Graduate Thesis Or Dissertation
 

Regulation of expression of four baculovirus genes and the immunocytochemical characterization of their products

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  • 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.
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