INTRODUCTION

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Ty1 is one of five endogenous retrotransposons of the yeast Saccharomyces cerevisiae. The life cycle of Ty1 begins with transcription of the genomic element and translation of the resulting mRNA. Like retroviruses, Ty1 encodes a protease (PR) that processes the element-encoded proteins (1, 18, 23). Ty1 Gag and Gag-Pol proteins, together with Ty1 mRNA, form virus-like particles (VLPs), which are essential transposition intermediates (8, 9). Reverse transcription of Ty1 mRNA takes place in VLPs. The newly synthesized cDNA enters the nucleus and then the yeast genome via an integration reaction (3, 11, 17).

Viruses and the closely related retrotransposons are under selective pressure to maintain a small genome. A number of unique strategies have evolved to ensure that all of the functions necessary for completion of their life cycle are carried out. Host protein functionalities may be usurped, obviating the need for a similar, element-encoded protein.

A genome streamlining strategy used by most long terminal repeat-containing retroelements is programmed ribosomal frameshifting. An mRNA that contains a frameshifting signal can encode multiple proteins, typically Gag and Gag-Pol, in a predetermined stoichiometric ratio that is determined by the efficiency of the frameshift signal. A frameshift-containing retroelement does not require separate promoters to drive the transcription of two messages and thereby increases the information content of its genome with minimal expense. In addition, the common sequences present in Gag and Gag-Pol direct the assembly of the Pol proteins into the virion or VLP.

A 7-nucleotide signal in Ty1 mRNA is necessary and sufficient for directing ribosomal frameshifting (2) and can function translationally in various heterologous contexts. This frameshift signal is required for retrotransposition and is thought to secure the appropriate Gag and Gag-Pol stoichiometry. The frameshifting signal is approximately 5 to 10% efficient and therefore results in a 10- to 20-fold excess of the structural protein Gag with respect to Gag-Pol (6, 7, 21). The Gag-Pol polyprotein contains the enzymatic components required for Ty1 replication.

Ty1 mRNA can have two translational fates. In the more common scenario, in which the frameshift signal is read through by the translating ribosome, a 49-kDa Gag species is made and processed in trans to a 45-kDa Gag species with concomitant liberation of its carboxy-terminal 40 amino acids. The 49-kDa precursor and 45-kDa (CA) processed forms migrate with apparent molecular masses of 58 and 54 kDa, respectively. For a complete description of our Ty1 protein nomenclature, see the study by Merkulov et al. (14). The liberated 40-mer, called the p4 peptide, was suspected to serve some role in particle formation and/or maturation, because mutations in this region result in smaller-than-normal VLPs and severe transposition defects (15). When frameshifting occurs, a Gag-Pol species is synthesized. Proteolytic processing of Gag-Pol liberates Ty1 PR, integrase (IN), and reverse transcriptase (RT) from the precursor polyprotein (10). A 45-kDa Gag species, CA, identical to that made from Gag is also produced, and we show here that it is through this processing event that the amino terminus of the PR is defined (Fig. 1). Most of the p4 peptide sequence is therefore common to the amino terminus of the PR and the carboxy terminus of 49-kDa Gag.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Schematic of the p4 region. The Gag and Gag-Pol polyproteins as well as the relevant amino acids are shown. The amino acids encoded fully or in part by the frameshift are underlined. The PR active site residues are in boldface. The PR cleavage sites in Gag and Gag-Pol are indicated by arrows. The p4 peptide is hatched. The large black dot represents the site of +1 translational frameshifting. The stop codon present in Gag is represented by an asterisk. Block substitution mutations s4 to s12 are shown as bars underneath the amino acids replaced by the peptide sequence AAGSAA. The transpositional competence of elements bearing these mutations is indicated.

Certain mutations in the p4 region common to Gag, p4, and PR block transposition. However, interpretation of this mutant phenotype is complicated by the fact that these mutations simultaneously alter Gag, PR, and p4 sequences. The transposition defect observed in these mutants could be secondary to a loss or gain of function in any or all of these proteins. The functional independence of the frameshift signal suggests a unique manner by which the critical Ty1 protein(s) affected by these mutations can be identified.

In this study, we describe the creation of frameshift transplant mutants of Ty1. These mutants enable us to specifically and unambiguously attribute the deleterious effects of mutations in the p4 region to the Ty1 PR. The p4 peptide, previously thought to be required for transposition, is shown here to be dispensable. We also physically define the amino terminus of the PR by microsequencing and thereby confirm the location of the Gag-PR cleavage site. We confirm that the mutations in this region affect the N terminus of the Ty1 PR; frameshift transplant mutants combined in cis with P4 region mutations are as defective as the same mutants in an otherwise wild-type Ty1 element. Frameshift transplantation is a novel approach that permits the isolated study of one or more proteins encoded by a single mRNA. This approach should prove useful in the study of viruses that are under selective pressure to maintain small, information-dense genomes.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Strains and plasmids. All experiments were performed with S. cerevisiae strain YH51 (MATa ura3-52 his4-539 lys2-801 spt3-202). All of the yeast Ty1 expression plasmids were constructed in the context of plasmid pJEF1105, in which the Ty1 element is marked with the Tn903 neomycin phosphotransferase (neo) gene and carried on a 2µm, URA3-marked plasmid (5).

Expression and purification of Ty1 PR. Escherichia coli strain BL21(DE3), containing various pET vector derivatives, was inoculated and grown to an optical density at 600 nm (OD₆₀₀) of 0.6 in Luria-Bertani medium containing 100 µg of ampicillin per ml and 1 mM isopropyl--D-thiogalactopyranoside. Protein expression was induced for 4 h at 26°C. Following induction, the cells were pelleted and lysed via sonication in binding buffer (0.5 M NaCl, 20 mM Tris-HCl [pH 7.9], 5 mM imidazole, 10% glycerol). The lysate was cleared by centrifugation in an SS-34 rotor at 19,000 rpm for 30 min. The His₆-tagged protein was purified under native conditions over Ni-nitrilotriacetic acid resin (Qiagen). The 0.75-ml column was washed with 10 volumes of binding buffer. Ty1PRHis₆ fusion protein was eluted in binding buffer containing 300 mM imidazole.

cDNA synthesis assays. Yeast cells harboring the indicated plasmid were grown for 24 h at 24°C in uracil-negative SC medium containing 2% raffinose (SC ura, 2% raffinose). The cells were then diluted to an OD₆₀₀ of 0.6 and induced with the addition of galactose to a final concentration of 2%. Five milliliters of cells at an OD₆₀₀ of 2.0 was then pelleted and washed once in water. Nucleic acids were extracted from the cell pellet by bead beating with glass beads in 400 µl of DNA extraction buffer (0.5 M NaCl, 20 mM Tris-HCl [pH 7.5], 1 mM EDTA, 1% sodium dodecyl sulfate), 200 µl of buffer-saturated phenol, and 200 µl of chloroform. The lysate was centrifuged for 10 min in an Eppendorf microcentrifuge at 14,000 rpm. The supernatant was extracted with phenol chloroform and then with chloroform before the nucleic acids were ethanol precipitated. Approximately 10 µg of nucleic acid was digested with 20 U of EcoRI and 2 µg of RNase A in a 15-µl reaction mixture.

Immunoblotting. Ty1 expression was induced for 24 h at 24°C as described above. The cell pellets from 2 ml of culture with an OD₆₀₀ of 1.0 to 1.5 were lysed in 40 µl of buffer B-EDTA (20 mM Tris [pH 7.9], 15 mM KCl, 0.1 mM EDTA) with PR inhibitors and phenylmethylsulfonyl fluoride (1 mM) with a Mini-Bead Beater (Biospec Products) at a high setting for 1 min. The lysate was cleared of cell debris by centrifugation in an Eppendorf centrifuge at 14,000 rpm. The protein concentration was determined by the method of Bradford. Samples (5 µg) were then used for immunoblotting. After electrophoresis, proteins were transferred to Immobilon membranes at 300 mA for 2 h. Membranes were blocked in 3% milk-0.1% Tween 20 in phosphate-buffered saline (PBS). Primary antibody R2-F was applied in blocking solution for 1 h at 1:10,000 dilution. The blots were washed three times for 10 min in PBS-0.1% Tween 20. Secondary antibody (antirabbit-horseradish peroxidase conjugate [Amersham]) was applied at a 1:7,500 dilution for 40 min. The membranes were washed as described above and developed with the Amersham ECL enhanced chemiluminescent system as per the manufacturer's instructions.

Transposition assays. Yeast cells, grown as patches, were replica plated from SC ura, 2% glucose to SC ura, 2% galactose and grown at 26°C for 4 days. The patches were then replica plated to SC ura, 2% glucose and grown overnight at 30°C. The patches were replica plated to yeast-peptone-dextrose (YPD) and, after overnight growth at 30°C, replica plated to SC plus 0.1% 5-fluoroorotic acid. A portion of the patch was then diluted and plated on both YPD and YPD plus 75 µg of G418 per ml. The transposition frequency of the wild-type element is defined as 100%.

	RESULTS

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Figure 1 gives a schematic representation of the p4 region of Ty1. This region is defined at its amino terminus by the Gag-p4 cleavage site and at its carboxy terminus by the Ty1 frameshift signal. The p4 peptide shares sequences with Gag and PR. Block substitution mutations in the region have been described previously: two of them, s7 and s8, block transposition (15).

The sequence of the carboxy terminus of processed Ty1 CA (capsid) protein has previously been determined from yeast VLP preparations to be TARAH (15). This places the CA-p4 cleavage site between amino acids 401 and 402. The p4 peptide liberated by the proteolytic maturation of Gag to CA has not been detected in yeast lysates or VLPs, however, which raises the possibility that additional proteolytic events may be responsible for shaping the amino terminus of the PR. If this is the case, linker insertion mutants in the p4 region that alter the primary structure of the protein might block such further processing and thereby compromise transposition.

Definition of the Ty1 PR N terminus. To determine the identity of the amino terminus of Ty1 PR, as defined by autoproteolytic processing, we expressed it in E. coli. Two series of constructs were prepared (Fig. 2A). The first contained the entire Gag ORF in frame with the PR ORF, and the endogenous frameshift signal was "erased," via the fs mutation, to permit expression in E. coli, in which the frameshift signal is nonfunctional. A carboxy-terminal hexahistidine tag was affixed to facilitate purification. Control constructs in which an active site mutation (DSG to EAA) was introduced in the PR were also prepared.

View larger version (33K): [in this window] [in a new window]   FIG. 2.   Expression of Gag-PR in E. coli and demonstration of autoprocessing. (A) Schematic representation of the expression constructs used to generate the underlying blots. The carboxy-terminal hexahistidine tag is shown in black. Relevant amino acid positions are shown. (B) Immunoblot of bacterial lysates in which the Gag-PR constructs were expressed. VLPs from yeast were separated in lane 1. (C) Appearance of the PR following expression of the PPR precursor protein. Polyclonal antibody R2-F, used in the immunoblot shown in panel B, recognizes Ty1 Gag. Antipeptide antibody JH695 (15), directed against an epitope in the p4 region, was used in the immunoblot shown in panel C.

Frameshift signal transplantation. The endogenous +1 frameshift signal is essential for Ty1 transposition and is believed to secure the appropriate stoichiometry of Gag and the Gag-Pol polyprotein. It is located in a region of the Ty1 genome that encodes the carboxy terminus of Gag and the amino terminus of the PR (Fig. 1). The minimal frameshift signal is only 7 nucleotides long and can function independently of the surrounding context.

View larger version (42K): [in this window] [in a new window]   FIG. 3.   A schematic representation of the frameshift transplant constructs. The wavy line represents Ty1 mRNA. The transposition efficiency in strain YH51 is shown. The sequences surrounding the PR cleavage sites are shown in the wild-type (WT or wt) construct.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Frameshift transplant mutants produce the expected Gag proteins. A Western blot probed with polyclonal antibody R2-F, which recognizes Gag, is shown. Five micrograms of total yeast lysate from strain YH51 was loaded in each lane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (9% polyacrylamide) was used to separate the Gag species. Lysates from a PR active site mutant (derived from pGM17) (16) are shown for comparison. pRS refers to plasmid pRS326, a 2µm, URA3-marked plasmid. The Gag immunoreactive band in the fst1 lane migrates as a tight doublet. The doublet cannot be resolved to a singlet with phosphatase treatment. The mobilities of the upper band in the fst1 doublet and of the 56-kDa fst2 species are consistent with the predicted mobilities of the unprocessed Gag species for these mutants. WT, wild type.

View larger version (91K): [in this window] [in a new window]   FIG. 5.   The frameshift transplant mutants reverse transcribe Ty1-neo mRNA. Total yeast nucleic acids from strain YH51 were digested with EcoRI, treated with RNase A, and separated on a 1% agarose gel. This gel was probed with 32P-labeled DNA generated from an XhoI-HindIII fragment of the Tn903 neo gene via random hexamer labeling. The lane background results from Ty1-neo insertions into yeast genomic DNA. WT, wild type.

View larger version (32K): [in this window] [in a new window]   FIG. 6.   Schematic of frameshift transplant s7 and s8 double mutants. Transposition frequencies in strain YH51 are indicated. The transposition frequencies reported are typical and did not vary by more than 5% in any independent experiment. WT or wt, wild type.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

This study describes a novel technique called frameshift transplantation. This technique allows a previously unattainable insight into the relationship between the carboxy terminus of Ty1 CA and the amino terminus of the Ty1 PR and the p4 peptide. The amino-terminal sequence of the Ty1 PR is generated by the same cleavage that generates the carboxy terminus of Ty1 CA. Ty1 CA protein can be made in one of two ways: as the product of Gag-p49 processing or via processing of Gag-Pol-p199. We have shown that the cleavage site in both of these cases is the same. The p4 peptide liberated by the proteolysis of Gag-p49 to CA (Gag-p45) is shown to be dispensable for transposition. Mutations in the p4 region that block transposition are conclusively demonstrated to be uniquely attributable to an effect on the PR through the use of the frameshift transplant technique.

The exact positions of the Ty1 PR cleavage sites between PR and integrase (PR/IN) and integrase and reverse transcriptase (IN/RT) have previously been reported. The positions of these cleavage sites were recently confirmed by analysis of PR cleavage site mutants (14). The Gag-PR cleavage site was mapped based on the carboxy-terminal sequencing of endogenous Gag protein and confirmed by mass spectroscopic analysis (15). However, because we have never detected the p4 peptide in cell lysates or VLPs, it remained possible that additional events (such as multiple proteolytic cleavages) shaped the amino terminus of the PR. Heterologous expression of two different constructs confirms that the N terminus of the PR corresponds to its predicted position based on the C terminus of CA.

In this paper, the p4 region was shown to be dispensable for transposition both as part of the carboxy terminus of Gag and as an independent peptide. This was done by relocating the frameshift signal such that the p4 peptide sequence is expressed exclusively as part of the PR. The p4 peptide is similar in size to other retroviral nucleocapsid proteins, and it was thought that it might serve in a similar capacity. Recent evidence from our laboratory has shown the N-terminal segment of PR may facilitate at least one nucleocapsid-like function (J.F.L., G.M., and J.D.B., submitted for publication).

Ty1 cDNA can enter the host genome via targeted integration, an integrase-dependent event, or by homologous integration, a RAD52-dependent event (19). If the p4 region was necessary for targeted integration, it might have been possible for Ty1 cDNA synthesized by these mutants to enter the host genome via homologous recombination. However, we found that the frameshift transplant constructs were capable of transposition even in a rad52 spt3 strain background. This effectively rules out any role for the p4 region of Ty1 Gag and the p4 peptide in targeted integration.

Frameshift signals are found in a majority of retroviruses. In fact, the human T-cell leukemia virus type 1 and the mouse mammary tumor virus contain two independent frameshift signals. Many retroviruses position their frameshift signal between the structural and enzymatic components of the virus. The positioning typically ensures that structural components are synthesized in excess of the enzymatic components. The fact that we are able to relocate the frameshift signal and retain a functional retroelement raises interesting questions regarding how frameshifts arose in retroelement evolution. Both functional frameshift transplant constructs transpose at slightly reduced efficiencies, suggesting that the position of the native frameshift sequence may have evolved to maximize transposition frequency.

Among yeast retrotransposons, Ty1, the closely related Ty2 element, and the more distantly related Ty3 and Ty4 contain +1 frameshifting signals. Interestingly, only Ty1, Ty2, and Ty4 use the conserved 7-nucleotide motif CTT AGG C. The stop codon in Ty2 is predicted to produce a similar 4-kDa peptide by proteolysis. However, in Ty4, the Gag stop codon is located downstream of the PR active site residues. The peptide released from the Ty4 Gag C terminus is therefore considerably longer than its analogs in Ty1 and Ty2.

It has been shown that mutations in the C-terminal Gag-p6 peptide in human immunodeficiency virus might affect particle release from the cell, although little effort was made to distinguish between the effects of these mutations on the p6 and overlapping sequences (24). The generality of the frameshift transplant approach suggests that it can be used to genetically separate the effects of mutations that affect sequences present in multiple viral proteins.