Ty3 Capsid Mutations Reveal Early and Late Functions of the Amino-Terminal Domain

Abstract

The Ty3 retrotransposon assembles into 50-nm virus-like particles that occur in large intracellular clusters in the case of wild-type (wt) Ty3. Within these particles, maturation of the Gag3 and Gag3-Pol3 polyproteins by Ty3 protease produces the structural proteins capsid (CA), spacer, and nucleocapsid. Secondary and tertiary structure predictions showed that, like retroviral CA, Ty3 CA contains a large amount of helical structure arranged in amino-terminal and carboxyl-terminal bundles. Twenty-six mutants in which alanines were substituted for native residues were used to study CA subdomain functions. Transposition was measured, and particle morphogenesis and localization were characterized by analysis of protein processing, cDNA production, genomic RNA protection, and sedimentation and by fluorescence and electron microscopy. These measures defined five groups of mutants. Proteins from each group could be sedimented in a large complex. Mutations in the amino-terminal domain reduced the formation of fluorescent Ty3 protein foci. In at least one major homology region mutant, Ty3 protein concentrated in foci but no wt clusters of particles were observed. One mutation in the carboxyl-terminal domain shifted assembly from spherical particles to long filaments. Two mutants formed foci separate from P bodies, the proposed sites of assembly, and formed defective particles. P-body association was therefore found to be not necessary for assembly but correlated with the production of functional particles. One mutation in the amino terminus blocked transposition after cDNA synthesis. Our data suggest that Ty3 proteins are concentrated first, assembly associated with P bodies occurs, 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.

Ty3, a Position-specific Retrotransposon in Budding Yeast

“…Although specific contacts for Gag3 subdomains in uncoating have not been identified, mutations in CA NTD and SP block retrotransposition subsequent to cDNA synthesis (56,58). This phenotype is consistent with an uncoating defect.…”

“…Assembly was particularly sensitive to disruption of the N-terminal 100 aa (58). Only a few individual mutations completely abrogated particle formation and in one case this was accompanied by appearance of extensive cytoplasmic filaments.…”

“…Only a few individual mutations completely abrogated particle formation and in one case this was accompanied by appearance of extensive cytoplasmic filaments. Visualization with RFP and GFP fusions showed that these filaments contained Gag3 and at least two components of processing bodies (PB) (58). Two-hybrid analysis showed that interactions occur between the NTD and NTD, as well as between the NTD and CTD and that introduction of mutations with effects on assembly disrupted these interactions (45,58).…”

“…The simplest interpretation is that the VLP shell not only triggers processing to activate protein domain functions, but also selectively sequesters, concentrates and protects enzymes and substrates. Ty3 PR and RT active site mutants, and WT VLP populations have been characterized by transmission electron microscopy and atomic force microscopy (44,(56)(57)(58). Immature VLPs generated from Ty3 PR active site mutants are relatively uniform, and appear roughly spherical with diameters measured by atomic force microscopy of 44 to 53 nm.…”

“…They share N-terminal domain (NTD) and C-terminal domain (CTD) bundles of alpha helices (61,62) with an intervening linker. Modeling suggests that, similar to retroviral CA domains, the CA domain of Ty3 Gag3 forms alpha helical bundles with the NTD contributing the outer surface of the VLP (58). The N-terminal 20-aa of Ty3 Gag3 contains a late domain motif (YPXL).…”