Retroviruses vary widely in their ability to cause neoplastic transformation or immunodeficiency. In addition, retroviruses are used as a backbone for constructing nonpathogenic vectors for gene transfer applications. However, in all cases, retroviruses recruit host cell proteins to achieve cytoplasmic expression of their unspliced genome-length RNA. We have identified sequences adjacent to the 5'cap of spleen necrosis virus (SNV) that facilitate Rev/Rev responsive element (RRE)-independent expression of HIV-1 unspliced reporter RNA. The RU5 region of the SNV long terminal repeat functions as a distinct position- and orientation-dependent cap-dependent translational enhancer of intron-containing HIV-1 gag RNA as well as nonviral luciferase (luc) RNA. Designated the SNV post-transcriptional control element (PCE). polysome analyses indicate that its functional mechanism is to stimulate translation initiation, although the PCE is not an internal ribosome entry sequence. Recently, we identified PCE activity in the 5'RNA terminus of two divergent retroviruses and in a cellular protooncogene mRNA. Our novel hypothesis is that 5'PCEs are a feature shared among divergent retroviruses and selected cellular mRNAs to achieve efficient translation in the face of multiple barriers to efficient cytoplasmic expression. Combined results of site-directed mutagenesis, RNA affinity chromatography and MALDI-TOF mass spectroscopy have determined redundant stem-loop motifs are necessary for PCE activity and that the structural features of the PCE present unpaired nucleotides for interaction with RNA helicase A (RHA). Knockdown of endogenous RHA by RNA silencing eliminates PCE translation stimulation and demonstrates that RHA is necessary for PCE activity. Our findings have generated the following essential questions: i) What conserved motifs necessaryfor translation stimulation are shared among retroviral PCEs? ii) What residues in RHA are necessary for interaction with the PCE and with translation factors or auxiliary proteins that mediate PCE activity? iii)What step of translation is stimulated? The overall goal of this proposal is to characterize the biochemical mechanism of PCE-RHA translational enhancement. Three integrated Specific Aims for this proposal are:1) to define conserved features in PCEs among divergent retroviruses;2) to define the domains of RNA helicase A necessary for PCE translational enhancement;and 3) to evaluate the function of the PCE-RHA interaction in translation initiation. Our results will illuminate a unique control mechanism of eukaryotic post-transcriptional gene expression and define virus-host interactions that are important for viral replication and progression to disease. Our new fundamental knowledge of translational control will define the process by which cellular mRNAs become committed to cytoplasmic expression and produce new strategies to optimize vector systems for diverse gene transfer applications.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA108882-05
Application #
7996196
Study Section
Virology - A Study Section (VIRA)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
2006-02-03
Project End
2012-12-31
Budget Start
2010-01-01
Budget End
2012-12-31
Support Year
5
Fiscal Year
2010
Total Cost
$193,069
Indirect Cost
Name
Ohio State University
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
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Jacob, Aishwarya G; O'Brien, Dennis; Singh, Ravi K et al. (2013) Stress-induced isoforms of MDM2 and MDM4 correlate with high-grade disease and an altered splicing network in pediatric rhabdomyosarcoma. Neoplasia 15:1049-63
Sharma, Amit; Boris-Lawrie, Kathleen (2012) Determination of host RNA helicases activity in viral replication. Methods Enzymol 511:405-35
Sharma, Amit; Yilmaz, Alper; Marsh, Kim et al. (2012) Thriving under stress: selective translation of HIV-1 structural protein mRNA during Vpr-mediated impairment of eIF4E translation activity. PLoS Pathog 8:e1002612
Ranji, Arnaz; Shkriabai, Nikolozi; Kvaratskhelia, Mamuka et al. (2011) Features of double-stranded RNA-binding domains of RNA helicase A are necessary for selective recognition and translation of complex mRNAs. J Biol Chem 286:5328-37
Hayes, Amy M; Qian, Shuiming; Yu, Lianbo et al. (2011) Tat RNA silencing suppressor activity contributes to perturbation of lymphocyte miRNA by HIV-1. Retrovirology 8:36
Jin, Jianyu; Jing, Wei; Lei, Xin-Xiang et al. (2011) Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes. Nucleic Acids Res 39:3724-34
Ranji, Arnaz; Boris-Lawrie, Kathleen (2010) RNA helicases: emerging roles in viral replication and the host innate response. RNA Biol 7:775-87
Bolinger, Cheryl; Sharma, Amit; Singh, Deepali et al. (2010) RNA helicase A modulates translation of HIV-1 and infectivity of progeny virions. Nucleic Acids Res 38:1686-96
Bolinger, Cheryl; Boris-Lawrie, Kathleen (2009) Mechanisms employed by retroviruses to exploit host factors for translational control of a complicated proteome. Retrovirology 6:8

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