Proteolytic activation of the NiV F protein depends on clathrin-mediated endocytosis and subsequent cleavage at the monobasic cleavage site (R109
) by a pH-dependent endosomal protease (18
). Cathepsin L has been conclusively shown to act as an F cleavage enzyme using Vero cells as a model cell line (53
). Here, we show that in contrast to Vero cells, F activation in MDCK cells does not depend on cathepsin L but rather requires active cathepsin B. Since MDCK cells did not display any detectable cathepsin L activity, the different protease usage in these cells strongly argues for a redundant or alternative functional role of cathepsin B in NiV activation. The finding that functional F cleavage and NiV infection in MDCK cells were not prevented by nocodazole and the prominent colocalization of internalized F and cathepsin B with EEA-1, Rab4, and Rab11 strengthen the idea that NiV activation by cathepsin B occurs within early and recycling endosomes of MDCK cells and does not require trafficking through late endosomal compartments.
Despite a pronounced activity of cathepsin B in Vero cells, we did not see a significant effect of the cathepsin B inhibitor NS134P on NiV F cleavage () and virus release (). This is in principal agreement with the finding of Pager et al. (53
), reporting that cathepsin L is the only protease responsible for F activation in Vero cells. In contrast to MDCK cells, in which cathepsin B was found to functionally process NiV F0
, the readily expressed cathepsin B appeared not to be involved in NiV activation in Vero cells. As proposed by Pager et al. (53
), this might be due to an incorrect cleavage by cathepsin B in Vero cells. An alternative explanation could be a different compartmentalization of cathepsin B in Vero cells that does not allow sufficient contact with the NiV F protein or pH conditions in endosomes that prevent a proteolytic processing by cathepsin B at R109
Cathepsins L and B are ubiquitous cysteine proteases of the papain superfamily not only involved in bulk protein turnover in the lysosomal compartment but also critically engaged in proteolytic processing of specific substrates in endosomes like hormones, growth factors, and protein antigens (see reference 59
and references therein). Even though these processing steps require precise cleavage, cathepsins exhibit a broad substrate and cleavage site specificity, albeit some prefer certain amino acids to others in the target sequence. For example, cathepsin L prefers hydrophobic amino acids in P2 and basic residues in P1 (56
). In contrast, cathepsin B preferentially cleaves after two basic amino acid residues in P1 and P2, but efficient cleavage still takes place if P2 consists of a hydrophobic amino acid (23
). Analysis of the P4-P2′ NiV F cleavage site by the software tool PEPS (Prediction of Endopeptidase Substrates [37
]), which is based on known cathepsin B or cathepsin L cleavage sites in full-length protein substrates, revealed prediction scores of the NiV F cleavage site of 0.165 and 0.2 for cathepsin L and cathepsin B, respectively. Both scores indicate a more than 1,000-fold likelihood of NiV F cleavage by cathepsin B or L than by a random amino acid sequence. The major difference in the structures of cathepsins L and B is that the latter contains a so-called occluding loop that covers part of the catalytic center, thereby limiting access of substrate proteins for endoproteolytic cleavage. Recently, the cleavage specificities of cathepsins B and L on naturally occurring peptides have been reassessed by the proteomic approach PICS (proteomic identification of protease cleavage sites [5
]). Remarkably, the PICS cleavage site logo at pH 6 for cathepsin B contains 6 of the 8 amino acids present in the P4-P4′ NiV F cleavage site. The P3′ glycine in the NiV F site is especially typical for the cathepsin B endoprotease activity, because this small amino acid is not sterically hindered by the occluding loop. This enables access of the NiV F protein to the catalytic center of the protease and determines the specific cleavage site. In contrast, the PICS profile for cathepsin L highlights only 4 of the 8 residues present in the P4-P4′ NiV F cleavage site. These structure-substrate considerations indicate that cathepsin B, in addition to cathepsin L, is well suited for selective processing of NiV F. Although the amino acid sequence of the substrate itself strongly dictates its cleavage, specificity of cleavage is orchestrated by the biochemical environment, the set of cleaving enzymes, the time that a protein substrate resides within the same compartment, and the change of endosomal pH from almost neutral to acidic (29
). Due to different pH optima, proteases are active only at certain stages in the endolysosomal compartment (32
). While specific cleavage by cathepsin L is optimal at a slightly acidic pH of 5.5 to 6 and the protease is unstable under neutral conditions (43
), cathepsin B has a wider pH optimum (pH 4.5 to 7.0) (61
). Activity of cathepsin B endopeptidase is maximal between pH 6 and 7 (3
), thereby strongly supporting our model that cathepsin B-mediated NiV F cleavage occurs in early and recycling endosomes (pH 5.9 to 6.5) rather than in late endosomal compartments (pH 5.0 to 6.0).
Even though the substrate specificity and pH dependence of cathepsins L and B are not fully identical, they share many substrates. The idea of a partial redundancy of both enzymes was supported by studies with knockout mice. Here, the loss of one protease can be compensated for by the remaining protease (9
). Knockout mice deficient in only cathepsin B or cathepsin L presented only a few phenotypic alterations (58
), whereas double-knockout mice lacking both cathepsin L and cathepsin B died early due to CNS atrophy (21
). We also observed a redundant function of cathepsins L and B when we infected MEFs from knockout mice. Even though syncytium formation and release of infectious NiV in cathepsin L-knockout MEFs were drastically reduced and the reduction was more pronounced than that in MEFs lacking cathepsin B, productive virus replication was completely blocked only in the absence of both cathepsin L and cathepsin B. This again supports our view that both cathepsins have the ability to produce fusion-active NiV F proteins and further strengthens our model that cathepsin B can play an important role in NiV activation in cells lacking sufficient cathepsin L activity.
Redundant functions of cathepsins L and B, especially if one cathepsin is lacking, were also reported for the proteolytic processing of other viral proteins. For example, Ebola virus (EBOV) entry into Vero cells and MEFs requires glycoprotein digestion by cathepsins B and L (11
). 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
). From this it was concluded that both cathepsin B and cathepsin L can support MLV infection, with cathepsin B being the favored protease. These data are consistent with our idea of an alternative usage of cathepsins L and B for NiV activation, depending on cell type and availability of the respective protease. Recent studies on reovirus infections also highlighted distinct roles of cathepsins in vivo
. While reoviruses are known to utilize cathepsins B, S, and L for disassembly of the outer capsid to initiate productive replication, only cathepsin L was required for efficient virus growth in mouse brains in vivo
). The contribution of the two cathepsins for cell or organ tropism of NiV infection in vivo
might be an interesting question for the future.
Currently, no approved antiviral treatment for NiV infection is available, even though a variety of approaches have been investigated (for a review, see reference 76
). A more detailed understanding of NiV activation by host cell proteases could be a further starting point for developing antivirals acting against cellular targets. Due to the impact of several cathepsins in pathophysiological processes, including cancer, osteoarthritis, and autoimmune disorders, some promising results for the effectiveness of cathepsin inhibitors in tumor invasion, in osteoporosis prevention, and as immunomodulators have been obtained (15
). Short-term inhibition of cathepsins in the context of a potentially lethal Nipah virus infection might be a clinically feasible option. Since our data indicate that NiV can principally use cathepsins L and B and as it is not yet clear which protease is predominantly used in in vivo
infections, antiviral strategies should include inhibitors blocking both cathepsins.
Besides the restricted expression of cellular receptors, limited expression of activating proteases is an important factor for virus spread and cell tropism, thereby determining pathogenesis in vivo
). Our results showing that NiV can use two broadly expressed cathepsins, however, suggest that protease expression does not restrict NiV spread in vivo
. The ability to utilize either cathepsin L or cathepsin B likely allows the virus to principally replicate in any receptor-positive cell type.