Ty3 nucleocapsid controls localization of particle assembly

Area of Science:

• Molecular Biology
• Genetics
• Virology

Background:

• Budding yeast retrotransposon Ty3 produces virus-like particles (VLPs) and retrotransposes.
• The Ty3 Gag3 protein is processed into capsid, spacer, and nucleocapsid (NC) during VLP maturation.
• The Ty3 NC protein contains a zinc-binding motif crucial for retroviral NC proteins.

Purpose of the Study:

• Investigate the role of the Ty3 nucleocapsid (NC) domain in P-body clustering and VLP assembly.
• Determine how NC mutations affect Ty3 RNA packaging, protein localization, and particle formation.

Main Methods:

• Generated and analyzed 15 Ty3 NC Ala substitution and deletion mutants.
• Utilized transposition, immunoblot, RNA protection, cDNA synthesis, and multimerization assays.
• Characterized microscopic localization of Ty3 proteins and VLPs.

Main Results:

• Mutations in the zinc-binding motif abolished Ty3 RNA packaging.
• Disruption of conserved residues in the zinc-binding motif led to loss of P-body clustering and particle formation.
• NC domain mutations altered Gag3 nuclear localization and particle states.

Conclusions:

• The Ty3 NC domain mediates the formation of Ty3 RNA foci and association with P bodies.
• Ty3 assembly is linked to P-body components, with NC acting as a molecular switch.
• NC domain mutations impact Gag3 conformational states, affecting assembly and localization.