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 α–importin β 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 α–importin β, 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.
Highlights
Adenoviruses consist of an icosohedral particle containing a double-stranded DNA genome, which is condensed by association with adenovirus ‘core’ proteins called terminal protein, V, VII and Mu, all of which are contained within a capsid shell
We show that transportin is the primary import receptor for protein VII and the viral DNA and that www.traffic.dk 1313 transportin must be available for the correct subnuclear localization of protein V to occur
We investigated a much wider panel of import receptors and found that preVII and VII were bound by transportin but not by importins b, 4 or 5 (Figure 1Ab)
Summary
Adenoviruses consist of an icosohedral particle containing a double-stranded DNA genome, which is condensed by association with adenovirus ‘core’ proteins called terminal protein, V, VII and Mu, all of which are contained within a capsid shell. While preVII is involved in condensing the viral DNA during particle assembly, it is mature protein VII that is present in the viral DNA-core protein complex that enters the cell nucleus. Having identified import factors able to bind the core proteins V, preVII and VII, we used a permeabilized cell import assay and known competitors of the identified import receptors to confirm their import activity. This approach revealed striking differences in the import properties of preVII compared with mature VII. We show that transportin is the primary import receptor for protein VII and the viral DNA and that www.traffic.dk 1313 transportin must be available for the correct subnuclear localization of protein V to occur
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