Graduate Thesis Or Dissertation
 

OpMNPV p32, a baculovirus polyhedral envelope-associated protein : genetic location, nucleotide sequence, transcriptional mapping and immunocytochemical characterization

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  • Baculoviruses comprise a diverse group of pathogens infectious for members of the insect orders Lepidoptera, Hymenoptera and Diptera. Of the three subgroups, two (subgroup A, nuclear polyhedrosis virus (NPV) and subgroup B, granulosis virus (GV) are occluded in a crystalline protein structure while the remaining subgroup (subgroup C, non-occluded virus) is not. These viruses have a large double-stranded, supercoiled DNA genome (88-160 kb) packaged within a rod-shaped, enveloped nucleocapsid. Two viral phenotypes are observed during the life cycle of the virus. The polyhedra-derived virus (PDV) is packaged in polyhedra within the cell nucleus late in infection and is responsible for primary infection of the insect. The nucleocapsid of the budded virus (BV) is assembled in the nucleus and acquires an envelope as it is budded from the cell early in infection. This form of the virus is responsible for spread of infection from cell to cell in the insect. Very few baculovirus structural proteins have been well characterized as to their genetic location, expression and function. Characterization of viral and virally-associated structural proteins will lead to a better understanding of virus structure and function and determination of phenotype specific proteins will allow a better understanding of phenotype regulation. The purpose of this study was to determine the genetic location of a structural protein and charcterize its expression and location during infection and possibly elucidate its function. Using a polyclonal mouse antiserum produced against purified virions of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV), I identified two immunoreactive lambda gtll clones containing non-overlapping insert DNAs which mapped to a single open reading frame (ORF) in the HindIII-M fragment. Analysis of nucleotide sequence data indicates that this ORF encodes a protein with a MW of 32.4 kDa. A trpE-p32 gene fusion containing the entire p32 ORF was constructed, and the fusion protein was purified and used to immunize rabbits. Western blot analysis and immunofluorescence studies using the anti-TrpE-p32 antiserum detected a polyhedra-derived virus (PDV)-associated protein of 32 kDa at 24 hours post infection (hpi). The protein was observed in the cytoplasm and nucleus at 24 hpi and became concentrated in the cytoplasm late in infection. Western blot analysis and immunofluorescent microscopy of polyhedra solubilized under various conditions, indicated that p32 is associated with the polyhedral envelope. The predicted amino acid sequence for p32 showed 58% amino acid identity with the predicted amino acid sequence for an ORF (ORF 3) in a similar region of the genome of the MNPV of Autographa californica (AcMNPV) (Oellig et al., 1987). The solubility properties of the p32 protein and reciprocal immunoblotting experiments indicate the OpMNPV p32 gene encodes a protein which is homologous to the polyhedral envelope-associated phosphoprotein, pp34, recently reported by Whitt and Manning (1988). In addition, the sequenced HindIII-M region of the OpMNPV genome was transcriptionally mapped and compared to the homologous region in the AcMNPV genome. Five ORFs (including the above mentioned ORF 3) were identified and compared between AcMNPV and OpMNPV and amino acid homologies of 25-70% were observed. The comparison revealed a number of major differences in the genomes of the two viruses. Discontinuous homology between the two viruses of the ORF 1 gene led to the identification of a putative transposable element in the AcMNPV ORF 1 sequence reported by Oellig et al (1987). In addition, it was found that a region corresponding to the 4 kb HindIII-K/EcoRl-S region of AcMNPV was not present in the OpMNPV genome. Although the ORFs characterized in the OpMNPV HindIII-M region all were expressed as late genes and all contained the conserved ATAAG putative promoter/mRNA start site sequence, primer extension analysis indicated that use of this signal for transciptional initiation may vary between different ORFs.
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