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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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Virus DNA import into permeabilized cells. Permeabilized HeLa cells were incubated at 30°C with complete import mixtures containing 2 × 108 plaque-forming units purified virus. In duplicate experiments, 2 mm inhibitor (GST-SV40 or GST-M9) and/or 1 mm transportin was added to the import mix as indicated. Following incubation, the cells were fixed, permeabilized and processed for detection of the viral DNA by FISH. Cells were costained for lamin A/C using mouse anti-lamin (Santa Cruz) and were mounted using vectashield mounting medium containing DAPI (Vector). A) A two-colour confocal laser micrograph image with lamin in green and viral DNA in red together with a merged image. In each subsequent pair of images, B) DAPI or C–H) lamin A/C staining is shown on the left and DNA is on the right. Bars represent 10 μm.
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fig06: Virus DNA import into permeabilized cells. Permeabilized HeLa cells were incubated at 30°C with complete import mixtures containing 2 × 108 plaque-forming units purified virus. In duplicate experiments, 2 mm inhibitor (GST-SV40 or GST-M9) and/or 1 mm transportin was added to the import mix as indicated. Following incubation, the cells were fixed, permeabilized and processed for detection of the viral DNA by FISH. Cells were costained for lamin A/C using mouse anti-lamin (Santa Cruz) and were mounted using vectashield mounting medium containing DAPI (Vector). A) A two-colour confocal laser micrograph image with lamin in green and viral DNA in red together with a merged image. In each subsequent pair of images, B) DAPI or C–H) lamin A/C staining is shown on the left and DNA is on the right. Bars represent 10 μm.

Mentions: Import of the viral DNA genome was investigated using fluorescent in situ hybridization (FISH), which was carried out on permeabilized cells subjected to import assays using purified virus that had not been heat treated (we were unable to detect the DNA from heat-treated particles). Following the same approach and criteria as the initial studies of adenovirus DNA import (10), we took colocalization of viral DNA and lamin A/C, a component of the inner nuclear membrane, to indicate that import had taken place. In a complete import assay, DNA was seen to be concentrated in discrete foci overlapping the lamin staining (Figure 6A). As the lamin appeared to undergo some structural disintegration in cells that had undergone import, compared with negative controls, we also stained the nuclear DNA with 4′,6-diamidino-2-phenylindole (DAPI) to confirm that the viral DNA was in the nucleus (Figure 6B) and found that this was indeed the case. Viral DNA import was inhibited by omission of RRL (Figure 6C), ATP (Figure 6D) or by addition of WGA (Figure 6E) to the import reaction mixture. Addition of excess GST-SV40 to the import reaction mixture did not effect DNA import (Figure 6F), an observation that contradicts previous studies (10). In our assay, the transportin inhibitor GST-M9 prevented DNA import completely (Figure 6G) and addition of excess recombinant transportin to the import reaction overcame this block (Figure 6H). Following addition of purified V or GST-VII to the import reaction mixture, viral DNA was still seen in the nucleus (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)

Virus DNA import into permeabilized cells. Permeabilized HeLa cells were incubated at 30°C with complete import mixtures containing 2 × 108 plaque-forming units purified virus. In duplicate experiments, 2 mm inhibitor (GST-SV40 or GST-M9) and/or 1 mm transportin was added to the import mix as indicated. Following incubation, the cells were fixed, permeabilized and processed for detection of the viral DNA by FISH. Cells were costained for lamin A/C using mouse anti-lamin (Santa Cruz) and were mounted using vectashield mounting medium containing DAPI (Vector). A) A two-colour confocal laser micrograph image with lamin in green and viral DNA in red together with a merged image. In each subsequent pair of images, B) DAPI or C–H) lamin A/C staining is shown on the left and DNA is 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

fig06: Virus DNA import into permeabilized cells. Permeabilized HeLa cells were incubated at 30°C with complete import mixtures containing 2 × 108 plaque-forming units purified virus. In duplicate experiments, 2 mm inhibitor (GST-SV40 or GST-M9) and/or 1 mm transportin was added to the import mix as indicated. Following incubation, the cells were fixed, permeabilized and processed for detection of the viral DNA by FISH. Cells were costained for lamin A/C using mouse anti-lamin (Santa Cruz) and were mounted using vectashield mounting medium containing DAPI (Vector). A) A two-colour confocal laser micrograph image with lamin in green and viral DNA in red together with a merged image. In each subsequent pair of images, B) DAPI or C–H) lamin A/C staining is shown on the left and DNA is on the right. Bars represent 10 μm.
Mentions: Import of the viral DNA genome was investigated using fluorescent in situ hybridization (FISH), which was carried out on permeabilized cells subjected to import assays using purified virus that had not been heat treated (we were unable to detect the DNA from heat-treated particles). Following the same approach and criteria as the initial studies of adenovirus DNA import (10), we took colocalization of viral DNA and lamin A/C, a component of the inner nuclear membrane, to indicate that import had taken place. In a complete import assay, DNA was seen to be concentrated in discrete foci overlapping the lamin staining (Figure 6A). As the lamin appeared to undergo some structural disintegration in cells that had undergone import, compared with negative controls, we also stained the nuclear DNA with 4′,6-diamidino-2-phenylindole (DAPI) to confirm that the viral DNA was in the nucleus (Figure 6B) and found that this was indeed the case. Viral DNA import was inhibited by omission of RRL (Figure 6C), ATP (Figure 6D) or by addition of WGA (Figure 6E) to the import reaction mixture. Addition of excess GST-SV40 to the import reaction mixture did not effect DNA import (Figure 6F), an observation that contradicts previous studies (10). In our assay, the transportin inhibitor GST-M9 prevented DNA import completely (Figure 6G) and addition of excess recombinant transportin to the import reaction overcame this block (Figure 6H). Following addition of purified V or GST-VII to the import reaction mixture, viral DNA was still seen in the nucleus (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