Host Cell Cathepsins Potentiate Moloney Murine Leukemia Virus Infection

Abstract

The roles of cellular proteases in Moloney murine leukemia virus (MLV) infection were investigated using MLV particles pseudotyped with vesicular stomatitis virus (VSV) G glycoprotein as a control for effects on core MLV particles versus effects specific to Moloney MLV envelope protein (Env). The broad-spectrum inhibitors cathepsin inhibitor III and E-64d gave comparable dose-dependent inhibition of Moloney MLV Env and VSV G pseudotypes, suggesting that the decrease did not involve the envelope protein. Whereas, CA-074 Me gave a biphasic response that differentiated between Moloney MLV Env and VSV G at low concentrations, at which the drug is highly selective for cathepsin B, but was similar for both glycoproteins at higher concentrations, at which CA-074 Me inhibits other cathepsins. Moloney MLV infection was lower on cathepsin B knockout fibroblasts than wild-type cells, whereas VSV G infection was not reduced on the B ؊/؊ cells. Taken together, these results support the notion that cathepsin B acts at an envelope-dependent step while another cathepsin acts at an envelope-independent step, such as uncoating or viral-DNA synthesis. Virus binding was not affected by CA-074 Me, whereas syncytium induction was inhibited in a dose-dependent manner, consistent with cathepsin B involvement in membrane fusion. Western blot analysis revealed specific cathepsin B cleavage of SU in vitro, while TM and CA remained intact. Infection could be enhanced by preincubation of Moloney MLV with cathepsin B, consistent with SU cleavage potentiating infection. These data suggested that during infection of NIH 3T3 cells, endocytosis brings Moloney MLV to early lysosomes, where the virus encounters cellular proteases, including cathepsin B, that cleave SU.

Activation of the Nipah Virus Fusion Protein in MDCK Cells Is Mediated by Cathepsin B within the Endosome-Recycling Compartment

“…However, cathepsin L-deficient human monocyte-derived dendritic cells also supported EBOV entry, thus indicating that cathepsin L is dispensable for infection in these cells (41). In Moloney murine leukemia virus (MLV) infection, virus entry is facilitated by cathepsin B, which cleaves the surface unit SU in the early endosome (36). While inhibition of cathepsin L had no effect on infection in cells expressing both cathepsin B and cathepsin L, a block of cathepsin L significantly inhibited MLV infection in cathepsin B-deficient NIH 3T3 cells (81).…”

“…Besides cathepsin L, the only other cysteine protease known to be widely expressed in the endolysosomal compartment and shown to process substrates at basic cleavage sites is cathepsin B (2). Since cathepsins B and L have been shown to have redundant or synergistic functions in several viral infections (11,19,36,64), we wanted to evaluate the role of cathepsin B in NiV infection. In agreement with reports that cathepsin B is the most abundant cysteine protease in most cell types (32,73), we detected high cathepsin B activities in MDCK cells (Fig.…”

Critical Role of Leucine-Valine Change in Distinct Low pH Requirements for Membrane Fusion between Two Related Retrovirus Envelopes

“…Upon receptor binding, a conformational change occurs in the SU subunit of Env, leading to large scale conformational rearrangements of TM that drive membrane fusion (3). Although most retroviruses are currently believed to fuse directly at the plasma membrane (8), several retroviruses have been found to require a low pH (8 -13), low pH-dependent protease activities (14), or receptor priming plus low pH (15,16) for fusion activation. The underlying mechanisms for these pHdependent fusion activation pathways are currently not well defined.…”

Receptor Binding and Low pH Coactivate Oncogenic Retrovirus Envelope-Mediated Fusion

“…Most retroviruses use a pH-independent pathway for entry, during which receptor binding relieves the ability of SU to restrain TM, resulting in conformational changes in TM and subsequent fusion with the cell membrane (11). Interestingly, increasing numbers of retroviruses have recently been shown to require a low pH (3,15,24,28,31) or pH-dependent protease activities to trigger fusion (18); the latter property has also been demonstrated for some other enveloped viruses (2,14,18,26,27,33,34). Among these, avian sarcoma leukosis virus (ASLV) is unique in that it uses a two-step mechanism for fusion, in which receptor binding primes the second trigger of low pH (24).…”