Primary evidence for ssGP expression was obtained from SDS-PAGE analysis (Fig. secretion of ssGP in tissue culture during EBOV contamination. Biochemical analysis of recombinant ssGP characterized this protein as a disulfide-linked homodimer that was exclusively N glycosylated. In conclusion, we have identified and characterized a new EBOV nonstructural glycoprotein, which is usually expressed as a Pungiolide A result of transcriptional editing of the GP gene. While ssGP appears to share comparable structural properties with sGP, it does not appear to have the same anti-inflammatory function on endothelial cells as sGP. INTRODUCTION (ZEBOV) is the type species of the genus in the family (11) and a causative agent Pungiolide A of a severe hemorrhagic fever in primates, with case fatality rates as high as 90% in humans (12). All Ebola viruses (EBOV) possess a nonsegmented, negative-sense RNA genome with seven linear genes that encode seven structural proteins. The nucleoprotein (NP), virion protein 30 (VP30) and VP35, and RNA-dependent RNA polymerase (L) are components of the nucleocapsid structures, which are the active transcription/replication complexes. VP24, VP40, and the transmembrane glycoprotein (GP1,2) are associated with the viral membrane. GP1,2 is the only surface protein and forms trimeric spikes that facilitate virus entry by receptor binding and fusion with target cells (39). EBOV undergoes site-specific transcriptional editing of the glycoprotein (GP) gene (see Fig. 1A), comparable to the RNA editing that is commonly observed in the phosphoprotein (P) genes of viruses from the family (16, 24). The primary product of the Rabbit Polyclonal to OR51G2 GP gene is the soluble glycoprotein (sGP), a nonstructural secreted glycoprotein, which is usually expressed from unedited RNA transcripts (40, 51). GP1,2 is usually expressed only following transcriptional editing, which occurs at a series of seven uridine residues within the genomic RNA, resulting in an additional adenosine (A) residue in the transcript (40, 51). The subsequent +1 shift results in an extended open reading frame (ORF). The expression of an additional, as yet unidentified nonstructural protein, designated small soluble glycoprotein (ssGP), has long been proposed. This product would occur as a result of transcriptional editing that leads to a +2 shift, resulting in a truncated ORF. Thus, all GP gene products have identical N-terminal primary sequences of 295 amino acids (aa) but differ at their C-terminal portions Pungiolide A following the transcriptional editing site (14, 39) (Fig. 1A). Open in a separate window Fig. 1. Ebola virus glycoprotein gene RNA editing results in multiple gene products. (A) Organization of the Ebola virus glycoprotein gene. (Top) Putative open reading frames (ORFs) for the different GP gene products (sGP, GP1,2, and ssGP). (Bottom) The primary structures of glycoprotein gene products are shown in an alignment of the primary amino acid sequences of sGP, GP1,2, and ssGP. All three proteins share the first 295 aa, including the signal peptide (aa 1 to 32), but differ in their carboxy-terminal portions. (B) Detection of ssGP transcripts and and ZEBOV replication and exhibited that ssGP was expressed and secreted during contamination. In addition, we decided that transcriptional editing was the mechanism of expression. Thus, we have identified a new EBOV nonstructural glycoprotein. Biochemical and structural characterization identified ssGP as a secreted homodimer made up of N-linked carbohydrates. Therefore, ssGP appears to have biochemical and structural features similar to those of sGP (1, 2, 10); however, ssGP did not rescue barrier function following treatment of endothelial cells with tumor necrosis factor alpha (TNF-)a function that has been described previously for sGP (56). This result suggests that these proteins do not share biological functions. (This work is usually part of the Ph.D. thesis of M. Mehedi, Department of Medical Microbiology, University of Manitoba, Winnipeg, Canada.) MATERIALS AND METHODS Virus, cell culture, and animals. ZEBOV strain Mayinga (GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”AF086833″,”term_id”:”10141003″,”term_text”:”AF086833″AF086833) was used for all infectious experiments. Vero E6 (a monkey kidney cell line), Huh7 (a human liver cell line), and 293T (a human embryonic kidney cell.