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Cover for MECHANISMS CONTROLLING HUMAN NOROVIRUS POLYPROTEIN PROCESSING
dc.contributor.advisorKorba, Brenten
dc.creatoren
dc.date.accessioned2015-06-01T16:35:15Zen
dc.date.available2015-06-01T16:35:15Zen
dc.date.created2015en
dc.date.issueden
dc.date.submitted01/01/2015en
dc.identifier.otherAPT-BAG: georgetown.edu.10822_760908.tar;APT-ETAG: 9cf48a1c544216461e127303ee6d5329; APT-DATE: 2017-10-30_10:34:15en-US
dc.identifier.urien
dc.descriptionPh.D.en
dc.description.abstractNoroviruses (NoV) are the prevailing cause of nonbacterial acute gastroenteritis worldwide and pose a significant financial burden on healthcare systems. The human NoV ORF1 encodes a 200 kDa polyprotein which is cleaved by the viral 20 kDa 3C-like protease (Pro, NS6) into 6 non-structural proteins necessary for viral replication. NoV ORF1 polyprotein is processed in a specific order with `early' sites (NS1/2-3, NS3-4) being cleaved rapidly, and three 'late' sites (NS4-5, NS5-6, NS6-7) processed subsequently and less efficiently. The mechanisms controlling polyprotein processing order have not been established and we sought to determine the factors controlling this process. We have bacterially expressed and purified GI.1 Pro, GII.4 Pro, and GII.4 ProPol and characterized their buffer, pH, ion, and detergent requirements. Using short, FRET peptides representing the 5 polyprotein cleavage sites of ORF1, we have determined the enzyme kinetics of Pro and ProPol which directly correlated with the observed processing order in vitro. Enzyme turnover, kcat, was the primary determinant of enzyme efficiency while binding affinity Km showed modest influence. We have demonstrated that ProPol is equivalent or superior to mature Pro in cleaving all ORF1 cleavage site peptides, implicating ProPol as a capable protease in the viral lifecycle. Previously, the core sequence of amino acids surrounding the scissile bonds responsible for governing the relative processing order had not been determined. Using both FRET-based peptides and full-length NoV polyprotein, we have successfully demonstrated the core sequences spanning positions P4-P2' surrounding the NS2-3, NS4-5, NS5-6, and NS6-7 cleavage sites contain all of the structural information necessary to control processing order in in vitro translation assays. We also provide insight into a previously overlooked role for the NS2-3 P3 residue in enzyme efficiency. Our models predict that the favorable electrostatic and hydrogen-bonding interactions formed between the side chains of the P3-Glu, Lys-162 of the protease, and P1-Gln residues promote the formation of a productive enzyme:transition-state complex, thus increasing enzyme turnover. Our work provides significant additional insight into understanding viral polyprotein processing and has important implications for improving the design of inhibitors targeting the NoV protease.en
dc.formatPDFen
dc.format.extent127 leavesen
dc.languageenen
dc.publisherGeorgetown Universityen
dc.sourceGeorgetown University-Graduate School of Arts & Sciencesen
dc.sourceMicrobiology & Immunologyen
dc.subjectNorovirusen
dc.subjectPolyprotein processingen
dc.subjectProteaseen
dc.subject.lcshVirologyen
dc.subject.lcshMicrobiologyen
dc.subject.lcshMolecular biologyen
dc.subject.otherVirologyen
dc.subject.otherMicrobiologyen
dc.subject.otherMolecular biologyen
dc.titleMECHANISMS CONTROLLING HUMAN NOROVIRUS POLYPROTEIN PROCESSINGen
dc.typethesisen


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