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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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Related in: MedlinePlus

Minimal import assay for protein V. Cells were incubated at 30°C with minimal import reactions containing protein V from 2 × 108 plaque-forming units heat-treated virus, 2 mm GTP and 2 mm Ran. Either 1 mm transportin or 1 mm each of both importin α and importin β were added as indicated. As a control, 200 ng/mL WGA was also added to duplicate experiments where indicated. Following incubation, the cells were fixed, permeabilized and stained for protein V using rabbit anti-V (6) and alexafluor-conjugated secondary antibody. Bars represent 10 μm.
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fig05: Minimal import assay for protein V. Cells were incubated at 30°C with minimal import reactions containing protein V from 2 × 108 plaque-forming units heat-treated virus, 2 mm GTP and 2 mm Ran. Either 1 mm transportin or 1 mm each of both importin α and importin β were added as indicated. As a control, 200 ng/mL WGA was also added to duplicate experiments where indicated. Following incubation, the cells were fixed, permeabilized and stained for protein V using rabbit anti-V (6) and alexafluor-conjugated secondary antibody. Bars represent 10 μm.

Mentions: We next used minimal import assays in which specific import receptors plus Ran, GTP and an energy-regenerating system were added to the permeabilized cells to further examine the import of viral protein V. The results demonstrated that protein V could be imported by transportin only (Figure 5A) or by importins α plus β (Figure 5C); however, V did not accumulate in the nucleolus in either case. WGA inhibited the import of protein V in both assays, confirming that the import observed was dependent on active transport through the NPC (Figure 5B,D).


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

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

Minimal import assay for protein V. Cells were incubated at 30°C with minimal import reactions containing protein V from 2 × 108 plaque-forming units heat-treated virus, 2 mm GTP and 2 mm Ran. Either 1 mm transportin or 1 mm each of both importin α and importin β were added as indicated. As a control, 200 ng/mL WGA was also added to duplicate experiments where indicated. Following incubation, the cells were fixed, permeabilized and stained for protein V using rabbit anti-V (6) and alexafluor-conjugated secondary antibody. Bars represent 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171040&req=5

fig05: Minimal import assay for protein V. Cells were incubated at 30°C with minimal import reactions containing protein V from 2 × 108 plaque-forming units heat-treated virus, 2 mm GTP and 2 mm Ran. Either 1 mm transportin or 1 mm each of both importin α and importin β were added as indicated. As a control, 200 ng/mL WGA was also added to duplicate experiments where indicated. Following incubation, the cells were fixed, permeabilized and stained for protein V using rabbit anti-V (6) and alexafluor-conjugated secondary antibody. Bars represent 10 μm.
Mentions: We next used minimal import assays in which specific import receptors plus Ran, GTP and an energy-regenerating system were added to the permeabilized cells to further examine the import of viral protein V. The results demonstrated that protein V could be imported by transportin only (Figure 5A) or by importins α plus β (Figure 5C); however, V did not accumulate in the nucleolus in either case. WGA inhibited the import of protein V in both assays, confirming that the import observed was dependent on active transport through the NPC (Figure 5B,D).

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