Tao, Yizhi Jane2016-01-072016-01-072014-122015-01-15December 2Collier, Aaron Michael. "Initiation of RNA polymerization and polymerase encapsidation by a Picobirnavirus." (2015) Diss., Rice University. <a href="https://hdl.handle.net/1911/87757">https://hdl.handle.net/1911/87757</a>.https://hdl.handle.net/1911/87757During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How these RdRPs molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions that have highly variable answers depending on the virus family being examined. In this study, we have determined a 2.4 Å resolution structure of an RdRP from a human infecting strain of picobirnavirus (PBV). In addition to a conserved polymerase fold, the PBV RdRP possesses a unique, highly flexible 24-aa loop structure (aa495-518) located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays have shown that the wild-type RdRP is capable of initiating RNA synthesis using a de novo mechanism, while a mutant RdRP lacking the loop structure could only synthesize RNA through back-priming, suggesting that the loop likely functions as a platform for the priming nucleotide to bind. Unexpectedly, co-expression of the PBV RdRP with its respective capsid protein (CP) indicated that the PBV RdRP could not be incorporated into recombinant capsids in the absence of the viral genome. Additionally, the PBV RdRP exhibited a high affinity towards the conserved 5’-terminal sequence of the viral genome, suggesting that PBV RdRP molecules are likely packaged through their specific binding to viral RNA during assembly.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.double-stranded RNA virusRNA-dependent RNA polymerasevirus assemblypicobirnavirusgastroenteritisRNA replicationRNA transcriptioninitiation of RNA polymerizationback-primingterminal nucleotidyl transferaseThesis2016-01-07