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J. Biol. Chem., Vol. 279, Issue 5, 3837-3851, January 30, 2004

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Cathepsin B Is Inhibited in Mutant Cells Selected during Persistent Reovirus Infection^*

Daniel H. Ebert, Sarah A. Kopecky-Bromberg¶||, and Terence S. Dermody¶**

From the Departments of Microbiology and Immunology and ^¶Pediatrics and Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Persistent reovirus infections of murine L929 (L) fibroblast cells select mutant (LX) cells that do not support proteolytic disassembly of reovirus virions within the endocytic pathway. To better understand the function and regulation of endocytic proteases, we conducted experiments to define the block to reovirus disassembly displayed by LX cells. In contrast to parental L cells, mutant LX cells harbor defects that interfere with the maturation and activity of cathepsin B and cathepsin L but not cathepsin H. The cDNAs encoding cathepsin B and cathepsin L in L cells are identical to those in LX cells, indicating that LX cells manifest an extrinsic block to the function of these enzymes. Mixed lysates of L cells and LX cells lack activity of both cathepsin B and cathepsin L, suggesting the presence of an inhibitor of cathepsin function in LX cells. A cathepsin B-green fluorescent protein (GFP) fusion protein expressed in L cells and purified by immunoprecipitation retains cathepsin B activity, whereas cathepsin B-GFP expressed in LX cells does not. However, activity of cathepsin B-GFP expressed in LX cells can be recovered by incubating the immunoprecipitate with L cell lysate followed by immunoprecipitation, providing further evidence that LX cells express a cathepsin inhibitor. Native-gel electrophoresis and gel filtration chromatography demonstrate that, in both cell lines, the double-chain form of cathepsin B is sequestered in a large molecular weight complex that renders this form of the enzyme inactive. Alteration of this sequestration complex appears to be responsible for inhibition of cathepsin B in LX cells. These findings suggest that cathepsins can be regulated within the endocytic pathway. Moreover, this regulation influences host cell susceptibility to intracellular pathogens.

Received for publication, September 10, 2003

^* This work was supported by the Elizabeth B. Lamb Center for Pediatric Research and Public Health Service Awards T32 GM07347 from the National Institute of General Medical Sciences for the Vanderbilt Medical Scientist Training Program (to D. H. E.) and T32 AI07474 (to S. A. K.-B.) and R01 AI32539 from the National Institute of Allergy and Infectious Diseases. Additional support was provided by Public Health Service Awards CA68485 for the Vanderbilt-Ingram Cancer Center and DK20593 for the Vanderbilt Diabetes Research and Training Center. 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.

^|| Current address: Dept. of Microbiology, Mount Sinai School of Medicine, New York, NY 10029.

^** To whom correspondence should be addressed: Lamb Center for Pediatric Research, D7235 MCN, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-343-9943; Fax: 615-343-9723; E-mail: terry.dermody{at}vanderbilt.edu.

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