Characterization of a baculovirus nuclear localization signal domain in the late expression factor 3 protein.
The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) single-stranded DNA binding protein LEF-3 is a multi-functional protein that is required to transport the helicase protein P143 into the nucleus of infected cells where they function to replicate viral DNA. The N-terminal 56 amino acid region of LEF-3 is required for nuclear transport. In this report, we analyzed the effect of site-specific mutagenesis of LEF-3 on its intracellular distribution. Fluorescence microscopy of expression plasmid-transfected cells demonstrated that the residues 28 to 32 formed the core nuclear localization signal, but other adjacent positively-charged residues augmented these sequences. Comparison with other group I Alphabaculoviruses suggested that this core region functionally duplicated residues including 18 and 19. This was demonstrated by the loss of nuclear localization when the equivalent residues (18 to 20) in Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) LEF-3 were mutated. The AcMNPV LEF-3 nuclear localization domain was also shown to drive nuclear transport in mammalian cells indicating that the protein nuclear import systems in insect and mammalian cells are conserved. We also demonstrated by mutagenesis that two conserved cysteine residues located at 82 and 106 were not essential for nuclear localization or for interaction with P143. However, by using a modified construct of P143 that localized on its own to the nucleus, we demonstrated that a functional nuclear localization domain on LEF-3 was required for interaction between LEF-3 and P143.
Au
Victoria
V
Department of Microbiology and Immunology, Queen's University, Kingston, ON, Canada.
Yu
Mei
M
Carstens
Eric B
EB
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D016415:Sequence Alignment; D017384:Sequence Deletion; D018411:Spodoptera; D014709:Vero Cells; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D019913:Nuclear Localization Signals; D014764:Viral Proteins; C094647:late expression factor 3, baculovirus