Retroviruses and retrotransposons assemble intracellular immature core particles around a RNA genome, and nascent particles collect in association with membranes or as intracellular clusters. How and where genomic RNA are identified for retrovirus and retrotransposon assembly, and how translation and assembly processes are coordinated is poorly understood. To understand this process, the subcellular localization of Ty3 RNA and capsid proteins and virus-like particles was investigated. We demonstrate that mRNAs, proteins, and virus-like particles of the yeast Ty3 retrotransposon accumulate in association with cytoplasmic P-bodies, which are sites of mRNA translation repression, storage, and degradation. Deletions of genes encoding P-body proteins decreased Ty3 transposition and caused changes in the pattern of Ty3 foci, underscoring the biological significance of the association of Ty3 virus-like protein components and P-bodies. These results suggest the hypothesis that P-bodies may serve to segregate translation and assembly functions of the Ty3 genomic RNA to promote assembly of virus-like particles. Because Ty3 has features of a simple retrovirus and P-body functions are conserved between yeast and metazoan organisms, these findings may provide insights into host factors that facilitate retrovirus assembly.

A collection of 4457 Saccharomyces cerevisiae mutants deleted for nonessential genes was screened for mutants with increased or decreased mobilization of the gypsylike retroelement Ty3. Of these, 64 exhibited increased and 66 decreased Ty3 transposition compared with the parental strain. Genes identified in this screen were grouped according to function by using GOnet software developed as part of this study. Gene clusters were related to chromatin and transcript elongation, translation and cytoplasmic RNA processing, vesicular trafficking, nuclear transport, and DNA maintenance. Sixty-six of the mutants were tested for Ty3 proteins and cDNA. Ty3 cDNA and transposition were increased in mutants affected in nuclear pore biogenesis and in a subset of mutants lacking proteins that interact physically or genetically with a replication clamp loader. Our results suggest that nuclear entry is linked mechanistically to Ty3 cDNA synthesis but that host replication factors antagonize Ty3 replication. Some of the factors we identified have been previously shown to affect Ty1 transposition and others to affect retroviral budding. Host factors, such as these, shared by distantly related Ty retroelements and retroviruses are novel candidates for antiviral targets.

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.

Expression of the budding yeast retrotransposon Ty3 results in production of viruslike particles (VLPs) and retrotransposition. The Ty3 major structural protein, Gag3, similar to retrovirus Gag, is processed into capsid, spacer, and nucleocapsid (NC) during VLP maturation. The 57-amino-acid Ty3 NC protein has 17 basic amino acids and contains one copy of the CX 2 CX 4 HX 4 C zinc-binding motif found in retrovirus NC proteins. Ty3 RNA, protein, and VLPs accumulate in clusters associated with RNA processing bodies (P bodies). This study investigated the role of the NC domain in Ty3-P body clustering and VLP assembly. Fifteen Ty3 NC Ala substitution and deletion mutants were examined using transposition, immunoblot, RNA protection, cDNA synthesis, and multimerization assays. Localization of Ty3 proteins and VLPs was characterized microscopically. Substitutions of each of the conserved residues of the zinc-binding motif resulted in the loss of Ty3 RNA packaging. Substitution of the first two of four conserved residues in this motif caused the loss of Ty3 RNA and protein clustering with P bodies and disrupted particle formation. NC was shown to be a mediator of formation of Ty3 RNA foci and association of Ty3 RNA and protein with P bodies. Mutations that disrupted these NC functions resulted in various degrees of Gag3 nuclear localization and a spectrum of different particle states. Our findings are consistent with the model that Ty3 assembly is associated with P-body components. We hypothesize that the NC domain acts as a molecular switch to control Gag3 conformational states that affect both assembly and localization.Ty3 is a long-terminal-repeat (LTR) retrotransposon in Saccharomyces cerevisiae that encodes a Gag3 protein with domains similar to those of retrovirus capsid (CA) and nucleocapsid (NC) (51, 59). Our previous studies showed that Ty3 protein, RNA, and viruslike particles (VLPs) accumulate in association with RNA processing bodies (P bodies), and these are proposed to be the sites of assembly (4). The present study was undertaken to determine the role of the Ty3 NC domain in the association of Ty3 protein and RNA with P bodies and in the assembly of VLPs.The similarity of retrovirus and Ty3 CA and NC domains suggests that they perform similar functions in their respective assembly contexts. The major structural proteins of retroviruses include matrix (MA), CA, and NC domains, as well as other less-conserved domains (12). The MA domain concentrates precursor polyproteins at the membrane (reviewed in references 1, 19, 46, 54, and 60) or the microtubule organizing center (65, 76) assembly sites. The Gag NC zinc-binding residues are directly implicated in the recognition of genomic RNA (17). In addition, the NC domain mediates less-specific interactions of Gag with RNA via patches of basic amino acids. Binding to RNA in turn facilitates Gag-Gag interactions within the CA domain that are required for assembly. Hence, these basic NC residues together with the spacer (SP) domain found between CA and NC domains ...

The retrovirus-like element Ty3 of Saccharomyces cerevisiae integrates at the transcription initiation region of RNA polymerase III. To identify host genes that affect transposition, a collection of insertion mutants was screened using a genetic assay in which insertion of Ty3 activates expression of a tRNA suppressor. Fifty-three loci were identified in this screen. Corresponding knockout mutants were tested for the ability to mobilize a galactose-inducible Ty3, marked with the HIS3 gene. Of 42 mutants tested, 22 had phenotypes similar to those displayed in the original assay. The proteins encoded by the defective genes are involved in chromatin dynamics, transcription, RNA processing, protein modification, cell cycle regulation, nuclear import, and unknown functions. These mutants were induced for Ty3 expression and assayed for Gag3p protein, integrase, cDNA, and Ty3 integration upstream of chromosomal tDNA Val(AAC) genes. Most mutants displayed differences from the wild type in one or more intermediates, although these were typically not as severe as the genetic defect. Because a relatively large number of genes affecting retrotransposition can be identified in yeast and because the majority of these genes have mammalian homologs, this approach provides an avenue for the identification of potential antiviral targets.