Ty1 proteolytic cleavage sites are required for transposition: all sites are not created equal.
The 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 trans by 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.
Merkulov
G V
GV
Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Lawler
J F
JF
Eby
Y
Y
Boeke
J D
JD
D001665:Binding Sites; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D015151:Immunoblotting; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D018626:Retroelements / Q000235:genetics* / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014771:Virion / Q000502:physiology; D019065:Virus Assembly
D015683:Gene Products, gag; D011498:Protein Precursors; D018626:Retroelements; D019426:Integrases; D012194:RNA-Directed DNA Polymerase; D010450:Endopeptidases