All viruses must take over the host's protein synthesis (translation) machinery. Cellular mRNAs require a 5'cap and poly(A) tail to recruit the ribosome and initiate translation in a regulated manner. Many viral RNAs avoid this control step, and avoid host defenses, by lacking a 5'cap or poly(A) tail. Instead, many viral mRNAs harbor sequences in the untranslated regions (UTRs) that facilitate highly efficient cap-independent translation. Understanding how viruses do this could lead to development of antiviral agents that specifically target unique viral translation mechanisms. This knowledge could also allow exploitation of viruses as gene therapy vectors in humans, or as expression vectors to produce custom pharmaceutical polypeptides in plants. This proposal focuses on the novel cap-independent translation element (BTE) in the 3'UTR of barley yellow dwarf (BYDV) and other viral RNAs that facilitates translation initiation at the 5'end of the RNA. This process requires long-distance base pairing between the 5'and 3'UTRs. Our goal is to determine how the BTE recruits the translational machinery.
In Aim I we will determine the sequence and structural requirements of the BTE at high resolution by high volume mutagenesis, and translation in cell-free wheat germ extracts and in plant protoplasts.
In Aim II we will dissect the role and structural requirements of translation initiation factors eIF4G and eIF4E, and possibly other factors that are required for BTE-mediated translation. We will observe binding of mutant factors with the BTE RNA by a variety of RNA-protein interaction assays. The functions of mutant factors will be discerned by reconstituting factor-depleted cell-free extracts, and in cells depleted of factors by virus-induced gene silencing.
In Aim III, the mechanism of ribosome entry on the RNA will be investigated by sucrose gradient centrifugation of RNA-ribosome complexes, toeprinting, and other approaches. Throughout the project, the role of the BTE and its interactors in virus replication will be assessed. This research on a model virus and major plant pathogen may contribute to understanding picornaviruses (e.g. polio) that also employ cap-independent translation regulated by interactions between the UTRs, and nidoviruses (e.g. SARS) and flaviviruses (e.g. dengue, West Nile) that regulate gene expression and replication by long-distance RNA base pairing. Finally, the research will provide fundamental insight on eukaryotic translation mechanisms.

Public Health Relevance

All viruses must take over the host's protein synthesis (translation) machinery. Viruses of plants and animals share many common mechanisms for translation. We are using a plant virus as a small, easy-to-use model to investigate the mechanisms by which an RNA virus interacts with the host translational apparatus. This research may contribute to understanding mechanisms by which medically important viruses such as poliovirus, common cold rhinoviruses, the SARS virus, dengue virus, and West Nile viruses regulate gene expression and replication. Finally, the research will provide fundamental insight on translation mechanisms in cells of all higher organisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067104-08
Application #
8114222
Study Section
Virology - A Study Section (VIRA)
Program Officer
Bender, Michael T
Project Start
2003-07-01
Project End
2013-01-31
Budget Start
2011-08-01
Budget End
2013-01-31
Support Year
8
Fiscal Year
2011
Total Cost
$221,964
Indirect Cost
Name
Iowa State University
Department
Other Basic Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
005309844
City
Ames
State
IA
Country
United States
Zip Code
50011
Zhao, Pei; Liu, Qiao; Miller, W Allen et al. (2017) Eukaryotic translation initiation factor 4G (eIF4G) coordinates interactions with eIF4A, eIF4B, and eIF4E in binding and translation of the barley yellow dwarf virus 3' cap-independent translation element (BTE). J Biol Chem 292:5921-5931
Miras, Manuel; Miller, W Allen; Truniger, VerĂ³nica et al. (2017) Non-canonical Translation in Plant RNA Viruses. Front Plant Sci 8:494
Miller, W Allen; Shen, Ruizhong; Staplin, William et al. (2016) Noncoding RNAs of Plant Viruses and Viroids: Sponges of Host Translation and RNA Interference Machinery. Mol Plant Microbe Interact 29:156-64
Sharma, Sohani Das; Kraft, Jelena J; Miller, W Allen et al. (2015) Recruitment of the 40S ribosome subunit to the 3'-untranslated region (UTR) of a viral mRNA, via the eIF4 complex, facilitates cap-independent translation. J Biol Chem 290:11268-81
Miller, W Allen; Jackson, Jacquelyn; Feng, Ying (2015) Cis- and trans-regulation of luteovirus gene expression by the 3' end of the viral genome. Virus Res 206:37-45
Smirnova, Ekaterina; Firth, Andrew E; Miller, W Allen et al. (2015) Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement. PLoS Pathog 11:e1004868
Miras, Manuel; Sempere, Raquel N; Kraft, Jelena J et al. (2014) Interfamilial recombination between viruses led to acquisition of a novel translation-enhancing RNA element that allows resistance breaking. New Phytol 202:233-46
Simon, Anne E; Miller, W Allen (2013) 3' cap-independent translation enhancers of plant viruses. Annu Rev Microbiol 67:21-42
Kraft, Jelena J; Treder, Krzysztof; Peterson, Mariko S et al. (2013) Cation-dependent folding of 3' cap-independent translation elements facilitates interaction of a 17-nucleotide conserved sequence with eIF4G. Nucleic Acids Res 41:3398-413
Fan, Qiuling; Treder, Krzysztof; Miller, W Allen (2012) Untranslated regions of diverse plant viral RNAs vary greatly in translation enhancement efficiency. BMC Biotechnol 12:22

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