Investigation of the proteolytic processing of the Hendra virus fusion protein

The Hendra virus (HeV) was initially identified following an outbreak of severe respiratory illness in 1994. HeV is closely related to another newly identified virus the Nipah virus (NiV). A large genome containing large untranslated regions is among the attributes which determined the incorporation of the two viruses into the new genus of the paramyxovirus subfamily, the Henipaviruses. The HeV genome consists of a single negative strand of RNA and encodes two surface glycoproteins, the attachment protein (G) and the fusion protein (F).;The Hendra virus fusion (HeV F) protein is cleaved C-terminal to a single basic amino acid in the sequence VGDVK109. Unlike the majority of paramyxovirus F proteins, HeV F is not cleaved by furin after a multibasic sequence of amino acids. Unlike other paramyxovirus fusion proteins cleaved after a single basic residue, the HeV F protein is cleaved in cultured cell lines and multiple rounds of HeV infection can be obtained without the addition of exogenously added trypsin. Substitution of the basic residue upstream of the cleavage site of other paramyxovirus F proteins results in the inhibition of cleavage. However, mutation of the lysine at position 109 to alanine fails to eliminate cleavage of the HeV F protein. Individual mutation of seven additional residues upstream of the cleavage site also did not ablate cleavage. Complete elimination of the sequence upstream of the cleavage site through simultaneous mutation of these eight residues to alanine failed to disrupt processing, indicating that proteolytic recognition and cleavage of HeV F protein is not dependent on a specific animo acid sequence.;Following the completion of mutational analysis studies, cathepsin L was identified as the protease involved in the processing of HeV F. To determine if mutant HeV F proteins could serve as a substrate for cathepsin L, uncleaved wildtype and mutant HeV F proteins were generated by incubating cells at 20°C following metabolic labeling. All mutant HeV F proteins underwent cleavage by purified catehpsin L similar to wildtype protein. Together these results indicate that recognition is not based on a specific sequence upstream of the activation site but on another determinating factor(s).