Subcellular localization and calcium and pH requirements for proteolytic processing of the Hendra virus fusion protein.
Proteolytic cleavage of the Hendra virus fusion (F) protein results in the formation of disulfide-linked F1 and F2 subunits, with cleavage occurring after residue K109 in the sequence GDVK/L. This unusual cleavage site and efficient propagation of Hendra virus in a furin-deficient cell line indicate that the Hendra F protein is not cleaved by furin, the protease responsible for proteolytic activation of many viral fusion proteins. To identify the subcellular site of Hendra F processing, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F were metabolically labeled and chased in the absence and presence of inhibitors of exocytosis. The addition of carbonyl-cyanide-3-chlorophenylhydrazone, monensin, brefeldin A, or NaF-AlCl3 or incubation of cells at 20 degrees C all inhibited processing of the Hendra F protein, suggesting that cleavage of Hendra F occurs either in secretory vesicles budding from the trans-Golgi network or at the cell surface. In contrast to proteolytic cleavage of the simian virus 5 (SV5) F protein by the Ca(2+)-dependent protease furin, proteolytic cleavage of the Hendra F protein was not significantly inhibited by decreases in Ca2+ levels following incubation with EGTA or A23187. However, in the presence of weak amines and H+ V-ATPase inhibitors, known to raise intracellular pH, cleavage of Hendra F protein was inhibited while processing of the SV5 F protein was not significantly affected. The subcellular location, sensitivity to pH changes, and decreased Ca2+ requirement suggest that the protease responsible for cleavage of Hendra F protein differs from proteases previously shown to be involved in the processing of other viral glycoproteins.
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0298, USA.
Cara Theresia
CT
Pager
Mark Allen
MA
Wurth
Rebecca Ellis
RE
Dutch
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D005089:Exocytosis; D045404:Hendra Virus / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011499:Protein Processing, Post-Translational; D022142:Secretory Vesicles / Q000378:metabolism; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000378:metabolism*
D014760:Viral Fusion Proteins; D002118:Calcium