CHARACTERIZING THE FUNCTIONAL DOMAINS OF THE BACULOVIRUS LATE EXPRESSION FACTOR 3 (LEF-3) INVOLVED IN NUCLEAR LOCALIZATION AND DNA REPLICATION
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Transient replication assays have identified a late expression factor 3, LEF-3, to be essential for DNA replication and late gene expression in the baculovirus species, AcMNPV. Although its specific role in these two processes has not been determined, this single-stranded DNA binding protein is multi-functional. LEF-3 forms a homo-oligomer, binds single-stranded DNA, interacts with components of the viral replication complex and is required to transport the helicase protein P143 into the nucleus of infected cells. Various regions within LEF-3 were deleted to determine the domain essential to its function and the N-terminal amino acids 1-125 were found to be sufficient for late gene expression. This N-terminal region includes the 56 amino acid region of LEF-3 required for nuclear transport. In order to define this domain, the effect of site-specific mutagenesis of LEF-3 on its intracellular distribution was determined. Fluorescence microscopy of expression plasmid transfected cells demonstrated that amino acids 14 to 37 formed the core nuclear localization signal (NLS), but the flanking amino acids may act as regulatory elements. Comparison with other group 1 Alphabaculoviruses suggested that this core region contained a functionally duplicated NLS. The AcMNPV LEF-3 also functioned in mammalian cells indicating that the protein nuclear import systems in insect and mammalian cells are conserved. Mutagenesis of two conserved cysteine residues located at amino acids 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, it was demonstrated that a functional nuclear localization domain on LEF-3 was required for an interaction between LEF-3 and P143.