Mitogen-activated protein kinases activate the nuclear localization sequence of human papillomavirus type 11 E1 DNA helicase to promote efficient nuclear import.
Human and animal papillomavirus DNA replicates as multicopy nuclear plasmids. Replication requires two viral proteins, the origin-recognition protein E2 and the replicative DNA helicase E1. Using genetic, biochemical, and immunofluorescence assays, we demonstrated that efficient nuclear import of the human papillomavirus (HPV) type 11 E1 protein depends on a codominant bipartite nuclear localization sequence (NLS) and on phosphorylation of the serine residues S89 and S93 by the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase, and c-Jun N-terminal protein kinase. The NLS and the MAPK substrates are located within a 50-amino-acid-long peptide near the amino terminus, previously designated the localization regulatory region (LRR). The downstream NLS overlaps the cyclin-binding motif RRL, which is necessary for phosphorylation by the cyclin-dependent kinases to inactivate a dominant nuclear export sequence, also in the LRR. Alanine mutations of the MAPK substrates significantly impaired nuclear import, whereas phospho-mimetic mutations partially restored nuclear import. We further identified two MAPK docking motifs near the C terminus of E1 that are conserved among E1 proteins of many HPVs and bovine papillomavirus type 1. Mutations of these MAPK docking motifs or addition of specific MAPK inhibitors significantly reduced nuclear import. Interestingly, a fraction of the NLS-minus E1 protein was cotransported with the E2 protein into the nucleus and supported transient viral DNA replication. In contrast, E1 proteins mutated in the MAPK docking motifs were completely inactive in transient replication, an indication that additional properties were adversely affected by those changes.
Yu
Jei-Hwa
JH
Department of Biochemistry and Molecular Genetics, McCallum Building, University of Alabama at Birmingham, 1918 University Boulevard, Birmingham, Alabama 35294-0005, USA.
Lin
Biing Yuan
BY
Deng
Wentao
W
Broker
Thomas R
TR
Chow
Louise T
LT
D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D002522:Chlorocebus aethiops; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052140:Human papillomavirus 11 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008856:Microscopy, Fluorescence; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics
D004268:DNA-Binding Proteins; C084304:E1 protein, Human papillomavirus type 11; D019913:Nuclear Localization Signals; D047428:Protein Kinase Inhibitors; D014764:Viral Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D004265:DNA Helicases