Cellular uptake and nuclear delivery of recombinant adenovirus penton base.
An Ad2 capsid component, the penton base, expressed as recombinant protein, was found to be capable of affecting the entire entry pathway of adenovirion in HeLa cells, i.e., cell attachment, endocytosis, vesicular escape, intracytoplasmic movement, and translocation through the nuclear pore complex. Data with pentamerization-defective mutants suggested that none of these successive steps depended upon penton base pentamer status, indicating that the peptide domains responsible for these functions were carried by the monomer. Observations performed with wild-type (WT) and an integrin-binding-site double-mutant (K288E340) suggested that the penton base could enter the cell via an alternative, RGD- and LDV-independent, pathway. Of three mutants that were found to be defective in nuclear addressing in insect cells, only one, W165H, was also altered in nuclear transport in HeLa cells. The other two, W119H and RRR547EQQ, showed a WT pattern of nuclear localization in HeLa cells, suggesting that the region including tryptophan-119 and the basic signal at position 547 did not act as a nuclear localization signal in the human cell context. The integrity of cellular structures and the cytoskeleton seemed to be required for the vectorial movement and nuclear import of WT penton base, as suggested by experiments using permeabilized HeLa cells, isolated nuclear membranes, and cytoskeleton-targeted drugs.
Hong
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Laboratoire de Virologie et Pathogénèse Virale, CNRS UMR 5537, Faculté de Médecine RTH Laennec, Rue Guillaume Paradin, Lyon, 69008, France.
Gay
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B
Karayan
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L
Dabauvalle
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MC
Boulanger
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P
D000260:Adenoviruses, Human / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D002213:Capsid / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D002451:Cell Compartmentation / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D018411:Spodoptera; D028901:alpha Karyopherins
D036022:Capsid Proteins; D004274:DNA, Recombinant; D004279:DNA, Viral; C490630:KPNA2 protein, human; D009687:Nuclear Proteins; D028901:alpha Karyopherins; C043322:penton protein, adenovirus