This is an application for renewal of a grant to study to the mechanism and fidelity of nucleotide incorporation by the replicative polymerase of RNA viruses, the RNA-dependent RNA polymerase (RdRp). Over the past ten years, the world has witnessed the emergence of SARS, the spread of West Nile encephalitis, and the fear of a global flu pandemic. These diseases are caused by RNA viruses. In addition, the threat of intentional release of RNA viruses as weapons or agents of terror has increased substantially. The long-term goal of this research program is to develop strategies to treat and/or prevent RNA virus infection by targeting the RdRp. Our program has employed a prototypical RNA virus, poliovirus (PV), and its RdRp (3Dpol) as our model system. The previous funding period was devoted to the interrogation of the chemical mechanism for 3Dpol- catalyzed nucleotidyl transfer, elucidation of the structural basis for 3Dpol incorporation fidelity, and development of the tools to solve a crystal structure for 3Dpol in complex with primed template and nucleotide. We have made outstanding progress towards completion of all our aims. We have obtained new insight into the chemical mechanism for nucleotidyl transfer. We discovered a link between RdRp incorporation fidelity and pathogenesis. We discovered a connection between RdRp dynamics and incorporation fidelity. Together, our studies lead to the very provocative hypothesis that RdRp incorporation fidelity is a target for antiviral and vaccine development that will be elaborated during the next funding period. We will pursue the following specific aims: (1) Elucidate additional roles for the general acid in polymerase function;(2) Identify novel determinants and mechanisms of polymerase fidelity;and (3) Establish dynamics-function relationships for the RdRp.
RNA viruses represent an existing and emerging threat to US public health. Achievement of the goals of the application will provide novel targets and mechanisms for development of vaccines and inhibitors to prevent and to treat infections by RNA viruses.
|Woodman, Andrew; Arnold, Jamie J; Cameron, Craig E et al. (2016) Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination. Nucleic Acids Res 44:6883-95|
|Cameron, C E; Moustafa, I M; Arnold, J J (2016) Fidelity of Nucleotide Incorporation by the RNA-Dependent RNA Polymerase from Poliovirus. Enzymes 39:293-323|
|Lee, Cheri A; August, Avery; Arnold, Jamie J et al. (2016) Polymerase Mechanism-Based Method of Viral Attenuation. Methods Mol Biol 1349:83-104|
|Li, Sixing; Ding, Xiaoyun; Mao, Zhangming et al. (2015) Standing surface acoustic wave (SSAW)-based cell washing. Lab Chip 15:331-8|
|Van Slyke, Greta A; Arnold, Jamie J; Lugo, Alex J et al. (2015) Sequence-Specific Fidelity Alterations Associated with West Nile Virus Attenuation in Mosquitoes. PLoS Pathog 11:e1005009|
|van der Linden, Lonneke; Vives-AdriÃ¡n, Laia; Selisko, Barbara et al. (2015) The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family. PLoS Pathog 11:e1004733|
|Liu, Xinran; Musser, Derek M; Lee, Cheri A et al. (2015) Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase. Viruses 7:5571-86|
|Liu, Yen-Chin; Kuo, Rei-Lin; Lin, Jing-Yi et al. (2014) Cytoplasmic viral RNA-dependent RNA polymerase disrupts the intracellular splicing machinery by entering the nucleus and interfering with Prp8. PLoS Pathog 10:e1004199|
|Alphonse, SÃ©bastien; Arnold, Jamie J; Bhattacharya, Shibani et al. (2014) Cystoviral polymerase complex protein P7 uses its acidic C-terminal tail to regulate the RNA-directed RNA polymerase P2. J Mol Biol 426:2580-93|
|Zafar, Maroof K; Ketkar, Amit; Lodeiro, Maria F et al. (2014) Kinetic analysis of human PrimPol DNA polymerase activity reveals a generally error-prone enzyme capable of accurately bypassing 7,8-dihydro-8-oxo-2'-deoxyguanosine. Biochemistry 53:6584-94|
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