AcMNPV lef-2 is essential for DNA replication and is involved in expression of late and very late gene promoters. In earlier work, the isolation of a virus mutant (VLD1) deficient only in very late protein synthesis was attributed to a point mutation in lef-2. DNA replication and late protein production remained largely unchanged. In this study, real time or quantitative polymerase chain reaction (q-PCR) was used to re-examine virus DNA replication by VLD1 and AcORF63260-1, a virus containing the same mutation within lef-2 derived by directed methods. The results showed that AcORF63260-1 DNA replication was comparable to wild type AcMNPV, but that VLD1 exhibited slightly delayed DNA replication, suggesting that this virus might have other gene mutations. A second mutation was discovered within lef-2, but when this was introduced into AcMNPV it had no apparent effect on DNA replication. The q-PCR was also used to examine late and very late gene expression in virus-infected cells. The expression of late (gp64 and capsid) genes was similar between AcORF63260-1 and AcMNPV whereas very late gene expression (p10 and polyhedrin) was down regulated in the mutant. This evidence supports a dual role for lef-2 in DNA replication and very late gene expression. Further analysis of the region of lef-2 containing the mutation in VLD1 revealed 5 cysteine residues highly conserved between examples of the gene from 37 different baculoviruses. Mutation of four of these cysteines into serines and construction of recombinant viruses showed that budded virus production was reduced from 0 — 48% of that seen in AcMNPV controls. This suggests an important role for the cysteine rich region of LEF-2 in AcMNPV replication. To facilitate the construction of a number of virus mutants in this study, a recombinant bacmid (AcΔlef-2.neo) was used that lacked the lef-2 coding region. This virus should not have been replication-competent but insect cells transfected with AcΔlef-2.neo DNA showed limited cytopathic effects. Subsequent titration of culture medium produced small, punctate plaques. The titres of these virus stocks were very low and made further characterisation difficult. Clearly, lef-2 is an important gene, but it can be deleted from the virus genome. To assess the ability of other baculovirus lef-2s to complement AcΔlef-2.neo, synthetic copies of genes with AcMNPV-specific flanking regions were constructed and used to rescue this virus. Only a lef-2 with at least 53% identity was able to produce a virus that replicated with 0.04% efficiency compared with wild type. Lef-2s with higher identity to that of AcMNPV are not yet available. This suggests that such examples of lef-2 may not be functional or more likely, that many other baculoviruses have yet to be identified.
Permanent link to this resource: https://doi.org/10.24384/pmdv-ns24
Allen, Clare
Supervisors: Possee, Bob
School of Biological and Medical SciencesFaculty of Health, Science and Technology
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