Limits...
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.

Show MeSH

Related in: MedlinePlus

Digitonin permeabilized cell import assay. Complete import assays were carried out using HeLa cells permeabilized by incubation with digitonin. Cells were incubated at 30°C with complete reaction mixtures containing A) 500 nm FITC-M9. Then B) 2 mm GST-M9 or C) 2 mm GST-SV40 were added to the reaction mixtures as indicated. Following incubation, the localization of the FITC-M9 was determined by fluorescence microscopy. Bars represent 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171040&req=5

fig02: Digitonin permeabilized cell import assay. Complete import assays were carried out using HeLa cells permeabilized by incubation with digitonin. Cells were incubated at 30°C with complete reaction mixtures containing A) 500 nm FITC-M9. Then B) 2 mm GST-M9 or C) 2 mm GST-SV40 were added to the reaction mixtures as indicated. Following incubation, the localization of the FITC-M9 was determined by fluorescence microscopy. Bars represent 10 μm.

Mentions: We used the digitonin permeabilized cell import assay (12) plus specific import receptor inhibitors to investigate the nuclear import of the adenovirus core. In order to confirm that the assay and nuclear import receptor inhibitors were functional in our hands, we first carried out import using fluorescein isothiocyanate (FITC)-labelled GST-M9 as a substrate. In a complete assay containing an energy-regenerating system and rabbit reticulocyte lysate (RRL) as a source of import receptors and other cellular proteins, FITC-GST-M9 was imported to the nucleus (Figure 2A). Addition of excess unlabelled GST-M9 inhibited this import (Figure 2B), whereas excess unlabelled GST-SV40 had no effect (Figure 2C). This confirmed that GST-M9 inhibits transportin-mediated import without affecting the classical import pathway. We also used FITC-GST-SV40 as import substrate and confirmed that addition of excess GST-SV40 inhibited its import, whereas addition of excess unlabelled GST-M9 did not (data not shown).


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

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

Digitonin permeabilized cell import assay. Complete import assays were carried out using HeLa cells permeabilized by incubation with digitonin. Cells were incubated at 30°C with complete reaction mixtures containing A) 500 nm FITC-M9. Then B) 2 mm GST-M9 or C) 2 mm GST-SV40 were added to the reaction mixtures as indicated. Following incubation, the localization of the FITC-M9 was determined by fluorescence microscopy. Bars represent 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig02: Digitonin permeabilized cell import assay. Complete import assays were carried out using HeLa cells permeabilized by incubation with digitonin. Cells were incubated at 30°C with complete reaction mixtures containing A) 500 nm FITC-M9. Then B) 2 mm GST-M9 or C) 2 mm GST-SV40 were added to the reaction mixtures as indicated. Following incubation, the localization of the FITC-M9 was determined by fluorescence microscopy. Bars represent 10 μm.
Mentions: We used the digitonin permeabilized cell import assay (12) plus specific import receptor inhibitors to investigate the nuclear import of the adenovirus core. In order to confirm that the assay and nuclear import receptor inhibitors were functional in our hands, we first carried out import using fluorescein isothiocyanate (FITC)-labelled GST-M9 as a substrate. In a complete assay containing an energy-regenerating system and rabbit reticulocyte lysate (RRL) as a source of import receptors and other cellular proteins, FITC-GST-M9 was imported to the nucleus (Figure 2A). Addition of excess unlabelled GST-M9 inhibited this import (Figure 2B), whereas excess unlabelled GST-SV40 had no effect (Figure 2C). This confirmed that GST-M9 inhibits transportin-mediated import without affecting the classical import pathway. We also used FITC-GST-SV40 as import substrate and confirmed that addition of excess GST-SV40 inhibited its import, whereas addition of excess unlabelled GST-M9 did not (data not shown).

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