| Summary: | 碩士 === 國立臺灣大學 === 植物病理與微生物學研究所 === 95 === The late expression factor 2 (lef-2) gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was originally identified by plasmid transient expression assays and shown to be important for expression of some virus-encoded late genes. Previous studies also suggested that lef-2 gene plays an essential but undefined role in viral DNA replication by using origin-dependent plasmid replication assays. To determine the role of lef-2 in AcMNPV life cycle, the lef-2 knockout (ko) mutant bacmid (bAc△lef2-hEG) was generated by homologous recombination in a wild-type (wt) bacmid (bAcwt-hEG) in Escherichia coli. Additionally, a lef-2 repair bacmid (bAclef2-Rep-hEG) was generated by transposing the lef-2 coding region along with its own promoter into the polyhedrin locus of lef-2 ko bacmid. To trace the movement of virion, DNA replication and gene expression, enhanced green fluorescence protein (eGFP) gene was inserted into bacmids, bAcwt-hEG, bAc△lef2-hEG and bAclef2-Rep-hEG. Budded viruses production of and viral spreading of bAclef2-Rep-hEG were similar to bAcwt-hEG by growth curve analyses and fluorescence microscopy, but bAc△lef2-hEG was defective in both processes. Slot blot analyses showed that the viral genome of lef-2 ko bacmid did not seem to replicate. However, by using a more sensitive replication assay based on DpnI digestion that can distinguish replicated from un-replicated DNA samples, it became clear that the lef-2 ko bacimd can replicate albeit very inefficiently. Further studies indicated that the replication level of lef-2 ko bacmid reduced substantially and onset of replication was delayed by about 48 hours. Due to late and very late gene expressions were depended on vial DNA replication, promoters from late genes, such as a capsid gene (vp39) and polyhedrin gene (polh), able to drive expression of reporter gene in the lef-2 ko background. Furthermore, LEF-2 protein was found to incorporated into the virions by using immunoblotting assay and immunogold electron microscopy, suggesting that virion-associated LEF-2 protein might be required immediately after entering cells for the initiation of viral DNA replication. However, in the absent of LEF-2, the formation of viral nucleocapsid derived from lef-2 ko bacmid appeared to be normal under transmission electron microscope, suggesting that the life cycle of lef-2 ko bacmid could proceed through DNA replication to viral pakckaging unimpededly. Finally, when the overall viral gene expression patterns were surveyed by viral cDNA microarray analyses in the absent of lef-2 gene, most of the late genes were affected. Thus, in contrast to prior studies using plasmid transient expression assay, current studies using lef-2 ko bacmid demonstrated that lef-2 was not an essential gene. lef-2 was not absolutely required for vial DNA replication and late gene expression, however without lef-2 gene in viral genome, the levels of replication and gene expression declined significantly.
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