NO ROLE FOR PEPSTATIN-A-SENSITIVE ACIDIC PROTEINASES IN REOVIRUS INFECTIONS OF L OR MDCK CELLS

Strong evidence indicates that virions of mammalian reoviruses undergo proteolytic processing by acid-dependent cellular proteinases as an e ssential step in productive infection. Proteolytic processing takes th e form of a series of cleavages of outer-capsid proteins sigma 3 and m u 1/mu 1C. Previous studies showed an effect of both NH4CI and E-64 on these cleavages, indicating that one or more of the acid-dependent cy steine proteinases in mammalian cells (cathepsins B and L, for example ) is required; however, these studies did not address whether acid-dep endent aspartic proteinases in those cells (cathepsin D, for example) may also be required. To determine the role of aspartic proteinases in reovirus entry, studies with pepstatin A, a specific inhibitor of asp artic proteinases, were performed. The results showed that pepstatin A neither blocks nor slows reovirus infection of L or MDCK cells. Exper iments using ribonuclease A and other proteins as cleavable substrates showed that cathepsin-D-like proteinases from these cells are inhibit ed within the tested range of pepstatin A concentrations both in vitro and within living cells. In other experiments, virion-bound sigma 3 p rotein was shown to be a poor substrate for cleavage by cathepsin D in vitro, consistent with the findings with inhibitors. In sum, the data indicate that cathepsin-D-like aspartic proteinases provide little or no activity toward proteolytic events required for infection of L or MDCK cells with reovirus virions. (C) 1998 Academic Press.