Characterization of Ty3-gypsy-like elements in Hibiscus syriacus

Southern blot analysis revealed a ubiquitous distribu-tion and high copy number of Ty3-gypsy-like elements in the genome of Hibiscus syriacus. Comparative phylogenetic analysis of the large subunit of Rubisco and the integrase region of Ty3-gypsy elements in various plant species indicated that the retrotransposon-like sequences have different evolutionary histories and their own unique polymorphism in the H. syriacus population. Sequence-tagged site-restriction fragment length polymorphisms (STS-RFLP) analysis also indicated great variability in the numbers and sequences of Ty3-gypsy-like elements within H. syriacus varieties. Ty3-gypsy-like elements may still be active within H. syriacus, since Northern analysis of wounded leaves of H. syriacus variety Saehan with a probe for the integrase domain gave strong hybridization signals. The sequence heterogeneity and ubiquity of the Ty3-gypsy-like elements in H. syriacus genomes could provide reliable DNA markers for line identification as well for the analysis of genetic diversity in H. syriacus.     

Characterization of a transpositionally active Ty3 element and identification of the Ty3 integrase protein.

Ty3 is a Saccharomyces cerevisiae retrotransposon associated with tRNA genes. Two Ty3 elements have been cloned and characterized. The complete nucleotide sequence for one element, Ty3-2, was reported previously (L. J. Hansen, D. L. Chalker, and S. B. Sandmeyer, Mol. Cell. Biol. 9:5245-5256, 1988). However, this element is incapable of autonomous transposition. The complete DNA sequence of a transpositionally competent Ty3 element, Ty3-1, is presented here. Its sequence translates into two overlapping open reading frames, TYA3-1 and TYB3-1, which encode proteins with homology to the proteins specified by the retroviral gag and pol genes, respectively. Comparison of the Ty3-1 nucleotide sequence to Ty3-2 suggests that the TYB3-2 open reading frame of Ty3-2 is truncated by the deletion of a single nucleotide, which causes a frameshift mutation. Restoration of the reading frame with insertion of a single adenine by site-directed mutagenesis converted Ty3-2 into a transpositionally active element, Ty3-2(+ A). Western blot analysis with antibodies made against synthetic peptides identified integrase (IN) proteins in viruslike particle preparations from cells expressing Ty3 elements. Cells expressing Ty3-1 and Ty3-2 (+A) produce antibody-reactive proteins with approximate molecular masses of 61 and 58 kilodaltons (kDa), while cells expressing Ty3-2 produce reactive proteins of approximately 52 and 49 kDa. Together, these data show that the 61- or 58-kDa protein, or both, provides the integrase function of Ty3.     

A Chimeric Ty3/Moloney Murine Leukemia Virus Integrase Protein Is Active In Vivo

This report describes the results of experiments to determine whether chimeras between a retrovirus and portions of Ty3 are active in vivo. A chimera between Ty3 and a Neo^r-marked Moloney murine leukemia virus (M-MuLV) was constructed. The C-terminal domain of M-MuLV integrase (IN) was replaced with the C-terminal domain of Ty3 IN. The chimeric retroviruses were expressed from an amphotrophic envelope packaging cell line. The virus generated was used to infect the human fibrosarcoma cell line HT1080, and cells in which integration had occurred were selected by G418 resistance. Three independently integrated viruses were rescued. In each case, the C-terminal Ty3 IN sequences were maintained and short direct repeats of the genomic DNA flanked the integration site. Sequence analysis of the genomic DNA flanking the insertion did not identify a tRNA gene; therefore, these integration events did not have Ty3 position specificity. This study showed that IN sequences from the yeast retrovirus-like element Ty3 can substitute for M-MuLV IN sequences in the C-terminal domain and contribute to IN function in vivo. It is also one of the first in vivo demonstrations of activity of a retrovirus encoding an integrase chimera. Studies of chimeras between IN species with distinctive integration patterns should complement previous work by expanding our understanding of the roles of nonconserved domains.     

Proteolytic processing of Ty3 proteins is required for transposition.

Ty3 is a retroviruslike element found in Saccharomyces cerevisiae. It encodes GAG3 and GAG3-POL3 polyproteins which are processed into mature proteins found in the Ty3 viruslike particle. In this study, the region encoding a protease that is homologous to retroviral aspartyl proteases was identified and shown to be required for production of mature Ty3 proteins and transposition. The Ty3 protease has the Asp-Ser-Gly consensus sequence found in copia, Ty1, and Rous sarcoma virus proteases, rather than the Asp-Thr-Gly found in most retroviral proteases. The Asp-Ser-Gly consensus is flanked by residues similar to those which flank the active sites of cellular aspartyl proteases. Mutations were made in the Ty3 active-site sequence to examine the role of the protease in Ty3 particle maturation and to test the functional significance of the Ser active-site variant in the consensus sequence. Mutation of the active-site Asp blocked processing of Gag3 and Gag3-Pol3 and allowed identification of a GAG3-POL3 polyprotein. This protein was turned over rapidly in cells expressing the mutant Ty3. Changing the active-site Ser to Thr caused only a modest reduction in the levels of certain Ty3 proteins. Five putative cleavage sites of this protease in Ty3 GAG3 and GAG3-POL3 polyproteins were defined by amino-terminal sequence analysis. The existence of an additional protein(s) of unknown function, encoded downstream of the protease-coding region, was deduced from the positions of these amino termini and the sizes of known Ty3 proteins. Although Ty3 protease cleavage sites do not correspond exactly to known retroviral protease cleavage sites, there are similarities. Residues P3 through P2' in the regions encompassing each of the five sites are uncharged, and no P1 position is occupied by an amino acid with a branched beta carbon.     

Ty3 nucleocapsid controls localization of particle assembly

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 CX₂CX₄HX₄C 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.     

Involvement of DNA End-Binding Protein Ku in Ty Element Retrotransposition

Saccharomyces cerevisiae Ty elements are retrotransposons whose life cycles are strikingly similar to those of retroviruses. They transpose via an RNA intermediate that is converted to linear double-stranded cDNA and then inserted into the host genome. Although Ty integration is mediated by the element-encoded integrase, it has been proposed that host factors are involved in this process. Here, we show that the DNA end-binding protein Ku, which functions in DNA double-strand break repair, potentiates retrotransposition. Specifically, by using a galactose-inducible Ty1 system, we found that in vivo, Ty1 retrotransposition rates were substantially reduced in the absence of Ku. In contrast, this phenotype was not observed with yeast strains containing mutations in other genes that are involved in DNA repair. We present evidence that Ku associates with Ty1 viruslike particles both in vitro and in vivo. These results provide an additional role for Ku and suggest that it might function in the life cycles of retroelements in other systems.