Baculoviruses are viruses of arthropods with large rod-shaped virions that contain supercoiled double-stranded DNA genomes. These viruses have been used as gene expression vectors and insect biological control agents, and have been studied as a virus model for the investigation of molecular mechanisms, such as apoptosis, gene expression, DNA replication, and virus-host interaction. Our current knowledge about baculovirus is largely based on the studies of the Autographa californica ucleopolyhedrovirus and the closely related species. In spite of the increasing interest of recombinant baculoviruses as gene expression and delivery vectors and bioinsecticides, the mechanisms of baculovirus DNA replication and virus-host interaction are still poorly understood. To take advantage of baculovirus diversity and their specific host-ranges, I studied the Spodoptera littoralis
nucleopolyhedrovirus (SpliNPV). Previous investigations indicated that SpliNPV possesses a unique host-range and genetic organization. In this dissertation, I studied the SpliNPV infection of an orthopteran cell line derived from the grasshopper, Melanopus sanguinipes, and provided evidence of viral DNA replication and production of viable
virus progeny. I next investigated SpliNPV infection in five cell lines derived from three lepidopteran families: Sf9, CLS79 and Se1 cell lines from Spodoptera (Noctuidae), Ld652Y cells from Lymantria dispar (Lymantriidae), and Md210 cells from Malacosoma disstria (Lasiocampidae), which represented permissive (Sf9, CLS79, and Se1), semipermissive (Ld652Y), and non-permissive (Md210) cell lines. SpliNPV infection in permissive cell lines resulted in viral gene expression, DNA replication, and production of viable progeny. While the semi-permissive cell line displayed reduced and delayed transcription of viral genes and supported limited viral DNA replication, the non-permissive cell line displayed dramatically reduced viral transcription and abolished viral progeny. Transient expression assays using SpliNPV early- or very late-promoter reporters suggested that non-productive infection of SpliNPV in semi- or non-permissive cell lines was a consequence of limited viral specific transcription at the early phase of viral infection.
Having documented the infection events in these cell lines, I investigated the
mechanism of SpliNPV DNA replication. Using transient replication assays I have
identified a non-hr origin of SpliNPV DNA replication. With limited sequence similarity to
other NPV non-hr origins, the putative SpliNPV origin consists of sequence motifs found in
other origins of virus DNA replication, such as imperfect palindromes, direct repeats, and
transcription factor binding sites. Transient expression assays indicated that the putative
non-hr origin represses the SpliNPV early gene, lef-3. Gel mobility shift analyses confirmed
that nuclear proteins from both infected and uninfected cells bound with specificity to the
putative origin.
After identification and characterization of the cis-acting factor involved in viral
DNA replication, I then identified a trans-acting factor involved in viral DNA replication.
I have sequenced a 6.4 kb DNA from SpliNPV genome that contains an ORF encoding a
predicated polypeptide of 998 amino acid sequences. Comparative sequence analyses demonstrated that the ORF encoded a DNA polymerase (dnapol) that consists of conserved exonuclease domains and DNA polymerase motifs found in other eukaryotic DNA viruses and in cellular DNA polymerases. The transcription initiation site of the 3 kb SpliNPV
dnapol transcript was mapped to an NPV early promoter element, ACGT. The transcript terminated at the polyadenylation signal AATAAA. Using E. coli and baculovirus expression systems, I over-expressed a 110 kDa full-length SpliNPV DNA polymerase (DNAPOL) and a truncated 96 kDa protein, in which the amino terminal 80 amino acids were deleted. Enzymatic analyses demonstrated that the DNA polymerase and 3'- 5’exonuclease activities are intrinsic to the SpliNPV DNAPOL. Deletion of the 80 amino
acid residues at the N-terminal of the DNAPOL did not affect DNA polymerase and exonuclease activities. Replication products from single-stranded M13 DNA revealed that SpliNPV DNAPOL possesses a proccessive activity. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/9080 |
| Date | 19 February 2018 |
| Creators | Huang, Jianhe |
| Contributors | Levin, David Bernard |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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