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dc.contributor.advisorWimmer, Eckard , Carter, Carolen_US
dc.contributor.authorWang, Chunlingen_US
dc.contributor.otherDepartment of Molecular Genetics and Microbiologyen_US
dc.date.accessioned2013-05-22T17:35:46Z
dc.date.available2013-05-22T17:35:46Z
dc.date.issued1-Dec-11en_US
dc.date.submitted11-Decen_US
dc.identifierWang_grad.sunysb_0771E_10746en_US
dc.identifier.urihttp://hdl.handle.net/1951/59907
dc.description199 pg.en_US
dc.description.abstractPolypeptide 2C<super>ATPase</super> is one of the most thoroughly studied but least understood proteins in the life cycle of poliovirus. Within the protein, multiple functional domains, important for uncoating, host cell membrane alterations, RNA replication and encapsidation have previously been identified. In this study, charged to alanine scanning mutagenesis was used to generate conditional-lethal mutations in hitherto uncharacterized domains of the 2C<super>ATPase</super> polypeptide, particularly those possibly involved in morphogenesis. Adjacent or clustered charged amino acids (2-4), scattered along the 2C<super>ATPase</super> coding sequence, were replaced with alanines. RNA transcripts of mutant poliovirus cDNA clones were transfected into HeLa cells. Subsequently, ten lethal, one severely temperature-sensitive, two quasi-infectious, and three wild type-like mutants were identified. Using a Renilla luciferase reporter virus, all lethal and quasi-infectious mutants demonstrated RNA replication defects. Temperature-sensitive mutants were defective in RNA replication only at the restricted temperatures. These mutants have led to the identification of several new sites within the 2C<super>ATPase</super> polypeptide that are required for RNA replication. Interestingly, I characterized a quasi-infectious mutant (K6A/K7A) that produced a suppressor mutation (G1R) and a novel 2B^2C<super>ATPase</super> cleavage site (Q^R). Surprisingly, this cleavage site mutation did not interfere with normal processing of the polyprotein. Furthermore, analysis of the suppressor mutants of one quasi-infectious mutant and a detailed mutagenic analysis of its flanking Cysteine Rich regions have revealed a new domain near the C-terminus of 2C<super>ATPase</super> that is involved in encapsidation possibly achieved through interacting with a spacer between A and B motifs of the NTP-binding domain of 2C<super>ATPase</super>. Most importantly, suppressor mutations were identified not only in PV nonstructural protein 2C<super>ATPase</super> but also in PV capsid proteins VP3 and VP1 - the first demonstration of genetic suppression of a 2C<super>ATPase</super> defect by capsid proteins in the background of PV genome. The data presented here reinforce our previous conclusion that an interaction between 2C<super>ATPase</super> and the capsid proteins is required for viral encapsidation. In addition, I performed a yeast two hybrid screening of a HeLa cell cDNA library and identified several cellular factors that may interact with PV non-structural protein 2C<super>ATPase</super>. The roles of these candidates in PV replication and/or assembly of PV viral particles, if any, need further investigations.en_US
dc.description.sponsorshipStony Brook University Libraries. SBU Graduate School in Department of Molecular Genetics and Microbiology. Charles Taber (Dean of Graduate School).en_US
dc.formatElectronic Resourceen_US
dc.language.isoen_USen_US
dc.publisherThe Graduate School, Stony Brook University: Stony Brook, NY.en_US
dc.subject.lcshVirology--Molecular biology--Health sciencesen_US
dc.subject.other2CATPase, alanine scanning mutagenesis, capsid proteins (VP1 and VP3), encapsidation, poliovirusen_US
dc.titleFunctional analysis of poliovirus protein 2CATPase in viral RNA replication and encapsidation using alanine scanning mutagenesisen_US
dc.typeDissertationen_US
dc.description.advisorAdvisor(s): Wimmer, Eckard ; Carter, Carol. Committee Member(s): Konopka, James ; Freimuth, Paul ; Grubman, Marvin.en_US
dc.mimetypeApplication/PDFen_US


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