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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

V import into permeabilized cells. Permeabilized HeLa cells were incubated at 30°C with complete import reaction mixtures containing either A–H) 500 nm bacterially expressed, 6His-tagged protein V or I–P) protein V derived from 2 × 108 plaque-forming units heat-treated virus. For control reactions, either RRL or ATP was omitted from the reaction or 200 ng/mL WGA was added as indicated. To assess the contribution of different import factors to the import of protein V, either 2 mm inhibitor (GST-SV40 or GST-M9) or 1 mm transportin was added as indicated. Following incubation, cells were fixed, permeabilized and stained for protein V using rabbit anti-V (6) and alexafluor-conjugated secondary antibody. Arrowheads in these images point to nucleolar accumulation of protein V. Bars represent 10 μm.
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fig04: V import into permeabilized cells. Permeabilized HeLa cells were incubated at 30°C with complete import reaction mixtures containing either A–H) 500 nm bacterially expressed, 6His-tagged protein V or I–P) protein V derived from 2 × 108 plaque-forming units heat-treated virus. For control reactions, either RRL or ATP was omitted from the reaction or 200 ng/mL WGA was added as indicated. To assess the contribution of different import factors to the import of protein V, either 2 mm inhibitor (GST-SV40 or GST-M9) or 1 mm transportin was added as indicated. Following incubation, cells were fixed, permeabilized and stained for protein V using rabbit anti-V (6) and alexafluor-conjugated secondary antibody. Arrowheads in these images point to nucleolar accumulation of protein V. Bars represent 10 μm.

Mentions: We wanted to determine if protein V showed a preference for import factors and to examine if, as in adenovirus-infected cells (6), protein V localized to the nucleolus. In complete import assays, bacterially expressed, purified V was imported into the nucleus and showed strong nucleolar staining in approximately 50% of cells (Figure 4A). Omission of RRL (Figure 4B), ATP (Figure 4C) or addition of WGA (Figure 4D) reduced import of bacterial V. Addition of GST only (Figure 4E) or GST-SV40 (Figure 4F) had very little effect on import; V was still imported and was nucleolar in around half of cells. However, addition of GST-M9 consistently ablated nucleolar accumulation of V without inhibiting its nuclear import (Figure 4G). Moreover, including additional recombinant transportin alongside GST-M9 restored nucleolar accumulation (Figure 4H).


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

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

V import into permeabilized cells. Permeabilized HeLa cells were incubated at 30°C with complete import reaction mixtures containing either A–H) 500 nm bacterially expressed, 6His-tagged protein V or I–P) protein V derived from 2 × 108 plaque-forming units heat-treated virus. For control reactions, either RRL or ATP was omitted from the reaction or 200 ng/mL WGA was added as indicated. To assess the contribution of different import factors to the import of protein V, either 2 mm inhibitor (GST-SV40 or GST-M9) or 1 mm transportin was added as indicated. Following incubation, cells were fixed, permeabilized and stained for protein V using rabbit anti-V (6) and alexafluor-conjugated secondary antibody. Arrowheads in these images point to nucleolar accumulation of protein V. Bars represent 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171040&req=5

fig04: V import into permeabilized cells. Permeabilized HeLa cells were incubated at 30°C with complete import reaction mixtures containing either A–H) 500 nm bacterially expressed, 6His-tagged protein V or I–P) protein V derived from 2 × 108 plaque-forming units heat-treated virus. For control reactions, either RRL or ATP was omitted from the reaction or 200 ng/mL WGA was added as indicated. To assess the contribution of different import factors to the import of protein V, either 2 mm inhibitor (GST-SV40 or GST-M9) or 1 mm transportin was added as indicated. Following incubation, cells were fixed, permeabilized and stained for protein V using rabbit anti-V (6) and alexafluor-conjugated secondary antibody. Arrowheads in these images point to nucleolar accumulation of protein V. Bars represent 10 μm.
Mentions: We wanted to determine if protein V showed a preference for import factors and to examine if, as in adenovirus-infected cells (6), protein V localized to the nucleolus. In complete import assays, bacterially expressed, purified V was imported into the nucleus and showed strong nucleolar staining in approximately 50% of cells (Figure 4A). Omission of RRL (Figure 4B), ATP (Figure 4C) or addition of WGA (Figure 4D) reduced import of bacterial V. Addition of GST only (Figure 4E) or GST-SV40 (Figure 4F) had very little effect on import; V was still imported and was nucleolar in around half of cells. However, addition of GST-M9 consistently ablated nucleolar accumulation of V without inhibiting its nuclear import (Figure 4G). Moreover, including additional recombinant transportin alongside GST-M9 restored nucleolar accumulation (Figure 4H).

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