Cellular factors and viral elements for parvovirus replication

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dc.contributor.authorDeville, Catherine Micheleen
dc.contributor.committeechairLederman, Muriel L.en
dc.contributor.committeememberBates, Robert C.en
dc.contributor.committeememberRogers, Patriciaen
dc.contributor.committeememberRutherford, Charles L.en
dc.contributor.committeememberStout, Ernest R.en
dc.contributor.committeememberTolin, Sue A.en
dc.contributor.committeememberTurner, Bruce J.en
dc.contributor.departmentBiologyen
dc.date.accessioned2014-03-14T21:22:15Zen
dc.date.adate2005-10-24en
dc.date.available2014-03-14T21:22:15Zen
dc.date.issued1994-04-15en
dc.date.rdate2005-10-24en
dc.date.sdate2005-10-24en
dc.description.abstractAutonomous parvoviruses, such as bovine parvovirus (BPV), need a factor present at the S-phase of the cell cycle for a productive infection, while dependent parvoviruses, the adenoassociated viruses (AAVs), require a helper virus to complete an infectious cycle. However, AAV can replicate autonomously in synchronized cells, suggesting that an S-phase factor substitutes for the helper virus. To investigate the nature of the cellular S-phase factor, we performed DNA retardation assays with uninfected nuclear extracts of S-phase cells, synchronized by hydroxyurea pretreatment, and radiolabeled parvoviral termini in their hairpinned conformation. We observed that proteins in HeLa cells, a tissue culture host for AAV, specifically interacted with the terminal sequences of this virus, which act as origins of replication (oris). These assays also showed specific binding between S-phase cellular proteins and termini (oris) of heterologous parvoviruses, for which the cells are not a natural host. For example, proteins from bovine fetal lung (BFL) cells, a tissue culture host for BPV, were able to bind to an AAV terminus and HeLa cell proteins interacted with both termini of the BPV genome. All DNA-protein complexes investigated appeared to be specific for S-phase synchronized cells. In order to begin to characterize the protein(s) involved in the complex formation, we performed SDS-PAGE electrophoresis of some retarded complexes. We report that a 54 kd protein was contained in the complex formed with the BPV left terminus and BFL cell extract. [Binding of BFL cell proteins to a BPV left terminus has been reported earlier]. Using a similar technique, we observed that two phosphoproteins of 55 and 90 kd were present in the retarded complex formed between a BPV left terminus and HeLa cell extract. An antibody directed against human p53, an anti-oncoprotein, was shown to compete binding of BFL cell extract and HeLa cell extract to the BPV left terminus. This antibody also competed the binding of HeLa cell extract to the AAV terminus. Our data suggest that proteins with similar characteristics, most probably among which is p53, are involved in the ori-binding complexes, possibly exerting a role as positive regulator of parvoviral replication. The secondary structure of the viral ends is remarkably conserved among parvoviruses. Of particular interest is the presence of mismatched/unpaired nucleotides, forming a bubble, in the stem of the left hairpin of almost all autonomous parvoviruses. To analyze the possible role of these unpaired/mismatched nucleotides in the BPV life cycle, two mutants clones lacking the bubble region were constructed and their replicative properties were analyzed after electroporation in permissive cells. Infectivity of the mutant clones was determined by three techniques: observation of cytopathic effect, detection of virally-coded proteins by indirect immunofluorescence, and transient DNA replication assays. We report that the mutant clone containing duplicate sequences of the (mismatched) nucleotides numbered 46 to 57 (BLOP) was defective for replication. The other bubbleless clone (BLOM), that contains duplicate sequences of the (mismatched) nucleotides 99 to 105, was able to replicate. The later clone produced monomer-length viral DNA at about 20% of the level of the infectious genomic clone of BPV, when electroporated as a linear excised sequence. This clone was infectious since it could be propagated by subsequent passage. Expression of viral structural proteins was seen by an indirect immunofluorescence assay using anti-capsid antibodies. Our results suggest that the bubble in the left hairpin of BPV is not required for the viral life cycle, but that specific sequences within the mismatched/unpaired region are necessary for viral replication.en
dc.description.degreePh. D.en
dc.format.extentix, 104 leavesen
dc.format.mediumBTDen
dc.format.mimetypeapplication/pdfen
dc.identifier.otheretd-10242005-174035en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-10242005-174035/en
dc.identifier.urihttp://hdl.handle.net/10919/40174en
dc.language.isoenen
dc.publisherVirginia Techen
dc.relation.haspartLD5655.V856_1994.D485.pdfen
dc.relation.isformatofOCLC# 30907479en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subject.lccLD5655.V856 1994.D485en
dc.subject.lcshParvoviruses -- Reproductionen
dc.titleCellular factors and viral elements for parvovirus replicationen
dc.typeDissertationen
dc.type.dcmitypeTexten
thesis.degree.disciplineBiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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