Positive-strand RNA viruses are serious pathogens causing encephalitis, hemorrhagic fever and hepatitis in humans and animals and devastating crop losses in plants. Despite extensive studies, replication of these viruses remains poorly understood. A major stumbling block in efforts to fully understand virus replication is the large size of viral genomes, which complicates efforts to link RNA structure with RNA function. We have discovered that a novel conformational switch activates (-)-strand synthesis in satC (356 nt) associated with the model virus Turnip crinkle (TCV;4054 nt). Since satC contains all sequences and structures necessary for replication by the TCV RdRp, studying its replication in the past has provided significant information subsequently found to be applicable to much larger viral genomes, including those that cause significant diseases in humans and animals. Analyses of satC and TCV replication elements has revealed astonishing complexities and differences in how satC and TCV use nearly identical sequences to replicate their genomes, which has important implications for interpretation of results using subviral RNA replicons. In this proposal, we will use biophysical and genetic approaches to define secondary and tertiary interactions that characterize the satC pre-active structure and structural transitions of wt and mutant satC. Full length and selected satC fragments will be analyzed by temperature gradient gel electrophoresis, UV melting curves, oligonucleotide accessibility and UV cross-linking. Site-specific mutagenesis and in vivo genetic selection (selex) will help define individual elements and the relationship between elements. We will also use mutagenesis approaches combined with RdRp binding analyses to explore TCV sequences that are uniquely important for (-)-strand synthesis although also found in satC. Finally, we will examine RdRp binding to specific satC and TCV hairpins and determine if elements that flank one satC hairpin affect satC replication while simultaneously interfering with TCV replication and repressing virion accumulation function through an interaction between the two viral RNAs. Successful completion of these experiments will provide new paradigms for virus replication and interactions between helper viruses and associated subviral RNAs and provide the most detailed understanding of RNA conformational switches and the steps that lead to initiation of (-)-strand synthesis for any RNA virus.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM061515-07S1
Application #
7904441
Study Section
Virology - A Study Section (VIRA)
Program Officer
Santangelo, George M
Project Start
2009-08-31
Project End
2011-12-31
Budget Start
2009-08-31
Budget End
2011-12-31
Support Year
7
Fiscal Year
2009
Total Cost
$309,971
Indirect Cost
Name
University of Maryland College Park
Department
Anatomy/Cell Biology
Type
Schools of Earth Sciences/Natur
DUNS #
790934285
City
College Park
State
MD
Country
United States
Zip Code
20742
Simon, Anne E; Miller, W Allen (2013) 3' cap-independent translation enhancers of plant viruses. Annu Rev Microbiol 67:21-42
Gao, Feng; Gulay, Suna P; Kasprzak, Wojciech et al. (2013) The kissing-loop T-shaped structure translational enhancer of Pea enation mosaic virus can bind simultaneously to ribosomes and a 5' proximal hairpin. J Virol 87:11987-2002
Yuan, Xuefeng; Shi, Kerong; Simon, Anne E (2012) A local, interactive network of 3' RNA elements supports translation and replication of Turnip crinkle virus. J Virol 86:4065-81
Gao, Feng; Kasprzak, Wojciech; Stupina, Vera A et al. (2012) A ribosome-binding, 3' translational enhancer has a T-shaped structure and engages in a long-distance RNA-RNA interaction. J Virol 86:9828-42
Stupina, Vera A; Yuan, Xuefeng; Meskauskas, Arturas et al. (2011) Ribosome binding to a 5' translational enhancer is altered in the presence of the 3' untranslated region in cap-independent translation of turnip crinkle virus. J Virol 85:4638-53
Chattopadhyay, Maitreyi; Shi, Kerong; Yuan, Xuefeng et al. (2011) Long-distance kissing loop interactions between a 3' proximal Y-shaped structure and apical loops of 5' hairpins enhance translation of Saguaro cactus virus. Virology 417:113-25
Guo, Rong; Meskauskas, Arturas; Dinman, Jonathan D et al. (2011) Evolution of a helper virus-derived, ribosome binding translational enhancer in an untranslated satellite RNA of Turnip crinkle virus. Virology 419:10-6
Yuan, Xuefeng; Shi, Kerong; Young, Megan Y L et al. (2010) The terminal loop of a 3' proximal hairpin plays a critical role in replication and the structure of the 3' region of Turnip crinkle virus. Virology 402:271-80
Zuo, Xiaobing; Wang, Jinbu; Yu, Ping et al. (2010) Solution structure of the cap-independent translational enhancer and ribosome-binding element in the 3' UTR of turnip crinkle virus. Proc Natl Acad Sci U S A 107:1385-90
Yuan, Xuefeng; Shi, Kerong; Meskauskas, Arturas et al. (2009) The 3' end of Turnip crinkle virus contains a highly interactive structure including a translational enhancer that is disrupted by binding to the RNA-dependent RNA polymerase. RNA 15:1849-64

Showing the most recent 10 out of 26 publications