Host Cell Cathepsins Potentiate Moloney Murine Leukemia Virus Infection

Moloney murine leukemia virus (MLV) has been widely used as the prototype ecotropic MLV in studies of retroviral replication. Its genome, structural and enzymatic proteins, and envelope protein (Env) also serve as the bases for many recombinant retroviral vectors. Infection begins with binding of virion Env to the high-affinity ecotropic virus receptor, which is thought to produce conformation changes in the envelope protein that lead to fusion of the viral and cellular membranes for penetration of the virion core. Studies of the pH dependence of Moloney MLV infection suggested that entry events may be more complex than a straightforward scenario of receptor triggering of Env. The seemingly paradoxical observations gave rise to the idea that a host cell protease might be involved in ecotropic MLV infection. The initial finding was Klaus Andersen's report that a protease inhibitor reduced infection (2). That same year, Anderson and Nexo reported that ecotropic MLV infection was inhibited by the weak base NH4Cl in SC-1 cells (3), a finding later confirmed by McClure and coworkers in NIH 3T3 and other normal mouse and rat cells (17). Paradoxically, McClure and coworkers found that low pH did not trigger Moloney MLV-induced cell-cell fusion; rather, the fusion occurred at neutral pH (17), suggesting that acidic pH was not involved directly. More puzzling still was their finding that unlike in other host cells, NH4Cl did not inhibit infection in rat XC cells (17). They reconciled these unusual findings by proposing that XC cells express a cell surface protease normally found in intracellular compartments that is important to infection and so are able to escape NH4Cl inhibition (17). In line with this possibility, limited trypsin exposure increased cell-cell fusion (4), and it was found that by 6 h postinfection, some of the surface subunit (SU) of the viral glycoprotein was cleaved into smaller fragments (1). However, the fate of SU at times less than 6 h postinfection was not determined, nor was it shown if the cleavage at 6 h was postentry degradation of the glycoprotein versus a beneficial event to the virus. To date, the question of cellular protease involvement in ecotropic MLV infection remains unanswered. No protease has been identified, and its role in infection has not been determined. We considered the suggestion of McClure et al. that XC cells may express the critical proteases inappropriately on their surfaces and searched the literature for clues as to what proteases these cells might overexpress. Originally derived from a sarcoma induced by Rous sarcoma virus, XC cells are highly transformed as a result of viral src gene expression (23, 26, 27). Notably viral-src-transformed cells secrete cellular proteases, particularly cathepsins (13, 25), suggesting that they comprise at least one type of protease that is secreted by XC cells and thus is accessible at the cell surface. Cathepsins are a large family of lysosomal proteases and include aspartyl, serinyl, and cysteinyl proteases that show the highest activity at pH 5 to 6 (29). This property and their location in the lysosomal compartment suggest that cathespins would be less active within NH4Cl- and other weak-base-treated cells but might remain highly active on the cell surface, where the weak bases have no effect. Thus, we examined the possibility that host cell cathepsins are involved in Moloney MLV infection. Here, we report results identifying cathepsin B as a host cell protease involved in the membrane fusion step of ecotropic Moloney MLV infection. Cathepsin B appears to act on the Moloney MLV envelope protein by cleaving SU, but not TM or CA, into specific fragments. Evidence is also presented that another as-yet-unidentified cathepsin is involved at a different step in infection that is envelope independent. This step is likely to be a postpenetration step, such as uncoating or viral-DNA synthesis.