Ty elements of Saccharomyces cerevisiae are long terminal repeat (LTR) retroelements related to retroviruses. Normal levels of Ty1 transposition require Dbr1p, a cellular enzyme that cleaves 2'-5' RNA bonds. We show that Ty1 RNAs lacking identifiable 5' ends accumulate in virus-like particles (VLPs) in dbr1 mutants. Debranching this RNA in vitro with Dbr1p creates an uncapped version of the normal Ty1 RNA 5' end. We show that the 5' nucleotide (nt) of Ty1 RNA forms a 2'-5' bond with a nt near the 3' end of the same RNA, creating a lariat. The properties of the lariat suggest it forms by a novel mechanism and that branching and debranching may play roles in Ty1 reverse transcription at the minus-strand transfer step.

Host cell cycle genes provide important functions to retroviruses and retroviruslike elements. To define some of these functions, the cell cycle dependence of transposition of the yeast retroviruslike element Ty3 was examined. Ty3 is unique among retroviruslike elements because of the specificity of its integration, which occurs upstream of genes transcribed by RNA polymerase III. A physical assay for Ty3 transposition which takes advantage of this position-specific integration was developed. The assay uses PCR to amplify a product of Ty3 integration into a target plasmid that carries a modified tRNA gene. By using the GALl upstream activating sequence to regulate expression of Ty3, transposition was detected within one generation of cell growth after Ty3 transcription was initiated. This physical assay was used to show that Ty3 did not transpose when yeast cells were arrested in G1 during treatment with the mating pheromone aL-factor. The restriction of transposition was not due to changes in transcription of either Ty3 or tRNA genes or to aspects of the mating pheromone response unrelated to cell cycle arrest. The block of the Ty3 life cycle was reversed when cells were released from G1 arrest. Examination of Ty3 intermediates during G1 arrest indicated that Ty3 viruslike particles were present but that reverse transcription of the Ty3 genomic RNA into double-stranded DNA had not occurred. In G1, the Ty3 life cycle is blocked after particle assembly but before the completion of reverse transcription.Retroviruses and long terminal repeat (LTR)-containing retrotransposons have similar structures and transposition cycles (58). The common steps of replication and integration for these elements, henceforth referred to collectively as retroviruslike elements, are (i) transcription of an integrated DNA copy of the element into RNA, (ii) translation of the RNA followed by assembly of particles composed of the RNA as well as the proteins encoded by the element, (iii) reverse transcription of the RNA into DNA, and (iv) integration of this DNA copy into the host cell genome. Retrotransposons are competent for transposition in the cell within which they reside, while retroviruses infect other cells.Retroviruslike elements have genomes of between 2 and 8 kbp which include LTRs of a few hundred base pairs and code for both major structural proteins of the viral particles and enzymes necessary for transposition. The LTRs contain information for initiating transcription of the element and for terminating and processing transcripts. The genes of retroviruses are generally classified as gag, pol, and env. The gag gene products include the capsid protein (CA), which is the main structural component of the retroviruslike element particle; the nucleocapsid protein (NC), which binds the genomic RNA of the element; and, in some cases, the protease (PR), which processes the polyprotein translation products of gag and pol. In other retroviruses, PR is encoded by the pol gene. The other poi gene products are the reverse transcriptase (...

Ty3, a member of the Metaviridiae family of long-terminal-repeat retrotransposons found in Saccharomyces cerevisiae, encodes homologs of retroviral Gag and Gag-Pol proteins, which, together with genomic RNA, assemble into virus-like particles (VLPs) that undergo processing and reverse transcription. The Ty3 structural proteins, capsid and nucleocapsid, contain major homology and nucleocapsid motifs similar to retrovirus capsid and nucleocapsid proteins, but Ty3 lacks a matrix-like structural domain amino terminal to capsid. Mass spectrometry analysis of Ty3 Gag3 processing products defined an acetylated Ser residue as the amino terminus of Gag3/p34, p27, and CA/p24 species and supported a model where p34 and p27 occur in phosphorylated forms. Using atomic force microscopy, VLPs were imaged from cells producing wild-type and protease and reverse transcriptase mutant Ty3. Wild-type VLPs were found to have a broad range of diameters, but the majority, if not all of the particles, exhibited arrangements of capsomeres on their surfaces which were consistent with icosahedral symmetry. Wild-type particles were in the range of 25 to 52 nm in diameter, with particles in the 42-to 52-nm diameter range consistent with T‫7؍‬ symmetry. Both classes of mutant VLPs fell into a narrower range of 44 to 53 nm in diameter and appeared to be consistent with T‫7؍‬ icosahedral symmetry. The smaller particles in the wild-type population likely correspond to VLPs that have progressed to reverse transcription or later stages, which do not occur in the protease and reverse transcriptase mutants. Ty3 VLPs did not undergo major external rearrangements during proteolytic maturation.This study was undertaken to investigate the virus-like particle (VLP) structure of the long-terminal-repeat (LTR) retrovirus like element Ty3 from Saccharomyces cerevisiae (75). The retrotransposon VLP is analogous to the nonenveloped core particle of retroviruses. Determination of detailed structures of retroviral cores is complicated, because cores are enveloped and heterogeneous and mature particles of many retroviruses are relatively fragile (20,79). It was of interest to determine whether the smaller size of the Ty3 structural domain would be reflected in a simplification of particle morphology. Retroviral cores are initially assembled from Gag together with lesser amounts of Gag-Pol to form spherical, immature cores. Naked (nonenveloped) cores range in average diameter from 79 nm for Rous sarcoma virus (RSV) (38) to 96 nm for murine leukemia virus (MLV) (86).

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The Saccharomyces cerevisiae DBR1 gene encodes a 2-5 phosphodiesterase that debranches intron RNA lariats following splicing. Yeast dbr1 mutants accumulate intron lariats and are also defective for mobility of the retrotransposons Ty1 and Ty3. We used a mutagenic PCR method to generate a collection of dbr1 mutant alleles to explore the relationship between the roles of DBR1 in transposition and debranching. Eight mutants defective for Ty1 transposition contained single amino acid changes in Dbr1p. Two mutations, G84A and N85D, are in a conserved phosphoesterase motif that is believed to be part of the active site of the enzyme, supporting a connection between enzymatic activity and Ty1 transposition. Two other mutations, Y68F and Y68D, occur at a potential phosphorylation site, and we have shown that Dbr1p is phosphorylated on tyrosine. We have developed an RNase protection assay to quantitate intron RNA accumulation in cells. The assay uses RNA probes that hybridize to ACT1 intron RNA. Protection patterns confirm that sequences from the 5 end of the intron to the lariat branch point accumulate in dbr1 mutants in a branched (lariat) conformation. RNase protection assays indicate that all of the newly generated dbr1 mutant alleles are also deficient for debranching, further supporting a role for 2-5 phosphodiesterase activity in Ty1 transposition. A Ty1 element lacking most of its internal sequences transposes independently of DBR1. The existence of Dbr1p-dependent Ty1 sequences raises the possibility that Dbr1p acts on Ty1 RNA.Saccharomyces cerevisiae Ty1 is a long terminal repeat (LTR) retroelement with a life cycle similar to that of retroviruses (2). Like retroviruses, Ty1 replicates via reverse transcription of an RNA intermediate, subsequently integrating element cDNA into the host genome. Retroviruses and LTR retrotransposons rely on their hosts to provide most of the factors necessary for propagation. We identified dbr1 mutants in a screen to identify host cell genes required for Ty1 transposition (15). The dbr1 mutant had been identified previously in a similar screen by others (6). Although dbr1 cells produce wild-type levels of Ty1 proteins, they accumulate Ty1 cDNA at a slower rate than do wild-type cells, suggesting that Dbr1p plays a role in Ty1 reverse transcription or cDNA stability (15).In its cellular role, Dbr1p acts at the end of the mRNA splicing process, cleaving the 2Ј-5Ј linkage at the branch point of intron RNA lariats released after exon ligation, converting them to linear RNAs that are rapidly degraded (6, 23). In a dbr1 mutant, intron RNAs accumulate as lariats (6). The uncleaved lariat branch point blocks the progression of 3Ј exonucleases, and there is no 5Ј end available on which a 5Ј exonuclease can act. DBR1 is highly conserved, and homologues have been cloned from Schizosaccharomyces pombe, S. cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Homo sapiens (6,17,18,24). Deletion of DBR1 has no significant effect on the growth rate of S. cerevisiae, but...