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Retroviruses in nondividing cells and yeast retrotransposons must transit the nuclear membrane in order for integration to occur. Mutations in a bipartite basic motif in the carboxyl-terminal domain of the Ty3 integrase (IN) protein were previously shown to block transposition at a step subsequent to 3'-end processing of Ty3 extrachromosomal DNA. In this work, the Ty3 IN was shown to be sufficient to target green fluorescent protein to the nucleolus. Mutations in the bipartite basic motif abrogated this localization. The region containing the motif was shown to be sufficient for nuclear but not subnuclear localization of a heterologous protein. Viruslike particles (VLPs) from cells expressing a Ty3 element defective for nuclear localization were inactive in an in vitro integration assay, suggesting that nuclear entry is required to form active VLPs or that this motif is required for post-nuclear entry steps. Ty3 inserts at transcription initiation sites of genomic tRNA genes and plasmid-borne 5S and U6 RNA genes transcribed by RNA polymerase III. In situ hybridization with Ty3- and Ty3 long terminal repeat-specific probes showed that these elements which are associated with tRNA genes do not colocalize with the ribosomal DNA (rDNA). However, a PCR assay of cells undergoing transposition showed that Ty3 insertion does occur into the 5S genes, which, in yeast, are interspersed with the rDNA and therefore, like Ty3 IN, associated with the nucleolus.

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Position-specific integration of the retroviruslike element Ty3 near the transcription initiation sites of tRNA genes requires transcription factors IIIB and IIIC (TFIIIB and TFIIIC). Using a genetic screen, we isolated a mutant with a truncated 95-kDa subunit of TFIIIC (TFIIIC95) that reduced the apparent retrotransposition of Ty3 into a plasmid-borne target site between two divergently transcribed tRNA genes. Although TFIIIC95 is conserved and essential, no defect in growth or transcription of tRNAs was detected in the mutant. Steps of the Ty3 life cycle, such as protein expression, proteolytic processing, viruslike particle formation, and reverse transcription, were not affected by the mutation. However, Ty3 integration into a divergent tDNA target occurred exclusively in one orientation in the mutant strain. Investigation of this orientation bias showed that TFIIIC95 and Ty3 integrase interacted in two-hybrid and glutathione S-transferase pulldown assays and that interaction with the mutant TFIIIC95 protein was attenuated. The orientation bias observed here suggests that even for wild-type Ty3, the protein complexes associated with the long terminal repeats are not equivalent in vivo.

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Ty3 is a gypsy-type, retrovirus-like element found in the budding yeast Saccharomyces cerevisiae. In cells overexpressing Ty3 under the GAL1 upstream activation sequence, Ty3 RNA, proteins, and DNA are made. Elucidation of the molecular masses and amino-terminal sequences of protease and reverse transcriptase indicated the existence of an additional intervening domain, designated J, in the Ty3 Gag3-Pol3p polyprotein. A region analogous to J can be found in many retrotransposable elements closely related to Ty3; however, J does not correspond to any of the highly conserved retroviral protein domains. Ty3 mutants deleted for the J-coding region showed moderately reduced transposition frequency but greatly reduced levels of Ty3 DNA. These results show that under galactose regulation, the Ty3 J domain is not absolutely essential.

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Ty3 integrates into the transcription initiation sites of genes transcribed by RNA polymerase III. It is known that transcription factors (TF) IIIB and IIIC are important for recruiting Ty3 to its sites of integration upstream of tRNA genes, but that RNA polymerase III is not required. In order to investigate the respective roles of TFIIIB and TFIIIC, we have developed an in vitro integration assay in which Ty3 is targeted to the U6 small nuclear RNA gene, SNR6. Because TFIIIB can bind to the TATA box upstream of the U6 gene through contacts mediated by TATA-binding protein (TBP), TFIIIC is dispensable for in vitro transcription. Thus, this system offers an opportunity to test the role of TFIIIB independent of a requirement of TFIIIC. We demonstrate that the recombinant Brf and TBP subunits of TFIIIB, which interact over the SNR6 TATA box, direct integration at the SNR6 transcription initiation site in the absence of detectable TFIIIC or TFIIIB subunit B". These findings suggest that the minimal requirements for pol III transcription and Ty3 integration are very similar.

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MOTIVATION: Computer-assisted methods are essential for the analysis of biosequences. Gene activity is regulated in part by the binding of regulatory molecules (transcription factors) to combinations of short motifs. The goal of our analysis is the development of algorithms to identify regulatory motifs and to predict the activity of combinations of those motifs. APPROACH: Our research begins with a new motif-finding method, using multiple objective functions and an improved stochastic iterative sampling strategy. Combinatorial motif analysis is accomplished by constructive induction that analyzes potential motif combinations. The hypothesis is generated by applying standard inductive learning algorithms. RESULTS: Tests using 10 previously identified regulons from budding yeast and 14 artificial families of sequences demonstrated the effectiveness of the new motif-finding method. Motif combination and classification approaches were used in the analysis of a sample DNA array data set derived from genome-wide gene expression analysis. AVAILABILITY: Programs will be available as executable files upon request. CONTACT: yhu@ics.uci.eduor yhu@cse.ttu.edu.tw

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SCFCdc4 (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development. Mass spectrometric analysis of proteins copurifying with Cdc53 identified the RING-H2 finger protein Hrt1 as a subunit of SCF. Hrt1 shows striking similarity to the Apc11 subunit of anaphase-promoting complex. Conditional inactivation of hrt1(ts) results in stabilization of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G1/S. Hrt1 assembles into recombinant SCF complexes and individually binds Cdc4, Cdc53 and Cdc34, but not Skp1. Hrt1 stimulates the E3 activity of recombinant SCF potently and enables the reconstitution of Cln2 ubiquitination by recombinant SCFGrr1. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality of hrt1Delta, and human HRT2 binds CUL-1. We conclude that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases.

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Ty3, a retroviruslike element of Saccharomyces cerevisiae, transposes into positions immediately upstream of RNA polymerase III-transcribed genes. The Ty3 integrase (IN) protein is required for integration of the replicated, extrachromosomal Ty3 DNA. In retroviral IN, a conserved core region is sufficient for strand transfer activity. In this study, charged-to-alanine scanning mutagenesis was used to investigate the roles of the nonconserved amino- and carboxyl-terminal regions of Ty3 IN. Each of the 20 IN mutants was defective for transposition, but no mutant was grossly defective for capsid maturation. All mutations affecting steady-state levels of mature IN protein resulted in reduced levels of replicated DNA, even when polymerase activity was not grossly defective as measured by exogenous reverse transcriptase activity assay. Thus, IN could contribute to nonpolymerase functions required for DNA production in vivo or to the stability of the DNA product. Several mutations in the carboxyl-terminal domain resulted in relatively low levels of processed 3' ends of the replicated DNA, suggesting that this domain may be important for binding of IN to the long terminal repeat. Another class of mutants produced wild-type amounts of DNA with correctly processed 3' ends. This class could include mutants affected in nuclear entry and target association. Collectively, these mutations demonstrate that in vivo, within the preintegration complex, IN performs a central role in coordinating multiple late stages of the retrotransposition life cycle.

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Retroviruses, including HIV-1 and the distantly related yeast retroelement Ty3, all encode a nucleoprotein required for virion structure and replication. During an in vitro comparison of HIV-1 and Ty3 nucleoprotein function in RNA dimerization and cDNA synthesis, we discovered a bipartite primer-binding site (PBS) for Ty3 composed of sequences located at opposite ends of the genome. Ty3 cDNA synthesis requires the 3' PBS for primer tRNAiMet annealing to the genomic RNA, and the 5' PBS, in cis or in trans, as the reverse transcription start site. Ty3 RNA alone is unable to dimerize, but formation of dimeric tRNAiMet bound to the PBS was found to direct dimerization of Ty3 RNA-tRNAiMet. Interestingly, HIV-1 nucleocapsid protein NCp7 and Ty3 NCp9 were interchangeable using HIV-1 and Ty3 RNA template-primer systems. Our findings impact on the understanding of non-canonical reverse transcription as well as on the use of Ty3 systems to screen for anti-NCp7 drugs.

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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.

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Expression of the auxiliary human immunodeficiency virus type 1 (HIV-1) protein Vpr causes arrest of primate host cells in G2. Expression of this protein in budding yeast has been previously reported to cause growth arrest and a large-cell phenotype. Investigation of the effect of Vpr expression in budding yeast, reported here, showed that it causes disruption of the actin cytoskeleton. Expression of HSP42, the gene for a small heat shock protein (sHSP), from a high-copy-number plasmid reversed this effect. The sHSPs are induced by exposure of cells to thermal, osmotic, and oxidative stresses and to mitogens. In animal cells, overexpression of sHSPs causes increased resistance to stress and stabilization of actin stress fibers. Yeast cells subjected to mild stress, such as shifting from 23 to 39 degrees C, arrest growth and then resume cell division. Growth arrest is accompanied by transient disorganization of the cytoskeleton. Yeast in which the HSP42 gene was disrupted and which was subjected to moderate thermal stress reorganized the actin cytoskeleton more slowly than did wild-type control cells. These results demonstrate that in yeast, as in metazoan cells, sHSPs promote maintenance of the actin cytoskeleton.

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The retrovirus-like element Ty3 of Saccharomyces cerevisae integrates into the yeast genomic DNA in a position specific manner. Ty3 integrates within 1-2 base pairs of the site of transcription initiation by RNA polymerase III. The human tRNA(Lys)3 gene was used as a target for transposition in a plasmid-based assay to determine whether Ty3 integration can be targeted to a human tRNA gene. Each transposition event observed was adjacent to the site of initiation of transcription of the human tRNA gene. Therefore, heterologous tRNA genes can serve as targets for Ty3 in yeast. This is a first step toward development of a system for targeted integrations in heterologous organisms.

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Ty3, a gypsylike retrotransposon of budding yeast, integrates at the transcription initiation site of genes transcribed by RNA polymerase III (pol III). It was previously shown that integration in vitro requires intact promoter elements and the pol III transcription factors TFIIIB and TFIIIC. In order to test the effect of pol III on integration, increasing amounts of a pol III-containing fraction were added to Ty3 in vitro integration reactions. The pol III-containing fraction was inhibitory to integration. These results are consistent with a model where the Ty3 integration complex and pol III recognize similar features of the stable transcription complex and compete with each other for access to the transcription initiation site.

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Ty3, a retroviruslike element in Saccharomyces cerevisiae, encodes an integrase (IN) which is essential for position-specific transposition. The Ty3 integrase contains the highly conserved His-Xaa(3-7)-His-Xaa(23-32)-Cys-Xaa(2)-Cys and Asp, Asp-Xaa(35)-Glu [D,D(35)E] motifs found in retroviral integrases. Mutations were introduced into the coding region for the Ty3 integrase to determine the effects in vivo of changes in conserved residues of the putative catalytic triad D,D(35)E and the nonconserved carboxyl-terminal region. Ty3 viruslike particles were found to be associated with significant amounts of linear DNA of the approximate size expected for a full-length reverse transcription product and with plus-strand strong-stop DNA. The full-length, preintegrative DNA has at each 3' end 2 bp that are removed prior to or during integration. Such 3'-end processing has not been observed for other retroviruslike elements. A mutation at either D-225 or E-261 of the Ty3 integrase blocked transposition and prevented processing of the 3' ends of Ty3 DNA in vivo, suggesting that the D,D(35)E region is part of the catalytic domain of Ty3 IN. Carboxyl-terminal deletions of integrase caused a dramatic reduction in the amount of Ty3 DNA in vivo and a decrease in reverse transcriptase activity in vitro but did not affect the apparent size or amount of the 55-kDa reverse transcriptase in viruslike particles. The 115-kDa viruslike particle protein, previously shown to react with antibodies to Ty3 integrase, was shown to be a reverse transcriptase-IN fusion protein. These results are consistent with a role for the integrase domain either in proper folding of reverse transcriptase or as part of a heterodimeric reverse transcriptase molecule.

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The Saccharomyces cerevisiae retroviruslike element Ty3 encodes the major structural proteins capsid (CA) and nucleocapsid in the GAG3 open reading frame. The Ty3 CA protein contains a sequence (QGX2EX5FX3LX3H, where H is a hydrophobic residue) which has not been observed in other retrotransposons but which is similar to the major homology region (MHR) described for retrovirus CA. In this study the effects of mutations in the Ty3 MHR on particle formation, processing, DNA synthesis, and transposition were examined. Each of the mutations tested resulted in severe defects in transposition, with disruption occurring prior to or at particle formation, subsequent to particle formation and prior to completion of DNA synthesis, and subsequent to DNA synthesis. Changing the Q in the motif to R had relatively little effect on particle formation but decreased transposition to about 13% of that of a wild-type element. Changing G to A or V almost completely eliminated the formation of intracellular particles, possibly by disruption of CA-CA interactions. Changes introduced at the position of E resulted in blocked processing, blocked DNA synthesis, or a block at some post-reverse transcription step, depending on the nature of the mutation introduced. These results showed that the integrity of the Ty3 MHR is required for multiple aspects of Ty3 replication involving CA. These functions are independent of extracellular budding and of infection, aspects of the retroviral life cycle which are not recapitulated in replication of the Ty3 retrotransposon.

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Many stress proteins and their cognates function as molecular chaperones or as components of proteolytic systems. Viral infection can stimulate synthesis of stress proteins and particular associations of viral and stress proteins have been documented. However, demonstrations of functions for stress proteins in viral life cycles are few. We have initiated an investigation of the roles of stress proteins in eukaryotic viral life cycles using as a model the Ty3 retrovirus-like element of Saccharomyces cerevisiae. During stress, Ty3 transposition is inhibited; Ty3 DNA is not synthesized and, although precursor proteins are detected, mature Ty3 proteins and virus-like particles (VLPs) do not accumulate. The same phenotype is observed in the constitutively stressed ssa1 ssa2 mutant, which lacks two cytoplasmic members of the hsp70 family of chaperones. Ty3 VLPs preformed under nonstress conditions are degraded more rapidly if cells are shifted from 30 degrees C to 37 degrees C. These results suggest that Ty3 VLPs are destroyed by cellular stress proteins. Elevated expression of the yeast UBP3 gene, which encodes a protease that removes ubiquitin from proteins, allows mature Ty3 proteins and VLPs to accumulate in the ssa1 ssa2 mutant, suggesting that, at least under stress conditions, ubiquitination plays a role in regulating Ty3 transposition.

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The yeast retroviruslike element Ty3 inserts at the transcription initiation sites of genes transcribed by RNA polymerase III (Pol III). An in vitro integration assay was developed with the use of Ty3 viruslike particles and a modified SUP2 tyrosine transfer RNA (tRNA(Tyr)) gene target. Integration was position-specific and required Ty3 integrase, Pol III transcription factor (TF) IIIB-, TFIIIC-, and Pol III-containing fractions showed that TFIIIB and TFIIIC, together, were sufficient for position-specific Ty3 integration, but not for transcription. This report demonstrates that in vitro integration of a retroelement can be targeted by cellular proteins.

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Ty3 is a retrotransposon of Saccharomyces cerevisiae that integrates just upstream of the transcription initiation site of genes transcribed by RNA polymerase III. Ty3 transcription is pheromone-inducible in haploid cells and is mating-type regulated in diploid cells. The specificity of Ty3 integration was exploited in the design of a novel target into which transposition of Ty3 elements could be selected. The target plasmid contains divergently oriented tRNA genes with 19 base pairs separating the two tRNA gene coding sequences. An inactive ochre suppressor tRNA(Tyr) gene with a modified transcription initiation region was used as the selectable marker and a tRNA(Val) (AAC) gene was used to direct Ty3 integration into the transcription initiation region of the suppressor tRNA(Tyr) gene. Integration of Ty3 activated expression of the suppressor tRNA gene, which resulted in suppression of ochre nonsense alleles ade2-101(0) and lys2-1(0) and allowed cell growth on selective medium. Based on the activity of this target, Ty3, under control of a galactose-inducible promoter and present on a high copy-number plasmid, was estimated to transpose into the genome at a rate of 5.6 x 10(-3) per cell division. We show here that induction of Ty3 transcription from its natural promoter results in transposition. Ty3 elements in strains of the a or alpha mating-type transposed efficiently to target plasmids in cells of the opposite mating-type. Thus, natural transposition of Ty3 is regulated temporally to occur in mating populations.

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