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Abstract
Ty1 is the most active long terminal repeat (LTR) retrotransposon in Saccharomyces cerevisiae and resembles retroviruses in genome organization and replication mechanisms. Ty1 encodes the capsid protein of virus-like particles (VLPs) called Gag and enzymes required for its protein processing, reverse transcription and integration. S. cerevisiae and its sister species S. paradoxus lack canonical transposition inhibition mechanisms like RNAi, yet maintain tight control over the Ty1 replication via the mechanism of copy number control (CNC). Work presented here focuses on the discovery of a new Ty1 protein named p22, which we show is both necessary and sufficient for CNC. This Ty1 restriction factor is encoded by a previously uncharacterized subgenomic Ty1 sense transcript termed Ty1i RNA. It initiates 800 bp downstream of Ty1 mRNA in GAG coding sequence and is translated in the samereading frame as GAG. Therefore, p22 shares protein sequence with the C-terminal half of Gag. Ty1i RNA and p22 are present in several wild type S. cerevisiae and S. paradoxus strains. Interestingly their levels increase in the cytoplasmic exoribonuclease xrn1 and Ty1 transcription factor spt3 mutants. Co-sedimentation analyses suggest that p22 associates with VLPs and co-immunoprecipitation of p22 and Gag suggests that this association is a result of p22 binding to Gag. This p22-VLP association leads to aberrant protein processing and abnormal VLP morphology as demonstrated by electron microscopy. However, the earliest step in the Ty1 life cycle that is affected by p22 is during formation of retrosomes, the cytoplasmic sites where VLP assembly takes place. Fluorescence in situ hybridization and immunoflouorescence experiments show that p22 disrupts retrosomes, perhaps leading to above mentioned defective VLPs. In summary, the work presented here focuses on the discovery of the novel Ty1 restriction factor p22 and how it alters Ty1 VLP structure and function, thereby bringing about Ty1 CNC.