Nuclear import of bovine papillomavirus type 1 E1 protein is mediated by multiple alpha importins and is negatively regulated by phosphorylation near a nuclear localization signal

Papillomavirus DNA replication occurs in the nucleus of infected cells and requires the viral El protein, which enters the nuclei of host epithelial cells and carries out enzymatic functions required for the initiation of viral DNA replication. In this study, we investigated the pathway and regulation of the nuclear import of the El protein from bovine papillomiavirus type I (BPV1). Using an in vitro binding assay, we determined that the El protein interacted with importins alpha 3, alpha 4, and alpha 5 via its nuclear localization signal (NLS) sequence. In agreement with this result, purified El protein was effectively imported into the nucleus of digitonin-permeabilized HeLa cells after incubation with importin alpha 3, alpha 4, or alpha 5 and other necessary import factors. We also observed that in vitro binding of El protein to all three alpha importins was significantly decreased by the introduction of pseudophosphorylation mutations in the NLS region. Consistent with the binding defect, pseudophosphorylated El protein failed to enter the nucleus of digitonin-permeabilized 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 wild-type El protein. Collectively, our data demonstrate that BPV1 E1 protein can be transported into the nucleus by more than one importin alpha and suggest that El phosphorylation by host cell kinases plays a regulatory role in modulating El nucleocytoplasmic localization. This phosphoregulation of nuclear El protein uptake may contribute to the coordination of viral replication with keratinocyte proliferation and differentiation.