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ABSTRACTRetrotransposition of the Ty1 element of Saccharomyces cerevisiaeis temperature sensitive. Transposition activity of Ty1 is abolished at temperatures above 34°C. In this report, we show that the major block to transposition at high temperature is the inhibition of processing of the Gag-Pol-p199 polyprotein and the concomitant reduction of reverse transcriptase (RT) activity. Expression of a Ty1 protease construct in Escherichia colishows that protease enzymatic activity is inherently temperature sensitive. In yeast, Gag processing is only partially inhibited at high temperature, while cleavage of the Gag-Pol polyprotein is completely inhibited. Sites of proteolytic processing are differentially susceptible to cleavage during growth at high temperature. Overall levels of the Gag-Pol polyprotein are reduced at high temperature, although the efficiency of the requisite +1 frameshifting event appears to be increased. RT activity is inherently relatively temperature resistant, yet no cDNA is made at high temperature and the amount of RT activity is greatly reduced in virus-like particles formed at high temperature. Taken together, these results suggest that alterations in Ty1 proteins that occur at high temperature affect both protease activity and RT activity, such that Ty1 transposition is abolished.

ABSTRACTTy1 is the most successful of the five endogenous yeast retrotransposons. The life cycle of Ty1 dictates that a number of nucleocapsid (NC)-facilitated events occur although the protein(s) responsible for these events has not been identified. The positioning of the NC peptide is conserved at the carboxy terminus of the Gag protein among most long terminal repeat (LTR)-containing retroelements. An analogous region of Ty1 that simultaneously encodes part of Gag, protease (PR), and the C-terminal p4 peptide was mutagenized. Some of these mutations result in smaller-than-normal virus-like particles (VLPs). The mutants were also found to impair an NC-like functionality contained within the amino terminus of the protease that is distinct and separable from its proteolytic activity. Remarkably, these mutants have distinct defects in reverse transcription.

ABSTRACTThe yeast retrotransposon Ty1 encodes a 7-nucleotide RNA sequence that directs a programmed, +1 ribosomal frameshifting event required for Gag-Pol translation and retrotransposition. We report mutations that block frameshifting, which can be suppressed in cisby “transplanting” the frameshift signal to a position upstream of its native location. These “frameshift transplant” mutants transpose with only a modest decrease in efficiency, suggesting that the location of the frameshift signal in a functional Ty1 element may vary. The genomic architecture of Ty1 is such that Gag, Ty1 PR (PR), and the Gag-derived p4 peptide share a common sequence. The functional independence of the movement of the frameshift signal to a new location within the Ty1 element is used to unambiguously attribute the effect of mutations deleterious to transposition in this region of overlapping coding sequences to effects on the Ty1 (PR). This work defines the amino terminus of the Ty1 PR and introduces a new technique for studying viral genome organization.

ABSTRACTThe retroviral protease is a key enzyme in a viral multienzyme complex that initiates an ordered sequence of events leading to virus assembly and propagation. Viral peptides are initially synthesized as polyprotein precursors; these precursors undergo a number of proteolytic cleavages executed by the protease in a specific and presumably ordered manner. To determine the role of individual protease cleavage sites in Ty1, a retrotransposon from Saccharomyces cerevisiae, the cleavage sites were systematically mutagenized. Altering the cleavage sites of the yeast Ty1 retrotransposon produces mutants with distinct retrotransposition phenotypes. Blocking the Gag/PR site also blocks cleavage at the other two cleavage sites, PR/IN and IN/RT. In contrast, mutational block of the PR/IN or IN/RT sites does not prevent cleavage at the other two sites. Retrotransposons with mutations in each of these sites have transposition defects. Mutations in the PR/IN and IN/RT sites, but not in the Gag/PR site, can be complemented in transby endogenous Ty1 copies. Hence, the digestion of the Gag/PR site and release of the protease N terminus is a prerequisite for processing at the remaining sites; cleavage of PR/IN is not required for the cleavage of IN/RT, and vice versa. Of the three cleavage sites in the Gag-Pol precursor, the Gag/PR site is processed first. Thus, Ty1 Gag-Pol processing proceeds by an ordered pathway.