The RNA genomes of important human pathogens such as poliovirus, hepatitis C and ebola virus are replicated by virally encoded RNA-dependent RNA polymerases (RdRp), an established anti-viral target. The molecular mechanisms of RdRp function will only be understood once we have both characterized its accessible structural states and delineated the transitions between these states as RdRp progresses through its catalytic cycle. This information would be critical for predicting fidelity-altering mutations that alter thi process, and/or for designing small molecule modulators of RdRp function that would interfere with the structural transitions. Crystal structures, by themselves, have been unable to capture the full range of structural rearrangements necessary for RdRp function, and give no information about the timescale of the conformational fluctuations. For example, active-site remote mutations that change RdRp fidelity and virus biology, do not lead to substantial structural differences, but rather, they change RdRp protein fluctuations over multiple timescales. In this grant application, we propose to use solution-state nuclear magnetic resonance (NMR) to watch the conformational rearrangements in an archetypal RdRp (in our case, from poliovirus) throughout its nucleotide addition cycle, contrast these conformational dynamics between wild-type and low/high fidelity-mutant RdRps, and delineate the molecular mechanisms of a novel class of nucleoside analogs, which include members under clinical trials. We predict that the fidelity-mutations and the nature of the incoming nucleotide ('correct' or 'incorrect' Watson-Crick base-pair) will change the kinetics and/or thermodynamics of structural rearrangements critical for RdRp function. We also propose that the fidelity-altering, remote-site mutations exert their effects through a long-range, amino acid network. We will delineate this network through kinetic and NMR studies of selected mutants, including mutants derived from the Sabin 1 vaccine strain (i.e. a clinically-used, orally bioavailable vaccine strain for poliovirus). We predict that RdRp mutations contribute to the Sabin attenuated phenotype through altering RdRp fidelity. Understanding the interactions for coordinating the structural rearrangements in RdRp will allow us to predict amino acid changes that interfere with these motions and alter polymerase function. Such mutations in RdRp would be predicted to change polymerase fidelity, and therefore may serve as the basis of novel vaccine strains. Small molecules may also be used to perturb the structural rearrangements in RdRps; our studies will serve as a basis for illuminating the poorly understood mechanisms of actions for these compounds. Structure and dynamics are highly conserved among RdRps, so these concepts will be applicable to RNA viruses in general.

Public Health Relevance

The RNA genomes of important human pathogens such as poliovirus, hepatitis C and ebola virus are replicated by virally encoded RNA-dependent RNA polymerases. Our proposal focuses on establishing relationships between polymerase function/fidelity and the structural rearrangements undergone by RNA-dependent RNA polymerases. These studies are important to human health because they will help establish new anti-viral strategies by (1) predicting polymerase mutations that change fidelity and therefore, can serve as a foundation towards the development of live, attenuated vaccine strains, and (2) illuminating the poorly understood mechanisms of action of small-molecule modulators of RdRp function, currently under clinical trials.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI104878-02
Application #
8892057
Study Section
Virology - A Study Section (VIRA)
Program Officer
Park, Eun-Chung
Project Start
2014-07-15
Project End
2018-06-30
Budget Start
2015-07-01
Budget End
2016-06-30
Support Year
2
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
003403953
City
University Park
State
PA
Country
United States
Zip Code
16802
Boehr, David D; D'Amico, Rebecca N; O'Rourke, Kathleen F (2018) Engineered control of enzyme structural dynamics and function. Protein Sci 27:825-838
Yang, Xiaorong; Liu, Xinran; Musser, Derek M et al. (2017) Triphosphate Reorientation of the Incoming Nucleotide as a Fidelity Checkpoint in Viral RNA-dependent RNA Polymerases. J Biol Chem 292:3810-3826
O'Rourke, Kathleen F; Gorman, Scott D; Boehr, David D (2016) Biophysical and computational methods to analyze amino acid interaction networks in proteins. Comput Struct Biotechnol J 14:245-51
Chan, Yan M; Moustafa, Ibrahim M; Arnold, Jamie J et al. (2016) Long-Range Communication between Different Functional Sites in the Picornaviral 3C Protein. Structure 24:509-517
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
van der Linden, Lonneke; Vives-Adrián, 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
Boehr, David D (2014) The ins and outs of viral RNA polymerase translocation. J Mol Biol 426:1373-6
Boehr, David D; Liu, Xinran; Yang, Xiaorong (2014) Targeting structural dynamics of the RNA-dependent RNA polymerase for anti-viral strategies. Curr Opin Virol 9:194-200
Moustafa, Ibrahim M; Korboukh, Victoria K; Arnold, Jamie J et al. (2014) Structural dynamics as a contributor to error-prone replication by an RNA-dependent RNA polymerase. J Biol Chem 289:36229-48
Liu, Xinran; Yang, Xiaorong; Lee, Cheri A et al. (2013) Vaccine-derived mutation in motif D of poliovirus RNA-dependent RNA polymerase lowers nucleotide incorporation fidelity. J Biol Chem 288:32753-65