Two-hybrid analysis of Ty3 capsid subdomain interactions

Two-hybrid analysis was used to understand the interactions that contribute to particle assembly. Gag3 interacted with itself as predicted based on its role as the major structural protein. The N-terminal subdomain (NTD) of the capsid was able to interact with itself and with the C-terminal subdomain (CTD) of the capsid, but interacted less well with intact Gag3. Mutations previously shown to block particle assembly disrupted Gag3 interactions more than subdomain interactions.The findings that the NTD interacts with itself and with the CTD are consistent with previous modeling and a role similar to that of the capsid in retrovirus particle structure. These results are consistent with a model in which the Gag3-Gag3 interactions that initiate assembly differ from the subdomain interactions that potentially underlie particle stability.The Ty3 retrotransposon in budding yeast forms virus-like particles (VLPs) comprised of precursor Gag3 and Gag3-Pol3 polyproteins [1,2]. Previous alanine-scanning mutagenesis indicated that the N-terminal domain (NTD) of the structural polyprotein Gag3 plays an important role in VLP formation [3]. During maturation, Gag3 is processed into 24 kDa capsid (CA), 27 kDa CA-spacer (SP), 3 kDa SP, and 7 kDa nucleocapsid (NC) protein by the Ty3 protease. Unlike most retrovirus cores, these cytoplasmic particles remain stable after proteolytic maturation.Two-hybrid analysis [4] was used to better understand the contributions of Gag3 subdomains to formation and stability of the Ty3 VLP. Fusions of Gag3 and derivatives to the C-terminus of the Gal4-BD tagged with c-Myc were expressed from the high-copy, TRP1-marked pGBK vector (Clontech, Palo Alto, CA, USA). Fusions of Gag3 and derivatives to the C-terminus of the Gal4-AD tagged with HA were expressed from the LEU2-marked high-copy plasmid pGAD T7 (pGAD). These fusions were constructed by amplifying the appropriate regions from Ty3 Gag3 subclones in pGEM (Invitrogen, Carlsbad, CA, USA) using polymer