Graduation date: 1996

Regulation of expression of three genes in the polyhedron envelope protein (PEP) gene region of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) was examined. These genes include open reading frame (ORF) 1 (encoding p21), ORF 2 (encoding gp16), and ORF 3 (encoding the polyhedron envelope protein). The effect of elimination of the late promoter elements of each ORF or both ORFs 1 and 2 on ORF 3 expression was examined by using an ORF 3 promoter-CAT gene fusion. The data indicated that the ORF 3 promoter was essential for the expression of the PEP. Destruction of ORF 1 caused no effect whereas destruction of ORF 2 promoter resulted in a 29% increase in CAT activity. To characterize the role of ORF 1 and 2 in the viral life cycle and the location of the proteins in virions and infected cells, antisera were produced against these proteins. The 21 kDa protein was present in both purified budded and occluded virions as demonstrated by Western blot analysis. Immunoelectron microscopy showed that the 21 kDa protein was a capsid-associated protein in both phenotypes. The ORF 2 gene encodes a 12 kDa protein that is N-glycosylated, migrates at a MW of 16 kDa, and is not present in budded or occluded virions. Immunoelectron microscopy indicated that gp16 is associated with lamellar-like membranous structures in close association with the nuclear membrane. It was also found associated with envelopes of virions that had budded from the nucleus into the cytoplasm. A gene that reportedly has a similar role to ORF 3 (polyhedron envelope protein) has been described in Autographa californica MNPV. This gene encodes a protein called the spheroidin-like protein (SLP) because of its sequence similarity to the spheroidin inclusion protein of the Choristoneura biennis entomopox virus. The gene was located, sequenced, transcriptionally mapped in OpMNPV and an antiserum was produced against a fusion protein containing most of the SLP ORF. Immunoelectron microscopy showed that the protein was concentrated in cytoplasmic inclusion bodies and was not associated with the polyhedron envelope structure in OpMNPV. It was found to be associated with polyhedra of AcMNPV, but no specific association with the polyhedron envelope was found. The role of the PEP and the p10 protein in polyhedron morphogenesis was examined using deletion mutants of OpMNPV and immunoelectron microscopy. The p10 deletion mutant produced polyhedra with patchy and poorly attached polyhedron envelopes, suggesting p10 has a direct or indirect role in the proper formation of the polyhedron envelope. The PEP deletion mutant showed that PEP was an essential component in the formation of the polyhedron envelope. The mutant with both p10 and PEP deleted had polyhedra that showed a distinct cubic morphology. These data suggest that these two proteins may affect polyhedra morphology. / Graduation date: 1993

The yeast two-hybrid system was used to examine interactions between the nine proteins involved in baculovirus DNA replication. From the six proteins required for DNA replication, four protein-protein interactions were identified, including an interaction between LEF-1 and LEF-2, LEF-3 and itself, LEF-3 and P143 (Helicase), and an interaction between IE-1 and itself. The replication factors LEF-1 and LEF-2 interacted in both yeast two-hybrid assays and glutathione S-transferase fusion affinity assays. Using the yeast two-hybrid system, we mapped the interaction domain of LEF-2 to amino acids between positions 20 and 60. Deletion analysis of LEF-1 failed to reveal an interaction domain, suggesting that either multiple interaction domains exists or the deletions disrupted secondary structures required for the interaction. All of the deletions which were unable to interact also failed to support significant levels of transient DNA replication, suggesting that this interaction plays a significant role in DNA replication. The baculovirus single-stranded DNA binding protein, LEF-3, interacts with itself in yeast two-hybrid assays and glutathione S-transferase fusion affinity assays. Deletions of LEF-3, which were unable to interact with full length LEF-3, also failed to support transient DNA replication, suggesting that this interaction is required for the proper function of LEF-3. LEF-3 was purified to apparent homogeneity and analyzed by analytical ultracentrifugation, native PAGE and MALDI mass spectrometry, identifying the oligomeric structure of LEF-3 as a homotrimer. In addition to interacting with itself, LEF-3 also interacts with P143 in yeast two-hybrid assays, immunoprecipitation experiments, and co-purification from a single-stranded DNA agarose column. The yeast two-hybrid system was used to map the LEF-3 interaction domain to the N-terminal 165 amino acids of LEF-3. Deletion analysis of P143 failed to reveal a delimited interaction domain. C-terminal deletions of LEF-3 containing amino acids 1 to 165 were unable to interact with full length LEF-3, indicating that the interaction of LEF-3 with itself (trimerization) is not required for the interaction between LEF-3 with P143. / Graduation date: 1998

Graduation date: 1995

Budded virions of AcMNPV can enter a variety of non-host cells, a characteristic likely due to the presence of GP64, an envelope protein found on a small subset of baculoviruses. Results show that AcMNPV's tropism for vertebrate cells can be restricted - a prerequisite for using AcMNPV for targeted in vivo gene delivery - by replacing the gp64 gene with SeF from SeMNPV. Unlike the relatively well characterized GP64 protein, the significance and function of the F homolog (Ac23, a pathogenicity factor), is poorly understood. How Ac23 might contribute to the faster speed of kill was examined by comparing occlusion bodies and occlusion-derived virions (ODV) of Ac23null mutant viruses with control viruses at the ultrastructural level. The results show that Ac23null mutant produces a significantly higher percentage of ODVs with single or lower number of nucleocapsids than controls, suggesting Ac23 may play a role in multicapsid envelopment of ODVs. / xiii, 101 leaves : ill. (some col.) ; 28 cm. --