The influenza polymerase is a complex of three subunits, polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1) and polymerase acidic protein (PA). It associates with the viral RNA segment and nucleoprotein (NP) to form a viral ribonucleoprotein (vRNP) complex which is important for transcription and replication of the viral genome. Concurrently, the previous three influenza pandemics viruses contain reassorted vRNP of different origins. This leads to the aim of study to investigate the role of polymerase in the pandemic viruses. By reconstitution of vRNPs in human cells, it was demonstrated that vRNPs of H2N2 and H3N2 pandemic viruses had higher polymerase activity than the H2N2 seasonal viruses in-between them. The recombinant virus with H2N2 pandemic vRNP also showed faster growth kinetics in the early stage of viral replication and better adaptability to the selective environment with neuraminidase inhibitor than the recombinant virus with H2N2 seasonal vRNP, which had a lower polymerase activity. Reconstitution of chimeric vRNPs of H2N2 pandemic and seasonal viruses revealed that PB2, PB1 and PA were responsible for the difference in polymerase activity between them. Five residues, one in PB2, three in PB1 and one in PA were identified to be significant for the polymerase activity change. These polymerase subunits and residues may act as part of the determining factors for the H2N2 pandemic virus. Furthermore, PB2-627 has been shown to have stringent host specificity and affect polymerase activity and viral replication. Recombinant viruses in mammalian and avian cells with random mutation were generated at this position. It showed that the amino acids at this position are not restricted to those appear in the nature for generating viable viruses. It was also observed that the avian-derived viruses generally had lower polymerase activity and reduced growth kinetics in mammalian cells, while part of the mammalian-derived viruses had lower polymerase activity and reduced growth kinetics in avian cells. This consolidated the role of PB2-627 on host specificity and demonstrated the possibility of some novel amino acids for this position, which may play a role in the future influenza pandemic. The 2009 H1N1 pandemic virus contains a reassorted vRNP with subunits of avian, human and swine origins. This prompts me to compare the polymerase activity of all the 81 possible combinations of chimeric vRNPs of three different origins. The results were statistically analyzed and several single subunit factors and interactions between vRNP subunits were identified to significantly affect the polymerase activity. In order to reduce the effort and resources required, a fractional factorial design of 27 experimental runs was developed to substitute the 81-combination full factorial design for identifying the significant single subunit factors that affect the polymerase activity. Overall, this study identified some factors that may contribute to a pandemic virus and allows us to have better understanding of the role of polymerase in a pandemic virus. These findings may contribute to evaluating the pandemic potential of the novel virus that emerges or may emerge in the nature and enhances the preparedness towards the next pandemic influenza. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

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Thesis (Ph.D.) -- McMaster University, 1998. / Includes bibliographical references (leaves 101-120). Also available via World Wide Web.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xvii, 179 p.; also includes graphics. Includes bibliographical references (p. 155-179). Available online via OhioLINK's ETD Center

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, August 1997. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2005. / "A dissertation in pharmaceutical sciences and chemistry." Advisor: Simon H. Friedman. Typescript. Vita. Description based on contents viewed June 23, 2006; title from "catalog record" of the print edition. Includes bibliographical references (leaves 327-353). Online version of the print edition.

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RNA polymerase II (RNAP II) is responsible for transcribing messenger RNAs (mRNAs) as well as non-coding RNAs such as small nuclear RNAs (snRNAs) and microRNAs in eukaryotic cells. Rpb1, the largest catalytic subunit of this complex, possesses a unique C-Terminal Domain (CTD) that consists of tandem heptad repeats (the number varying from 26 to 52 by organism) with the consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Y1S2P3T4S5P6S7). The CTD is extensively phosphorylated and dephosphorylated on non-proline residues during different steps of the transcription cycle, with roles for the threonine (Thr4) and tyrosine (Tyr1) attracting more attention. For example, in chicken cells, Thr4 functions in histone mRNA 3’ end formation, and Tyr1 phosphorylation is primarily associated with promoters and upstream antisense RNA formation, as well as preventing degradation of the polymerase, processes not found across all eukaryotes. A detailed introduction is described in Chapter 1. Taking advantage of the genetic tractability of yeast cells, we created a yeast (S. cerevisiae) strain with all CTD threonines substituted with valines (T4V) to study the role of CTD Thr4 in transcription in yeast, which prior to this study has been poorly characterized in S. cerevisiae. Using the T4V strain, we found that Thr4 was required for proper transcription of phosphate-regulated (PHO) and galactose-inducible (GAL) genes. We found genetic links between the T4V polymerase and genes encoding subunits of the Swr1 and Ino80 chromatin remodeling complexes, as well as the histone variant Htz1. We further provide evidence that CTD Thr4 is required for proper eviction of Htz1 by the Ino80 complex from genes requiring Thr4 for activation, presented in Chapter 2 of this thesis. Finally, Chapter 3 describes the functions of CTD Tyr1 in S. cerevisiae. Using a strategy similar to the T4V strain, I created a strain expressing an endogenous Rpb1 with all CTD tyrosine residues mutated to phenylalanine (Y1F). We found that this strain was viable, but with a severe slow-growth phenotype. We found genetic links between the Y1F polymerase and kinase/cyclin pair Srb10/Srb11, as well as an increase in occupancy on chromatin for the same. Further analysis indicated that RNA levels of genes associated with MAP Kinase associated stressors were dysregulated, and poly(A) site selection was biased towards distal poly(A) sites. Next, using an in vitro kinase assay, we showed Tyr1 phosphorylation on the CTD by MAP kinase Slt2, and in vivo CTD Tyr1 phosphorylation levels changed based on Slt2-associated stress response, as well as a decrease in in vivo Tyr1P-RNAP II from an Slt2 kinase-dead strain. Analysis of termination factors Nrd1 and Rtt103 showed transcription termination defects were likely the result of disruption of the interaction between the CTD interacting domains of these two proteins and the Y1F CTD. Extending this, we found additional disruptions in Slt2 recruitment to chromatin, increasing the depth of our knowledge of the interplay between induction of stress-associated genes, Slt2 function, and Nrd1-mediated termination.

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