| Ty3, a yeast retrotransposon, encodes homologs of retroviral Gag and Gag-Pol proteins, which, together with genomic RNA, assemble into virus-like particles (VLPs). Using atomic force microscopy, VLPs, imaged from cells producing wild-type and protease and reverse transcriptase mutant Ty3, were used to investigate whether proteolytic processing results in a dramatic change from immature particle to mature particle in morphology as occurs in lentiviruses, such as human immunodeficiency virus. Wild-type VLPs were found to have icosahedral symmetry features. Particle morphologies of protease and reverse transcriptase mutants were indistinguishable, suggesting Ty3 VLPs do not undergo major external rearrangements during proteolytic maturation. An Ala-scanning mutagenesis strategy was undertaken to study the Gag3 subdomain function. Mutagenesis study of the CA mutants suggested that Ty3 protein assembly occurs associated with P-bodies, and particle morphogenesis concludes with a post reverse transcription, CA-dependent step. Particle formation was generally resistant to localized substitutions, possibly indicating that multiple domains are involved. A follow-up study by yeast two-hybrid assay indicated that the Ty3 CA NTD contains a self interaction domain. Ty3 CA CTD can also influence the Gag3 interaction. We identified two interesting mutants from the mutagensis study of CA YPXL sequence, located at Ty3 CA extreme amino terminal domain. The K15A mutant had highly elevated transposition but wild-type Gag3 processing and cDNA synthesis. Fluorescence microscopy and electron microscopy showed that the K15A mutant formed much smaller clusters than wild type and has particles dispersed in the cytoplasm. The L16A mutant formed Ty3 protein clusters, but no particles. The phenotypes of these two mutants suggested that the Ty3 extreme N-terminal domain plays a very important role in cluster and particle formation. Ty3 Λp3 mutant formed particles, produced cDNA, but decreased in transposition. The phosphorylation candidate Ser residues in p3 were substituted with Ala. Mutation S218A had no effects on Ty3 transposition. However, S222A had deleterious effects on Ty3. An addition of S218A mutation to this mutant converted the deleterious phenotype back to wild type. Taken together, p3 may play a regulatory role in Ty3 life cycle. These results provide insights into structural domains that may influence intracellular assembly of retrotransposons, endogenous retroviruses, and retroviruses. |
