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

Pull down of adenovirus core proteins by immobilized import receptors. Purified recombinant import receptors or control proteins (BSA or purified recombinant adenovirus fibre protein) were immobilized on CN-Br-activated Sepharose beads. The immobilized proteins were then used to deplete cell extracts made from Ad2-infected HeLa cell cultures or from uninfected cell cultures as indicated (A and C). Alternatively, immobilized import factors were used to bind purified recombinant proteins GST-preVII (Ba), GST-VII (Bb) or 6His-V (D). Proteins bound to the immobilized import receptors were eluted and separated by SDS–PAGE. The adenovirus core proteins were detected by Western blotting using rat anti-VII (24) (A and B) and rabbit anti-V (6) (C and D). Secondary antibodies were horse radish peroxidase (HRP) conjugated and detection was by enhanced chemiluminescence. The position of preVII, mature VII and protein V is indicated. Imp., importin.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171040&req=5

fig01: Pull down of adenovirus core proteins by immobilized import receptors. Purified recombinant import receptors or control proteins (BSA or purified recombinant adenovirus fibre protein) were immobilized on CN-Br-activated Sepharose beads. The immobilized proteins were then used to deplete cell extracts made from Ad2-infected HeLa cell cultures or from uninfected cell cultures as indicated (A and C). Alternatively, immobilized import factors were used to bind purified recombinant proteins GST-preVII (Ba), GST-VII (Bb) or 6His-V (D). Proteins bound to the immobilized import receptors were eluted and separated by SDS–PAGE. The adenovirus core proteins were detected by Western blotting using rat anti-VII (24) (A and B) and rabbit anti-V (6) (C and D). Secondary antibodies were horse radish peroxidase (HRP) conjugated and detection was by enhanced chemiluminescence. The position of preVII, mature VII and protein V is indicated. Imp., importin.

Mentions: Purified recombinant import receptors were immobilized and used to deplete Ad2-infected and uninfected HeLa cell extracts. Following extensive washing, bound proteins were eluted and Western blotting performed using antibodies to the viral core proteins to identify interactions between import factors and viral proteins. Initially, we looked at the ‘classical’ pathway [which imports cargoes containing the simian virus 40 (SV40) T antigen nuclear localization signal (NLS)] by determining the ability of the adaptor protein, importin α, to bind the core proteins. We found that importin α was able to bind preVII but not VII (Figure 1Aa). We then investigated a much wider panel of import receptors and found that preVII and VII were bound by transportin (trans; which recognizes the M9 NLS) but not by importins β, 4 or 5 (Figure 1Ab). Finally, we determined that importins 7 and 13 also do not bind preVII or VII from infected cells (Figure 1Ac). We also used uninfected cell extracts as a pull-down control to ensure that the preparations of importin α and transportin did not fortuitously contain a protein that cross-reacted with the anti-VII antibody (Figure 1Ac).


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

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

Pull down of adenovirus core proteins by immobilized import receptors. Purified recombinant import receptors or control proteins (BSA or purified recombinant adenovirus fibre protein) were immobilized on CN-Br-activated Sepharose beads. The immobilized proteins were then used to deplete cell extracts made from Ad2-infected HeLa cell cultures or from uninfected cell cultures as indicated (A and C). Alternatively, immobilized import factors were used to bind purified recombinant proteins GST-preVII (Ba), GST-VII (Bb) or 6His-V (D). Proteins bound to the immobilized import receptors were eluted and separated by SDS–PAGE. The adenovirus core proteins were detected by Western blotting using rat anti-VII (24) (A and B) and rabbit anti-V (6) (C and D). Secondary antibodies were horse radish peroxidase (HRP) conjugated and detection was by enhanced chemiluminescence. The position of preVII, mature VII and protein V is indicated. Imp., importin.
© Copyright Policy
Related In: Results  -  Collection

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

fig01: Pull down of adenovirus core proteins by immobilized import receptors. Purified recombinant import receptors or control proteins (BSA or purified recombinant adenovirus fibre protein) were immobilized on CN-Br-activated Sepharose beads. The immobilized proteins were then used to deplete cell extracts made from Ad2-infected HeLa cell cultures or from uninfected cell cultures as indicated (A and C). Alternatively, immobilized import factors were used to bind purified recombinant proteins GST-preVII (Ba), GST-VII (Bb) or 6His-V (D). Proteins bound to the immobilized import receptors were eluted and separated by SDS–PAGE. The adenovirus core proteins were detected by Western blotting using rat anti-VII (24) (A and B) and rabbit anti-V (6) (C and D). Secondary antibodies were horse radish peroxidase (HRP) conjugated and detection was by enhanced chemiluminescence. The position of preVII, mature VII and protein V is indicated. Imp., importin.
Mentions: Purified recombinant import receptors were immobilized and used to deplete Ad2-infected and uninfected HeLa cell extracts. Following extensive washing, bound proteins were eluted and Western blotting performed using antibodies to the viral core proteins to identify interactions between import factors and viral proteins. Initially, we looked at the ‘classical’ pathway [which imports cargoes containing the simian virus 40 (SV40) T antigen nuclear localization signal (NLS)] by determining the ability of the adaptor protein, importin α, to bind the core proteins. We found that importin α was able to bind preVII but not VII (Figure 1Aa). We then investigated a much wider panel of import receptors and found that preVII and VII were bound by transportin (trans; which recognizes the M9 NLS) but not by importins β, 4 or 5 (Figure 1Ab). Finally, we determined that importins 7 and 13 also do not bind preVII or VII from infected cells (Figure 1Ac). We also used uninfected cell extracts as a pull-down control to ensure that the preparations of importin α and transportin did not fortuitously contain a protein that cross-reacted with the anti-VII antibody (Figure 1Ac).

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