Our data here demonstrate that the endosomal/lysosomal protease cathepsin L is involved in proteolytic maturation of the Hendra virus F protein. The incubation of Vero cells expressing Hendra virus F in the presence of nonspecific and specific cathepsin inhibitors reduced the cleavage of Hendra virus F (Fig. ). shRNA to cathepsin L suppressed cellular protein levels and enzymatic activity (Fig. ) and gave dramatically reduced proteolytic processing of the Hendra virus F protein (Fig. ). The low fusogenic activity of Hendra virus F in shRNA-expressing cells paralleled negligible amounts of cleaved Hendra virus F (Fig. ). Finally, we demonstrated that purified cathepsin L can digest precursor Hendra virus F0
to corresponding F1
subunits (Fig. ). These results fit with our previous characterization of Hendra virus F processing (25
), as cathepsin L does not require Ca2+
but is highly sensitive to increases in intracellular pH.
Lysosomal cysteine cathepsin proteases historically are thought of as destructive enzymes involved in the degradation of intracellular and endocytosed proteins (17
). Despite the adverse association of cathepsin L with tumor metastasis (29
) and osteoporosis (26
), cathepsin L plays a number of important physiological roles in the cell, including major histocompatibility complex class II antigen presentation in cortical epithelial cells of the thymus (24
), epidermal homeostasis (26
), hair differentiation (27
), and proteolytic processing of the CDP/CUX transcription factor (10
) and prohormones (34
). Cathepsins have also been reported to affect some viral processes. Following receptor-mediated uptake of the nonenveloped reovirus, cathepsins B and L digest the outer capsid proteins to form infectious subvirion particles which are then able to enter cells (8
). Proteolytic digestion of Ebola virus GP by endosomal proteases was recently described (4
). Viral entry following a secondary proteolytic event of Ebola GP was described to depend primarily upon cathepsin B digestion, with cathepsin L cleavage augmenting viral entry. Our finding of a role for cathepsin L in primary proteolytic processing of a viral glycoprotein suggests a novel activity for this important cellular protease. Unlike many cellular proteases, cathepsin L has no distinctive substrate recognition site, although the endoprotease is thought to favor hydrophobic amino acids at P2 and P3 sites (reviewed in reference 17
). The cleavage of Hendra virus or Nipah virus F protein is thought to occur after a monobasic residue in the G-D-V-K/R sequence (18
). However, single mutations of the basic residue as well as of amino acids upstream of the cleavage site to alanine failed to eliminate henipavirus F processing (5a
). Only when six residues immediately upstream of arginine 109 in Nipah virus F were deleted was the cleavage of Nipah virus F ablated (19
). These mutagenesis studies therefore demonstrate that no single amino acid is essential for processing of these F proteins. The apparent lack of specificity for henipavirus F cleavage aligns with the absence of a specific cleavage motif for cathepsin L.
Cathepsin L is localized primarily to the endosomal/lysosomal pathway and recently was reported to be in the secretory granules of the regulated secretory pathway (17
). A functional endocytosis motif is present within the cytoplasmic tails of the Hendra virus and Nipah virus F proteins (17a
). Mutagenesis of this endocytosis motif did not affect the intracellular trafficking of Hendra virus F to the plasma membrane; however, the rate of endocytosis and proteolytic processing of Hendra virus F was significantly decreased (17a
). This indicates a novel mechanism of proteolytic maturation of Hendra virus F compared to those of many viral fusion proteins, which are either processed during exocytic trafficking (e.g., Ebola virus GP, human immunodeficiency virus gp160, and Lassa virus GP [16
]) or later cleaved by an extracellular protease (28
). We propose that proteolytic processing of Hendra virus F by cathepsin L occurs during recycling of the F protein between the endosome and the cell surface and not in the secretory pathway during initial transport to the cell surface. Interestingly, purified Hendra virions are reported to contain both cleaved and uncleaved fusion proteins (18
), suggesting that a percentage of Hendra virus F protein has undergone at least one round of recycling prior to budding of the virion from the cell surface.
The proteolytic processing of Hendra virus F by an endosomal protease and the presence of both uncleaved and cleaved F proteins on the surfaces of Hendra virions suggest that Hendra virus can utilize several mechanisms of entry. Enveloped viruses are thought to enter cells by one of two well-characterized pathways (7
). The first, pH-independent entry occurs following fusion of the viral envelope with the plasma membrane. The second pathway requires endocytosis of the virus, where the low pH in the endosome serves to trigger conformational changes in the fusion protein, which stimulate fusion of the viral envelope with the endosomal membrane. As with other members of the paramyxovirus family, the presence of cleaved F protein on the surfaces of Hendra virions may permit fusion at the plasma membrane, especially since the Hendra virus F protein can promote fusion at neutral pH (1
). However, the Hendra virus could also enter cells via the endocytic pathway, where uncleaved F protein on the virion surface could be processed by cathepsin L, leading to fusion of the viral envelope with the endosomal membrane promoted by newly cleaved F protein. In this case, the pH of the environment would not be required to trigger membrane fusion, but rather the acidic environment would allow efficient enzymatic processing of the F protein. The potential ability of the virus to enter cells at the plasma membrane and/or the endocytic system would ensure viral infectivity and efficient propagation in the host. Future experiments examining the role of cathepsin L in Hendra virus infection as well as investigations into the mode(s) of Hendra virus entry will address these important possibilities.
Hendra virus was first identified as the etiological agent causing the deaths of 14 horses and 1 human due to severe respiratory illness during an outbreak in Australia in 1994 (23
). The fatal viral encephalitis outbreak attributed to Nipah virus in Malaysia in 1999 caused 105 fatalities out of 265 reported cases (5
). Currently, no antiviral therapies are available. Our finding that cathepsin L carries out a crucial processing event on the Hendra virus F protein, combined with knowledge of the cathepsin L crystal structure, the availability of cathepsin inhibitors, and studies examining the effectiveness of these inhibitors on tumor invasion (29
) and osteoporosis prevention (35
), suggests the possible use of cathepsin L inhibitors as therapy for Hendra virus infection.