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J. Biol. Chem., Vol. 280, Issue 39, 33206-33212, September 30, 2005

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Chemical Rescue of I-site Cleavage in Living Cells and in Vitro Discriminates between the Cytomegalovirus Protease, Assemblin, and Its Precursor, pUL80a^*

From the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Chemical rescue is an established approach that offers a directed strategy for designing mutant enzymes in which activity can be restored by supplying an appropriate exogenous compound. This method has been used successfully to study a broad range of enzymes in vitro, but its application to living systems has received less attention. We have investigated the feasibility of using chemical rescue to make a conditional-lethal mutant of the cytomegalovirus (CMV) maturational protease. The 28-kDa CMV serine protease, assemblin, has a Ser-His-His catalytic triad and an internal (I) cleavage site near its midpoint. We found that imidazole can restore I-site cleavage to mutants inactivated by replacing the critical active site His with Ala or with Gly, which rescued better. Comparable rescue was observed for counterpart mutants of the human and simian CMV assemblin homologs and occurred in both living cells and in vitro. Cleavage was established to be at the correct site by amino acid sequencing and proceeded at 11%/h in bacteria and 30%/h in vitro. The same mutations were unresponsive to chemical rescue in the context of the assemblin precursor, pUL80a. This catalytic difference distinguishes the two forms of the CMV protease.

Received for publication, June 24, 2005 , and in revised form, July 20, 2005.

^* This work was supported in part by Public Health Service Research Grants AI13718 and AI32957 (to W. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Supported by a Howard Hughes undergraduate summer research fellowship.

² Supported by a Johns Hopkins Post-baccalaureate Research Education Program award though Public Health Service Grant GM61424.

³ To whom correspondence should be addressed: Dept. of Pharmacology and Molecular Sciences, 725 North Wolfe St., Baltimore, MD 21205. Tel.: 410-955-8680; Fax: 410-955-3023; E-mail: wgibson{at}jhmi.edu.

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