Cathepsin B Is Inhibited in Mutant Cells Selected during Persistent Reovirus Infection
Journal of Biological Chemistry
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
... d 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 incubat- ing 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. The protease cascades required for complement activation, hemostasis, programmed cell death, and replication of some viruses illustrate that protease activity must be tightly regulated. Mechanisms of regulation include synthesis as zymogens, blockade by specific inhibitors, kinetic instability, and sequestration in localized environments. Endocytic proteases play important roles in propagating signals from the cell surface, hydrolysis of phagocytosed substrates, and generation of antigen-specific immune responses (1, 2). Endocytic proteases also act on internalized microorganisms, in some cases mediating their destruction but in others removing surface components that allow subsequent steps in the infectious cycle (3) . This study analyzes the defect in endocytic protease activity exhibited by mutant (LX) cells 1 selected during persistent reovirus infection to provide new insights into regulation of these enzymes. Studies of persistent viral infections have contributed significantly to an understanding of viral replication and viral pathogenesis (reviewed in Ref. 4). Persistent reovirus infections of murine L929 (L) cells select viral and cellular mutations that affect acid-dependent proteolysis of viral outer-capsid proteins during viral entry (5-8). Following attachment to cell surface receptors sialic acid (9, 10) and junctional adhesion molecule 1 (11), reovirus virions are internalized into cells by receptormediated endocytosis (12-15). Within the endocytic pathway, host proteases catalyze the conversion of virions to infectious subvirion particles (ISVPs) by cleavage of viral outer-capsid proteins (13, (15) (16) (17) . In murine fibroblasts, either of the endocytic proteases cathepsin B or cathepsin L are required for viral disassembly (3). Reovirus entry is completed as ISVPs penetrate endosomal membranes, releasing the transcriptionally active viral core particle into the cytoplasm (18 -22). The mutant LX cells studied here were derived from L cells persistently infected with reovirus strain type 3 Dearing for 230 days (8). During the first 4 days in culture, the persistently infected cell line underwent an intense period of crisis in which most of the cells in the culture were lysed. Over the next 2-3 weeks, small colonies of cells became apparent, and these colonies eventually reached sufficient density to permit passage. After stabilization, the cell line produced titers of infectious virus between 10 6 and 10 8 plaque forming units/ml throughout its maintenance. By passage in medium containing a reovirusspecific antiserum, the cultures were cured of viral infection as documented by plaque assay, infectious center assay, viral antigen staining, and reverse transcription-polymerase chain reaction for viral RNA (5,    . LX cells were cloned from the cured culture by two cycles of limiting dilution (8). These cells are not infected with virus and do not contain viral proteins or RNA (8).