Subcellular Localization and Calcium and pH Requirements for Proteolytic Processing of the Hendra Virus Fusion Protein

A number of enveloped viruses, including paramyxoviruses and retroviruses, enter host cells via a pH-independent mechanism that facilitates fusion of the viral envelope with a membrane of the target cell. In paramyxoviruses, this process is mediated by the fusion (F) protein. The F protein is a type I integral homotrimeric, N-glycosylated membrane protein that is synthesized as an inactive precursor protein (F0). F0 is proteolytically cleaved to the disulfide-bonded F1 and F2 forms. This proteolytic event is critical for exposure of the fusion peptide and production of the mature and fusogenic form of the F protein. Most paramyxovirus F proteins, including the simian virus 5 (SV5) F protein (20), the measles virus F protein (6), and the respiratory syncytial virus F protein (59), are proteolytically processed by furin. Furin is a secretory pathway protease that belongs to the family of mammalian subtilisin-like proprotein convertases (PCs). This membrane-associated, calcium (Ca2+)-dependent protease is known to process a wide range of precursor proteins within the exocytic and endocytic pathways at sites containing an Arg-X-Lys/Arg-Arg cleavage motif. Furin has additionally been described to proteolytically cleave a number of other viral glycoproteins, including the Ebola virus glycoprotein (Gp) (65), human immunodeficiency virus (HIV) gp160 (1, 28), the hemagglutinin (HA) protein of avian influenza virus strains H5 and H7 (58), and the cytomegalovirus glycoprotein B (63). Furthermore, a correlation of the efficiency of furin cleavage with virulence and pathogenicity has been described for both Newcastle disease virus (21) and Ebola virus (65). In contrast to intracellular proteolytic processing at a multibasic cleavage site by furin, a number of viral fusion proteins, such as Sendai virus F protein (31, 55) and H1, H2, and H3 subtypes of influenza virus HA protein (35), are cleaved at single basic residues by extracellular exogenous proteases. Cleavage of viral glycoproteins by proteases belonging to the pyrolysin branch of subtilisins has also been described (5, 37-39, 64). The Ca2+-dependent SKI-1/S1P protease was recently shown to proteolytically process the Lassa virus precursor protein (38, 39), the Crimean-Congo hemorrhagic fever virus glycoprotein (64), and the lymphocytic choriomeningitis virus glycoprotein (5, 37). SKI-1/S1P cleaves precursor proteins containing a conserved arginine at the fourth position and a hydrophobic amino acid at the second position prior to the cleavage motif and is predominantly active in the endoplasmic reticulum (ER), although the lymphocytic choriomeningitis virus glycoprotein is processed in a late Golgi compartment (5). Hendra virus is a newly emerged zoonotic paramyxovirus that was first isolated in 1994 in Australia during an outbreak of severe respiratory illness that resulted in the deaths of 14 horses and 1 human (48). A second human fatality occurred 13 months later due to viral encephalitis as a result of an initial Hendra virus infection (53). Nipah virus, a virus closely related to Hendra virus, was isolated as the etiological agent of the fatal viral encephalitis outbreak that caused 105 fatalities out of 265 human cases in Malaysia in 1999. More than one million pigs were culled in order to contain the Nipah virus epidemic (11). Fruit bats are suspected to be the natural hosts for both Hendra and Nipah viruses (12, 19). The high rates of mortality in humans and the ability of Hendra and Nipah viruses to infect a number of different hosts have contributed to their classification as biosafety level 4 agents. Genomic and phylogenetic studies demonstrated high homology between Hendra and Nipah viruses, yet limited identity with other viruses within the Paramyxovirinae subfamily. The broad host range and antigenic cross-reactivity between Hendra and Nipah viruses, together with genomic features such as the large genome, unique genome terminal sequences, and a unique motif within the L protein, have supported the creation and classification of Hendra and Nipah viruses into a new genus within the Paramyxovirinae subfamily, namely, Henipaviruses (29, 67). Hendra virus contains two glycoproteins, the attachment or G protein, which lacks both HA and neuraminidase activities, and the F protein. Similar to other paramyxoviruses, the Hendra virus F0 precursor protein is proteolytically cleaved into disulfide-linked subunits F1 and F2. The cleavage site (VGDVK109), predicted by amino acid sequence alignments, was confirmed by N-terminal sequencing of the F1 subunit (44). Cleavage of the closely related Nipah virus F protein similarly occurs after the basic residue arginine in the sequence VGDVR109 (28). Not only do the F proteins of both Hendra virus and Nipah virus lack the polybasic furin consensus motif, common to the majority of paramyxoviruses, but also the sequence at the site of proteolytic cleavage does not correspond to the recognition sequence of any known secretory protease. Successful growth of Hendra virus in the furin-deficient LoVo cell line confirmed that furin was not the protease involved in cleavage of the Hendra F protein (44). Furthermore, addition of exogenous trypsin did not affect propagation of Hendra virus in cell culture, indicating that an extracellular protease that cleaves at the basic residue is not required (44). In the present study, we have examined the subcellular location of cleavage as well as the Ca2+ and pH conditions required for efficient proteolytic processing of the Hendra F protein. We find that cleavage occurs either in the secretory vesicles budding from the trans-Golgi network (TGN) or at the cell surface. Cleavage is not significantly affected by decreases in cellular Ca2+ levels but is dramatically decreased by changes in intracellular pH. These differences suggest that the protease responsible for cleavage of the Hendra virus F protein differs from proteases previously shown to be involved in the processing of viral glycoproteins.