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Journal of Virology, January 2001, p. 638-644, Vol. 75, No. 2
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.2.638-644.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Ty1 Proteolytic Cleavage Sites Are Required for Transposition: All Sites Are Not Created Equal

Gennady V. Merkulov,dagger Joseph F. Lawler Jr., Yolanda Eby, and Jef D. Boeke*

Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received 7 June 2000/Accepted 28 September 2000

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.

* Corresponding author. Mailing address: Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, 617 Hunterian Bldg./725 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 955-0398. Fax: (410) 614-2987. E-mail: jboeke{at}jhmi.edu.

dagger Present address: Celera, Rockville, MD 20850.

Journal of Virology, January 2001, p. 638-644, Vol. 75, No. 2
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.2.638-644.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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