All retroviruses generate a full-length 7-9 kb primary RNA transcript, which is the unspliced mRNA for Gag and Gag-Pol polyproteins and the genomic RNA that is packaged into viral particles. This unspliced RNA is normally quite stable. However, a premature termination codon in the gag gene triggers nonsense-mediated mRNA decay (NMD) that rapidly degrades the RNA upon translation. This old result has become surprising in light of current popular models that propose that a downstream intronic sequence, marked during splicing by an exon-junction complex, is necessary to distinguish premature from natural termination codons in higher eukaryotes. In addition, Rous sarcoma virus, a simple avian retrovirus, has a 400 nt cis-acting stability element, which is located immediately downstream of the gag termination codon. Deletions in this 3'UTR region cause the RNA to undergo NMD, forcing the natural gag termination codon to be recognized as a premature termination codon by the cellular machinery. This suggests that the downstream context is important in determining proper translation termination and RNA stability. The stability element will also stabilize a PTC in gag, when inserted directly downstream of it. This stability element may be especially important for retroviral RNAs, since they have an unusually long 3'untranslated region, which usually destabilizes mRNA. In this proposal, both the mechanism of NMD and of immunity to NMD in retroviral RNA will be investigated. Differences in RNA structure or RNP composition are hypothesized to distinguish the coding region (faux UTR) from the 3'UTR downstream from the natural termination codon. Initial studies will be carried out with RSV but extended to a number of other retroviruses, including HIV-1. Intracellular sites of viral RNA decay will also be investigated.

Public Health Relevance

This proposal is focused on the basic gene expression mechanisms that discriminate natural stop codons from premature stop codons, which are subject to nonsense- mediated mRNA decay. It also explores a novel retroviral RNA element, which makes the viral RNA immune to this decay. Endogenous human retroviruses are over- expressed when this decay process is inhibited, suggesting it is important for viral RNA regulation. Further, the retroviral stability element may be important for design of improved retroviral vectors for gene therapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA048746-22
Application #
8258349
Study Section
Virology - B Study Section (VIRB)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
1988-12-01
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
22
Fiscal Year
2012
Total Cost
$328,590
Indirect Cost
$128,230
Name
Johns Hopkins University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Quek, Bao Lin; Beemon, Karen (2014) Retroviral strategy to stabilize viral RNA. Curr Opin Microbiol 18:78-82
Justice 4th, James; Beemon, Karen L (2013) Avian retroviral replication. Curr Opin Virol 3:664-9
Leblanc, Jason; Weil, Jason; Beemon, Karen (2013) Posttranscriptional regulation of retroviral gene expression: primary RNA transcripts play three roles as pre-mRNA, mRNA, and genomic RNA. Wiley Interdiscip Rev RNA 4:567-80
Withers, Johanna B; Beemon, Karen L (2010) Structural features in the Rous sarcoma virus RNA stability element are necessary for sensing the correct termination codon. Retrovirology 7:65
Weil, Jason E; Hadjithomas, Michalis; Beemon, Karen L (2009) Structural characterization of the Rous sarcoma virus RNA stability element. J Virol 83:2119-29
Yang, Feng; Xian, Rena R; Li, Yingying et al. (2007) Telomerase reverse transcriptase expression elevated by avian leukosis virus integration in B cell lymphomas. Proc Natl Acad Sci U S A 104:18952-7
LeBlanc, Jason J; Uddowla, Sabena; Abraham, Benjamin et al. (2007) Tap and Dbp5, but not Gag, are involved in DR-mediated nuclear export of unspliced Rous sarcoma virus RNA. Virology 363:376-86
Weil, Jason E; Beemon, Karen L (2006) A 3' UTR sequence stabilizes termination codons in the unspliced RNA of Rous sarcoma virus. RNA 12:102-10
LeBlanc, Jason J; Beemon, Karen L (2004) Unspliced Rous sarcoma virus genomic RNAs are translated and subjected to nonsense-mediated mRNA decay before packaging. J Virol 78:5139-46
Giles, Keith E; Caputi, Massimo; Beemon, Karen L (2004) Packaging and reverse transcription of snRNAs by retroviruses may generate pseudogenes. RNA 10:299-307

Showing the most recent 10 out of 28 publications