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 #
3R01CA048746-23S1
Application #
8665524
Study Section
Virology - B Study Section (VIRB)
Program Officer
Ogunbiyi, Peter
Project Start
1988-12-01
Project End
2015-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
23
Fiscal Year
2013
Total Cost
$66,065
Indirect Cost
$21,787
Name
Johns Hopkins University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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Justice 4th, James; Malhotra, Sanandan; Ruano, Miguel et al. (2015) The MET gene is a common integration target in avian leukosis virus subgroup J-induced chicken hemangiomas. J Virol 89:4712-9
Justice 4th, James F; Morgan, Robin W; Beemon, Karen L (2015) Common Viral Integration Sites Identified in Avian Leukosis Virus-Induced B-Cell Lymphomas. MBio 6:e01863-15
Malhotra, Sanandan; Justice 4th, James; Lee, Nathan et al. (2015) Complete genome sequence of an american avian leukosis virus subgroup j isolate that causes hemangiomas and myeloid leukosis. Genome Announc 3:
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
Bolisetty, Mohan; Blomberg, Jonas; Benachenhou, Farid et al. (2012) Unexpected diversity and expression of avian endogenous retroviruses. MBio 3:e00344-12
Withers, Johanna B; Ashvetiya, Tamara; Beemon, Karen L (2012) Exclusion of exon 2 is a common mRNA splice variant of primate telomerase reverse transcriptases. PLoS One 7:e48016
Withers, Johanna B; Beemon, Karen L (2011) The structure and function of the rous sarcoma virus RNA stability element. J Cell Biochem 112:3085-92

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