The function of the Gag protein during the process of normal and restricted Ty1 retrotransposition in Saccharomyces
Tucker, Jessica Mitchell
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The long terminal repeat (LTR) retrotransposon Ty1 of the budding yeast Saccharomyces is a mobile genetic element whose life cycle resembles retroviral replication. Ty1 elements contain GAG and POL genes, which are translated to produce Gag, the structural protein of virus-like particles, integrase, reverse transcriptase, and protease. This work focuses on the molecular interactions between host and retrotransposon gene products during the Ty1 life cycle in order to increase our understanding of Ty1 genomic RNA dynamics prior to VLP assembly and restriction of retrotransposon amplification in the host genome. Ty1 genome-length sense RNA serves as the template for reverse transcription and protein production in the cytoplasm. In the absence of Gag, Ty1 genomic RNA is not efficiently exported from the nucleus and is less stable in the cytoplasm, where it is degraded in the processing body. Ectopic Gag expression restores nuclear-cytoplasmic trafficking and stability of Ty1 genomic RNA, as well as virus-like particle assembly foci called retrosomes. Interestingly, the formation of retrosomes and virus-like particles are also disrupted in cells with elevated genomic copy numbers of Ty1 via an intrinsic restriction mechanism referred to as copy number control. High Ty1 copy number results in a dramatic decrease in Ty1 mobility and is mediated by a newly discovered 22 kilodalton protein (p22), which is translated from a subgenomic Ty1 sense RNA and shares sequence with the C-terminal half of the Gag protein. p22 has a trans-dominant negative effect on Ty1 retrotransposition and is both necessary and sufficient for copy number control (CNC). p22 inhibits Ty1 by interaction with Gag and its expression leads to disrupted retrosomes and lower yields of properly assembled and maturation-competent virus-like particles. Mutations in Gag cause resistance to CNC via the exclusion of p22 from assembled virus-like particles, indicating that the presence of p22 in virus-like particles is inhibitory to Ty1 replication. By studying the role of Ty1 Gag in the retrotransposon life cycle, the goal of this work is to inform future studies regarding retroelement replication and restriction in a variety of eukaryotes.