition of transportin results in aberrant localization of protein V and that transportin is necessary for protein V to accumulate in the nucleolus. Furthermore, inhibition of transportin results in inhibition of protein VII and DNA import, whereas disruption of the classical importin alpha-importin beta import pathway has little effect. We show that mature protein VII has different import preferences from the precursor protein, preVII from which it is derived by proteolytic processing. While bacterially expressed glutathione S-transferase (GST)-preVII primarily utilizes the pathway mediated by importin alpha-importin beta, bacterially expressed GST-VII favours the transportin pathway. This is significant because while preVII is important during viral replication and assembly only mature VII is available during viral DNA import to a newly infected cell. Our results implicate transportin as a key import receptor for the nuclear localization of adenovirus core.

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GST-preVII and GST-VII import into permeabilized cells. Complete import assays were carried out on HeLa cells permeabilized with digitonin. Cells were incubated at 30°C with complete reaction mixtures containing either (A–H) 500 nm GST-preVII or (I–P) GST-VII. For control reactions, either RRL or ATP was omitted from the reaction as indicated. Alternatively, 200 ng/mL WGA, 2 mm inhibitor (GST-SV40 or GST-M9), 2 mm GST, 1 mm importin α, 1 mm importin β or 1 mm transportin was added as indicated. Following incubation, the cells were fixed, permeabilized and stained for preVII and VII using rat anti-VII and costained for lamin A/C using mouse anti-lamin. Both were detected with alexafluor-conjugated secondary antibodies. In each pair of images, preVII/VII is shown on the left and lamin A/C on the right. Bars represent 10 μm.
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fig03: GST-preVII and GST-VII import into permeabilized cells. Complete import assays were carried out on HeLa cells permeabilized with digitonin. Cells were incubated at 30°C with complete reaction mixtures containing either (A–H) 500 nm GST-preVII or (I–P) GST-VII. For control reactions, either RRL or ATP was omitted from the reaction as indicated. Alternatively, 200 ng/mL WGA, 2 mm inhibitor (GST-SV40 or GST-M9), 2 mm GST, 1 mm importin α, 1 mm importin β or 1 mm transportin was added as indicated. Following incubation, the cells were fixed, permeabilized and stained for preVII and VII using rat anti-VII and costained for lamin A/C using mouse anti-lamin. Both were detected with alexafluor-conjugated secondary antibodies. In each pair of images, preVII/VII is shown on the left and lamin A/C on the right. Bars represent 10 μm.

Mentions: Because preVII and mature VII (and their bacterially expressed counterparts) showed import-factor-binding preferences, we wanted to determine whether these discrepancies were reflected in an in vitro import assay. We found that purified GST-preVII was imported into the nucleus in a complete import assay (Figure 3A), but omission of RRL (Figure 3B), ATP (Figure 3C) or addition of wheat germ agglutinin (WGA) (Figure 3D) blocked import. Also, addition of GST alone to the import assay did not affect GST-preVII import (Figure 3E). When recombinant import inhibitors were added to the reaction mixtures, we found that GST-SV40 reduced nuclear import of GST-preVII (Figure 3F), whereas addition of GST-M9 had little effect upon nuclear import (Figure 3G). Addition of excess recombinant importins α and β to the GST-SV40-containing reaction restored import of GST-preVII (Figure 3H). Comparable examination of GST-VII import (Figure 3I–P) showed that, in contrast to GST-preVII, import of GST-VII still took place in the presence of GST-SV40 (Figure 3N), whereas GST-M9 had an inhibitory effect (Figure 3O). Addition of excess recombinant transportin to the GST-M9-containing reactions restored nuclear import of GST-VII (Figure 3P).


A role for transportin in the nuclear import of adenovirus core proteins and DNA.

Hindley CE, Lawrence FJ, Matthews DA - Traffic (2007)

GST-preVII and GST-VII import into permeabilized cells. Complete import assays were carried out on HeLa cells permeabilized with digitonin. Cells were incubated at 30°C with complete reaction mixtures containing either (A–H) 500 nm GST-preVII or (I–P) GST-VII. For control reactions, either RRL or ATP was omitted from the reaction as indicated. Alternatively, 200 ng/mL WGA, 2 mm inhibitor (GST-SV40 or GST-M9), 2 mm GST, 1 mm importin α, 1 mm importin β or 1 mm transportin was added as indicated. Following incubation, the cells were fixed, permeabilized and stained for preVII and VII using rat anti-VII and costained for lamin A/C using mouse anti-lamin. Both were detected with alexafluor-conjugated secondary antibodies. In each pair of images, preVII/VII is shown on the left and lamin A/C on the right. Bars represent 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171040&req=5

fig03: GST-preVII and GST-VII import into permeabilized cells. Complete import assays were carried out on HeLa cells permeabilized with digitonin. Cells were incubated at 30°C with complete reaction mixtures containing either (A–H) 500 nm GST-preVII or (I–P) GST-VII. For control reactions, either RRL or ATP was omitted from the reaction as indicated. Alternatively, 200 ng/mL WGA, 2 mm inhibitor (GST-SV40 or GST-M9), 2 mm GST, 1 mm importin α, 1 mm importin β or 1 mm transportin was added as indicated. Following incubation, the cells were fixed, permeabilized and stained for preVII and VII using rat anti-VII and costained for lamin A/C using mouse anti-lamin. Both were detected with alexafluor-conjugated secondary antibodies. In each pair of images, preVII/VII is shown on the left and lamin A/C on the right. Bars represent 10 μm.
Mentions: Because preVII and mature VII (and their bacterially expressed counterparts) showed import-factor-binding preferences, we wanted to determine whether these discrepancies were reflected in an in vitro import assay. We found that purified GST-preVII was imported into the nucleus in a complete import assay (Figure 3A), but omission of RRL (Figure 3B), ATP (Figure 3C) or addition of wheat germ agglutinin (WGA) (Figure 3D) blocked import. Also, addition of GST alone to the import assay did not affect GST-preVII import (Figure 3E). When recombinant import inhibitors were added to the reaction mixtures, we found that GST-SV40 reduced nuclear import of GST-preVII (Figure 3F), whereas addition of GST-M9 had little effect upon nuclear import (Figure 3G). Addition of excess recombinant importins α and β to the GST-SV40-containing reaction restored import of GST-preVII (Figure 3H). Comparable examination of GST-VII import (Figure 3I–P) showed that, in contrast to GST-preVII, import of GST-VII still took place in the presence of GST-SV40 (Figure 3N), whereas GST-M9 had an inhibitory effect (Figure 3O). Addition of excess recombinant transportin to the GST-M9-containing reactions restored nuclear import of GST-VII (Figure 3P).

Bottom Line: We show that inhibition of transportin results in aberrant localization of protein V and that transportin is necessary for protein V to accumulate in the nucleolus.Furthermore, inhibition of transportin results in inhibition of protein VII and DNA import, whereas disruption of the classical importin alpha-importin beta import pathway has little effect.This is significant because while preVII is important during viral replication and assembly only mature VII is available during viral DNA import to a newly infected cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, Bristol, UK.

ABSTRACT
Adenoviruses target their double-stranded DNA genome and its associated core proteins to the interphase nucleus; this core structure then enters through the nuclear pore complex. We have used digitonin permeabilized cell import assays to study the cellular import factors involved in nuclear entry of virus DNA and the core proteins, protein V and protein VII. We show that inhibition of transportin results in aberrant localization of protein V and that transportin is necessary for protein V to accumulate in the nucleolus. Furthermore, inhibition of transportin results in inhibition of protein VII and DNA import, whereas disruption of the classical importin alpha-importin beta import pathway has little effect. We show that mature protein VII has different import preferences from the precursor protein, preVII from which it is derived by proteolytic processing. While bacterially expressed glutathione S-transferase (GST)-preVII primarily utilizes the pathway mediated by importin alpha-importin beta, bacterially expressed GST-VII favours the transportin pathway. This is significant because while preVII is important during viral replication and assembly only mature VII is available during viral DNA import to a newly infected cell. Our results implicate transportin as a key import receptor for the nuclear localization of adenovirus core.

Show MeSH
Related in: MedlinePlus