In the last issue of the MJM, Volume 10, Number 2, Victoria Au’s abstract should have been included among scientific abstracts from the CSLSR conference. We include her abstract below. The MJM apologizes for any inconvenience this error may have caused.
Characterizing the functional domains of baculovirus Late Expression Factor 3 (LEF-3)
VICTORIA AU AND DR. ERIC CARSTENS
DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY, QUEEN’S UNIVERSITY, KINGSTON, ONTARIO, CANADA
The Baculoviridae is a family of arthropod-specific viruses found in over 600 insect species. Baculovirus species have very specific host ranges and are unable to replicate in mammalian cells, making them ideal for use as biopesticides and vectors for gene therapy. AcMNPV is the best-studied member of the Baculoviridae family and most of the genes identified in this virus serve as a basis for comparison to other baculoviruses. Six proteins are essential for AcMNPV DNA replication: LEF-1, LEF-2, LEF-3, DNA Polymerase, P143, and IE-1. LEF-3 (407 aa, 45 kDa) is a single-stranded DNA binding protein that functions as a homotrimer. LEF-3 also transports P143, a helicase, to the nucleus. We predict that LEF-3 has several functional domains responsible for: ssDNA binding, interaction with P143, and interaction with host importin proteins for nuclear localization. Site directed mutagenesis revealed that the N terminal region (amino acids 5 to 56) are responsible for LEF-3 nuclear localization, while a region including 125 amino acids at the N-terminus is required for interaction with P143. To identify amino acids within the 5 to 56 region essential for nuclear transport, conserved amino acids were targeted for mutagenesis. The intracellular localization of mutated proteins expressed by plasmid-transfected cells was examined using fluorescence microscopy and biochemical fractionation. A similar approach will be used to identify the regions responsible for P143 interaction and ssDNA binding. Although the first fifty amino acids of AcMNPV LEF-3 contain characteristics similar to classic NLSs, the results of mutating 14 conserved amino acids or from deleting various regions within the 5 to 56 region of LEF-3 did not demonstrate any clear effect on nuclear localization. Therefore, the essential components of the domain remain to be determined and the transport of LEF-3 into the nucleus may be mediated by a novel NLS.