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J. Virol. doi:10.1128/JVI.01850-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Nuclear Import of BPV1 E1 Protein Is Mediated By Multiple Alpha Importins And Is Negatively Regulated By Phosphorylation Near A Nuclear Localization Signal

Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M Health Science Center, College Station, TX 77843-1114

^* To whom correspondence should be addressed. Email: wilson{at}medicine.tamhsc.edu.

	Abstract

Papillomavirus (PV) DNA replication occurs in the nucleus of infected cells and requires the viral E1 protein which enters the nuclei of host epithelial cells and carries out enzymatic functions required for initiation of viral DNA replication. In this study, we investigated the pathway and regulation of nuclear import of E1 protein from bovine papillomavirus type 1 (BPV1). Using an in vitro binding assay, we determined that E1 protein interacted with importins 3, 4 and 5 via its nuclear localization signal (NLS) sequence. In agreement with this result, purified E1 protein was effectively imported into the nucleus of digitonin-permeablized HeLa cells after incubation with importins 3, 4 or 5, and other necessary import factors. We also observed that in vitro binding of E1 protein to all 3 importins was significantly decreased by introduction of pseudophosphorylation mutations in the NLS region. Consistent with the binding defect, pseudophosphorylated E1 protein failed to enter the nucleus of digitonin-permeablized HeLa cells in vitro. Likewise, the pseudophosphorylation mutant showed aberrant intracellular localization in vivo and accumulated primarily on the nuclear envelope in transfected HeLa cells while the corresponding alanine replacement mutant displayed the same cellular location pattern as wildtype E1 protein. Collectively, our data demonstrate that BPV-1 E1 protein can be transported into the nucleus by more than one importin , and suggest that E1 phosphorylation by host cell kinases plays a regulatory role in modulating E1 nucleocytoplasmic localization. This phosphoregulation of E1 protein nuclear uptake may contribute to coordination of viral replication with keratinocyte proliferation and differentiation.

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