This is an application for renewal of a grant to study picornavirus genome replication. Picornaviruses represent an existing and emerging threat to US public health. Although protein factors and genetic elements required for picornavirus genome replication are known and appear to be conserved, a clear understanding of the mechanisms employed to produce picornaviral RNA is lacking. The long-term objective of this program is to reconstitute picornavirus genome replication in vitro from purified components. We have achieved all of the major objectives of the previous funding period. In addition, we have solved the first crystal structure for a picornaviral 3CD protein, developed the technology to study 3C-RNA interactions by using nuclear magnetic resonance spectroscopy, and discovered that the 3CD protein has both pre- and post-replication functions. During the next funding period, we will continue our studies of picornavirus genome replication as well as explore our newly discovered function for 3CD by pursuing the following specific aims: (1) Define the mechanism of assembly and structural organization of the picornavirus VPg uridylylation complex by using molecular genetic, biochemical and biophysical approaches;(2) Define the molecular basis for sequence- and structure-specific RNA recognition by 3C by using nuclear magnetic resonance spectroscopy;and (3) Elucidate the function of 3CD in formation of replication complexes.

Public Health Relevance

Picornaviruses 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 inhibitors to treat infections by picornaviruses, especially those for which vaccines are not available.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Study Section
Special Emphasis Panel (ZRG1-GGG-A (90))
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Park, Eun-Chung
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Pennsylvania State University
Schools of Arts and Sciences
University Park
United States
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Kolli, Swapna; Meng, Xiangzhi; Wu, Xiang et al. (2015) Structure-function analysis of vaccinia virus H7 protein reveals a novel phosphoinositide binding fold essential for poxvirus replication. J Virol 89:2209-19
Hsu, Nai-Yun; Ilnytska, Olha; Belov, Georgiy et al. (2010) Viral reorganization of the secretory pathway generates distinct organelles for RNA replication. Cell 141:799-811
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Shen, Miaoqing; Reitman, Zachary J; Zhao, Yan et al. (2008) Picornavirus genome replication. Identification of the surface of the poliovirus (PV) 3C dimer that interacts with PV 3Dpol during VPg uridylylation and construction of a structural model for the PV 3C2-3Dpol complex. J Biol Chem 283:875-88
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Marcotte, Laura L; Wass, Amanda B; Gohara, David W et al. (2007) Crystal structure of poliovirus 3CD protein: virally encoded protease and precursor to the RNA-dependent RNA polymerase. J Virol 81:3583-96
Korneeva, Victoria S; Cameron, Craig E (2007) Structure-function relationships of the viral RNA-dependent RNA polymerase: fidelity, replication speed, and initiation mechanism determined by a residue in the ribose-binding pocket. J Biol Chem 282:16135-45
Shen, Miaoqing; Wang, Qixin; Yang, Yan et al. (2007) Human rhinovirus type 14 gain-of-function mutants for oriI utilization define residues of 3C(D) and 3Dpol that contribute to assembly and stability of the picornavirus VPg uridylylation complex. J Virol 81:12485-95
Pathak, Harsh B; Arnold, Jamie J; Wiegand, Phillip N et al. (2007) Picornavirus genome replication: assembly and organization of the VPg uridylylation ribonucleoprotein (initiation) complex. J Biol Chem 282:16202-13
van Ooij, Mark J M; Vogt, Dorothee A; Paul, Aniko et al. (2006) Structural and functional characterization of the coxsackievirus B3 CRE(2C): role of CRE(2C) in negative- and positive-strand RNA synthesis. J Gen Virol 87:103-13

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