The yeast retrotransposon Ty3 forms stable virus-like particles. Gag3, the major structural protein, is composed of capsid, spacer and nucleocapsid domains. The capsid domain of Gag3 was previously modeled as a structure similar to retrovirus capsid.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.

Retroviruses and retrotransposons package genomic RNA into virus-like particles (VLPs) in a poorly understood process. Expression of the budding yeast retrotransposon Ty3 results in the formation of cytoplasmic Ty3 VLP assembly foci comprised of Ty3 RNA and proteins, and cellular factors associated with RNA processing body (PB) components, which modulate translation and effect nonsense-mediated decay (NMD). A series of Ty3 RNA variants were tested to understand the effects of read-through translation via programmed frameshifting on RNA localization and packaging into VLPs, and to identify the roles of coding and non-coding sequences in those processes. These experiments showed that a low level of read-through translation of the downstream open reading frame (as opposed to no translation or translation without frameshifting) is important for localization of full-length Ty3 RNA to foci. Ty3 RNA variants associated with PB components via independent determinants in the native Ty3 untranslated regions (UTRs) and in GAG3-POL3 sequences flanked by UTRs adapted from non-Ty3 transcripts. However, despite localization, RNAs containing GAG3-POL3 but lacking Ty3 UTRs were not packaged efficiently. Surprisingly, sequences within Ty3 UTRs, which bind the initiator tRNAMet proposed to provide the dimerization interface, were not required for packaging of full-length Ty3 RNA into VLPs. In summary, our results demonstrate that Gag3 is sufficient and required for localization and packaging of RNAs containing Ty3 UTRs and support a role for POL3 sequences, translation of which is attenuated by programmed frameshifting, in both localization and packaging of the Ty3 full-length gRNA.Highlights► Ty3 UTRs or GAG3-POL3 mediate RNA localization to Ty3 assembly foci. ► The Ty3 5′ UTR is sufficient for packaging of Ty3 GAG3 or GAG3-POL3 RNA into VLPs. ► The 5′–3′-bipartite PBS is not essential for packaging of Ty3 gRNA.

The yeast retrovirus-like element Ty3 GAG3 gene encodes a Gag3 polyprotein analogous to retroviral Gag. Gag3 lacks matrix, but contains capsid, spacer, and nucleocapsid domains. Expression of a Ty3 Gag3 or capsid domain optimized for expression in Escherichia coli was sufficient for Ty3 particle assembly. Virus-like ordered particles assembled from Gag3 were similar in size to immature particles from yeast and contained nucleic acid. However, particles assembled from the CA domain were variable in size and displayed much less organization than native particles. These results indicate that assembly can be driven through interactions among capsid subunits in the particle, but that the nucleocapsid domain, likely in association with RNA, confers order upon this process.