Retroviruses and long terminal repeat (LTR) retrotransposons comprise a family of mobile elements that encode reverse transcriptase and copy their RNA genome into a cDNA that is integrated into the host genome. These prolific genomic parasites constitute a significant fraction of virtually all eukaryotic genomes. Their success as parasites relies on an array of host functions, since retroviruses and retrotransposons have small genomes and complex modes of replication. Host factors that are required for retroelement replication are potential targets for small molecules to treat AIDS, yet few have been characterized thus far. The family of active Ty1 LTR-retrotransposons in the yeast, S. cerevisiae, provides a unique model system to explore the eukaryotic host-retrotransposon relationship. Using a synthetic genetic array screen and secondary molecular screens, my lab has identified 48 retrotransposition host factors (RHFs) that are required for accumulation of Ty1 cDNA. Many of these RHFs are global regulators of translation and mRNA localization and turnover, including ribosomal protein paralogs, ribosome biogenesis factors, a paralog of translation initiation factor eIF4G, nonsense-mediated decay proteins Upf1-Upf3, the activator of decapping, Dhh1, the decapping holoenzyme, Dcp1/Dcp2 and the 5'to 3'exoribonuclease, Xrn1. The RNA genome of Ty1, like that of retroviruses, functions as a template for both translation of virus- like particle (VLP) proteins and for reverse transcription within VLPs. We have recently found that eIF4G1 and two ribosomal protein paralogs are required for accumulation of TyA, the major capsid protein, while Xrn1 influences Ty1 RNA packaging into VLPs. Moreover, we have found that Ty1 RNA is translationally repressed in sucrose-dense ribonucleoprotein particles and that TyA is associated with cytoplasmic mRNA processing bodies (P bodies), where translational regulators and mRNA decay factors are concentrated. We propose to examine the mechanism of translational regulation of Ty1 RNA and determine whether this regulation plays a role in partitioning Ty1 RNA between translation and packaging in VLPs. In addition, we will examine the role of mRNA decay proteins in the formation of VLPs that are functional for reverse transcription.
The specific aims are: (1) to identify the steps in retrotransposition that are blocked in rhf mutants with reduced levels of Ty1 cDNA;(2) to examine the role of ribosomal protein paralogs, ribosome biogenesis factors and eIF4G1 in the translational regulation and localization of Ty1 RNA and protein;and, (3) to characterize Ty1 RNPs and determine whether mRNA decay factors influence Ty1 RNA or protein localization, RNA packaging in VLPs and/or reverse transcription. This project will elucidate specific mechanisms by which eukaryotic host factors promote the replication of retrotransposons and retroviruses.

Public Health Relevance

Retroviruses are a type of infectious virus that are the known or suspected causative agents in a variety of human diseases, including AIDS. This proposal uses a simple organism, budding yeast, to illuminate the role of cellular proteins that are necessary for replication of retroviruses in human cells. Cellular proteins that are required for retroviral replication are potential targets for the development of new drugs to treat AIDS.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM052072-17
Application #
8391697
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Janes, Daniel E
Project Start
1995-08-01
Project End
2014-11-30
Budget Start
2012-12-01
Budget End
2014-11-30
Support Year
17
Fiscal Year
2013
Total Cost
$309,168
Indirect Cost
$106,156
Name
Wadsworth Center
Department
Type
DUNS #
153695478
City
Menands
State
NY
Country
United States
Zip Code
12204
Bairwa, Narendra K; Mohanty, Bidyut K; Stamenova, Radostina et al. (2011) The intra-S phase checkpoint protein Tof1 collaborates with the helicase Rrm3 and the F-box protein Dia2 to maintain genome stability in Saccharomyces cerevisiae. J Biol Chem 286:2445-54
Dutko, James A; Kenny, Alison E; Gamache, Eric R et al. (2010) 5' to 3' mRNA decay factors colocalize with Ty1 gag and human APOBEC3G and promote Ty1 retrotransposition. J Virol 84:5052-66
Chalamcharla, Venkata R; Curcio, M Joan; Belfort, Marlene (2010) Nuclear expression of a group II intron is consistent with spliceosomal intron ancestry. Genes Dev 24:827-36
Beauregard, Arthur; Curcio, M Joan; Belfort, Marlene (2008) The take and give between retrotransposable elements and their hosts. Annu Rev Genet 42:587-617