No Role for Pepstatin-A-Sensitive Acidic Proteinases in Reovirus Infections of L or MDCK Cells

Abstract

Strong evidence indicates that virions of mammalian reoviruses undergo proteolytic processing by acid-dependent cellular proteinases as an essential step in productive infection. Proteolytic processing takes the form of a series of cleavages of outer-capsid proteins final sigma3 and mu1/mu1C. Previous studies showed an effect of both NH4Cl and E-64 on these cleavages, indicating that one or more of the acid-dependent cysteine proteinases in mammalian cells (cathepsins B and L, for example) is required; however, these studies did not address whether acid-dependent 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 aspartic proteinases, were performed. The results showed that pepstatin A neither blocks nor slows reovirus infection of L or MDCK cells. Experiments using ribonuclease A and other proteins as cleavable substrates showed that cathepsin-D-like proteinases from these cells are inhibited within the tested range of pepstatin A concentrations both in vitro and within living cells. In other experiments, virion-bound final sigma3 protein 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.

Adaptation of Reovirus to Growth in the Presence of Protease Inhibitor E64 Segregates with a Mutation in the Carboxy Terminus of Viral Outer-Capsid Protein ς3

“…5). Virions of wt reovirus are not susceptible to cathepsin D cleavage in vitro, and inhibition of cathepsin D does not affect reovirus entry in cell culture (18). As previously reported (18) (Fig.…”

“…To determine whether altered sensitivity to protease inhibitor E64 during endocytic processing of D-EA viruses correlates with altered susceptibility to protease treatment in vitro, virions of wt and D-EA viruses were treated with either of the endocytic proteases cathepsin L or cathepsin D. Cathepsin L is a cysteine protease that productively cleaves wt virions to functional ISVPs (2), whereas cathepsin D is an aspartic protease that does not digest wt virions (18). Virions were treated with purified recombinant human cathepsin L (8) for various intervals, and digestion of viral outer-capsid proteins was monitored by SDS-PAGE (Fig.…”

Cathepsin L and Cathepsin B Mediate Reovirus Disassembly in Murine Fibroblast Cells

“…Previous studies suggest that specific endocytic proteases mediate disassembly of reovirus virions to ISVPs. Cysteine protease inhibitor E64 blocks reovirus disassembly (24 -26), but aspartic protease inhibitor pepstatin A does not (29). Mutant cells selected during persistent reovirus infection do not support reovirus disassembly despite internalizing and transporting virions to acidified, perinuclear compartments.…”

“…Treatment of reovirus virions with purified cathepsin L leads to formation of particles that have the biochemical and growth properties of ISVPs generated in vitro by treatment of virions with intestinal proteases (27). In contrast, treatment of cells with the aspartic protease inhibitor pepstatin A (28) does not alter reovirus entry and growth (29), and treatment of virions with cathepsin D does not result in generation of ISVPs (26,29). These findings suggest that endocytic cysteine proteases but not aspartic proteases are required for reovirus disassembly.…”

Secretory ribonucleases are internalized by a dynamin-independent endocytic pathway

“…A19C RNase A and D16C ONC were purified as described for the wild-type proteins but with the following modifications (Kothandaraman et al, 1998). Refolding solutions were saturated with Ar(g) to remove O2(g).…”