Identification of the cellular protease involved in the proteolytic activation of henipavirus fusion proteins

Hendra and Nipah virus are two newly emergent paramyxoviruses associated with severe respiratory disease and encephalitis. Entry of these viruses into cells occurs via fusion of the viral envelope with a cellular membrane. This entry mechanism requires both the attachment (G) and fusion (F) glycoproteins. Proteolytic processing of the precursor F protein to the F1 + F 2 disulfide-linked heterodimer is essential to produce a mature and fusogenic form of the F protein. Although many paramyxovirus F proteins are proteolytically processed by the cellular protease furin at a multibasic cleavage motif, cleavage of Hendra and Nipah virus F proteins occurs by a previously unidentified cellular protease following a single lysine or arginine at residue 109 respectively, in the sequence GDVK/R↓L. This dissertation examines the subcellular location of Hendra F processing and identifies the endosomal protease cathepsin L as the protease involved in the maturation of both the Hendra and Nipah virus F proteins.;The subcellular location of cleavage as well as the Ca2+ and pH conditions required for efficient proteolytic processing of the Hendra F protein was examined in transfected Vero cells via metabolic labeling in the absence and presence of specific inhibitors. Cleavage of Hendra F was found to occur after budding of the secretory vesicles from the transGolgi network. Additionally proteolytic processing was not significantly affected by decreases in cellular Ca2+ levels but was dramatically decreased by changes in intracellular pH.;Protease inhibitors and small hairpin RNA oligonucleotides were used to identify cathepsin L as the protease involved in proteolytic processing of Hendra and Nipah virus F proteins. Purified cathepsin L was shown to activate Hendra and Nipah F in vitro and a knock-down of cathepsin L expression and activity affects cleavage and thus the fusion activity of both Hendra and Nipah F proteins. These studies introduce both a novel mechanism for primary proteolytic processing of viral glycoproteins, and also suggest a previously unreported biological role for cathepsin L.