title: A conserved N-terminal domain mediates required DNA replication activities and phosphorylation of the transcriptional activator IE1 of Autographa californica multicapsid nucleopolyhedrovirus.
abstract: IE1 is the principal transcriptional regulator of the baculoviruses. Like multifunctional transcription factors of other large DNA viruses, IE1 is an essential, site-specific DNA-binding phosphoprotein that activates virus gene expression and promotes genome replication. To define the poorly understood mechanisms by which IE1 achieves its diverse functions, we identified IE1 domains that contribute to productive infection of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), the baculovirus prototype. Site-directed mutagenesis revealed that the N-terminal 23 residues of IE1 are required for origin-specific DNA replication and AcMNPV propagation, but not for DNA-binding-dependent transcriptional activation. Within this defined replication domain, we identified an invariant TPXR/H motif that resembles a consensus cyclin-dependent kinase phosphorylation site. Amino acid substitutions of potential phosphorylation sites within or near this motif caused loss of IE1-mediated DNA replication activity. Remarkably, substitution of the single threonine (residue 15) within the TPXR/H motif caused complete loss of AcMNPV multiplication. The replication domain was required for IE1 phosphorylation. It was also sufficient for conferring phosphorylation of a heterologous protein. Importantly, IE1 hyperphosphorylation coincided exclusively with AcMNPV DNA replication. The temporal regulation of IE1 phosphorylation and the essential nature of the TPXR/H motif suggest that phosphorylation critically alters and possibly activates DNA replication activity of IE1 during infection. The striking conservation of the TPXR/H motif among IE1 proteins further suggests that this molecular switch may be a common mechanism by which the alphabaculoviruses coordinate DNA replication and gene expression by using a single regulator.
authors.affiliation: Institute for Molecular Virology, Department of Biochemistry, Graduate School and College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Taggart
authors.forename: Jonathan K
authors.initials: JK
authors.lastname: Mitchell
authors.forename: Paul D
authors.initials: PD
authors.lastname: Friesen
mesh_terms: D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D004261:DNA Replication; D004331:Drosophila melanogaster; D015967:Gene Expression Regulation, Viral; D017874:Immediate-Early Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D018411:Spodoptera; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: C069183:Ac-IE-1 protein, Autographa californica nucleopolyhedrovirus; D017874:Immediate-Early Proteins; D015534:Trans-Activators