In the present study we have investigated processing and maturation of the nonstructural small glycoprotein (sGP) of Ebola virus. When sGP expressed from vaccinia virus vectors was analyzed by pulse-chase experiments using SDS-PAGE under reducing conditions, the mature form and two different precursors have been identified. First, the endoplasmic reticulum form sGP(er), full-length sGP with oligomannosidic N-glycans, was detected, sGP(er) was then replaced by the Golgi-specific precursor pre-sGP, full-length sGP containing complex N-glycans. This precursor was finally converted by proteolysis into mature sGP and a smaller cleavage fragment, Delta-peptide. Studies employing site-directed mutagenesis revealed that sGP was cleaved at a multibasic amino acid motif at positions 321 to 324 of the open reading frame. Cleavage was blocked by RVKR-chloromethyl ketone. Uncleaved pre-sGP forms a disulfide-linked homodimer and is secreted into the culture medium in the presence of the inhibitor as efficiently as proteolytically processed sGP. In vitro treatment of pre-sGP by purified recombinant furin resulted in efficient cleavage, confirming the importance of this proprotein convertase for the processing and maturation of sGP. Delta-peptide is also secreted into the culture medium and therefore represents a novel nonstructural expression product of the GP gene of Ebola virus. Both cleavage fragments contain sialic acid, but only Delta-peptide is highly O-glycosylated.
Copyright 1999 Academic Press.