Expression and processing of proteins encoded by the Saccharomyces retrotransposon Ty5.

Ty5 is a Ty1/copia group retrotransposon of Saccharomyces (4, 30). Encoded between its long terminal repeats (LTRs) are homologues of retroviral gag and pol genes. For the retroviruses and LTR retrotransposons (collectively referred to as retroelements), gag specifies the structural proteins that form the viral core or the retrotransposon virus-like particle (VLP). Packaged within the particle are retroelement mRNAs and products of the pol gene. These include a protease (PR) that processes the retroelement polyproteins, a reverse transcriptase (RT) and its associated RNase H (RH) that synthesize a cDNA copy of the retroelement from the template mRNA, and an integrase (IN) that inserts the cDNA into a new site in the host chromosome. In addition to gag and pol, retroviruses have an env gene. The env gene product forms the viral envelope and allows the particle to exit the cell as a membrane-bound virion, which fuses with recipient cells during infection. Assembly of the viral core or virus-like particle requires an excess of “structural” Gag relative to “catalytic” Pol proteins (reviewed in references 9 and 18). For many viruses, the ratio of Gag to Pol approximates 15 to 1, and perturbing this ratio results in a significant loss in replication efficiency. Retroelements use a variety of expression strategies to regulate Gag and Pol expression, including two that are closely tied to protein synthesis—translational suppression and translational frameshifting. Translational suppression is employed by viruses such as murine leukemia virus, for which gag and pol are present in the same reading frame separated by a stop codon (33). Translation of murine leukemia virus mRNA predominantly produces Gag; however, occasionally the gag amber termination codon is decoded by a glutamine tRNA to produce a Gag-Pol fusion protein. Translational suppression is facilitated by downstream sequences that form a pseudoknot, which slows translation and enables the suppressor tRNA to better compete with translation release factors (17, 31). The second translational mechanism and most common strategy for regulating gag and pol expression is −1 and +1 frameshifting (reviewed in references 9 and 18). For retroelements that use frameshifting, the 3′ coding region of gag typically overlaps the 5′ coding region of pol in either the −1 or the +1 frame. Frameshifting is triggered by secondary structures (stem loops or pseudoknots) or rare codons in the mRNA that cause the ribosome to stall in the overlap region and then to slip forward or backward one nucleotide before continuing translation. Human immunodeficiency virus is an example of a virus that uses −1 ribosomal frameshifting (19). Retroelements that use +1 frameshifting include the yeast retrotransposons Ty1 and Ty3 (3, 10). The coding regions of some retrotransposons lack stop codons and internal frameshift sites. The ratio of Gag to Pol is nonetheless critical for these elements, and they use alternative strategies to regulate Gag and Pol expression. For example, the copia elements of Drosophila melanogaster produce a single genome-length mRNA that is subject to differential splicing (6, 34). The pol coding sequences are spliced from the majority of transcripts, leading to an abundance of gag mRNA and elevated levels of Gag protein. The Tf1 elements of Schizosaccharomyces pombe use a posttranslational strategy to regulate the stoichiometry of Gag and Pol (1, 25). A single Gag-Pol polyprotein is synthesized, which is processed to yield equimolar amounts of mature Tf1 proteins. As yeast cultures approach stationary phase, Pol is preferentially degraded, resulting in a 26:1 ratio of Gag to Pol. The degradation of Pol triggers Tf1 cDNA synthesis. Like copia and Tf1, Ty5 encodes a single open reading frame (ORF) and is the only Saccharomyces retrotransposon for which gag and pol are not separated by a +1 frameshift (36). Ty5 and copia are members of the Ty1/copia group of elements (Pseudoviridae), most of which contain a single ORF (4). In previous work, we demonstrated that Ty5 expresses a single mRNA, suggesting that differential mRNA splicing is not used by Ty5 to regulate Gag and Pol levels (36). In this study, we set out to characterize the Ty5 proteins and to assess possible mechanisms that regulate their expression.