Enveloped viruses must fuse the viral and cellular membranes to enter the cell. Understanding how
viral fusion proteins mediate entry will provide valuable information for
antiviral intervention to combat associated disease. The
avian sarcoma and leukosis virus envelope
glycoproteins, trimers composed of surface (SU) and transmembrane heterodimers, break the fusion process into several steps. First, interactions between SU and a
cell surface receptor at neutral pH trigger an initial conformational change in the viral
glycoprotein trimer followed by exposure to low pH enabling additional conformational changes to complete the fusion of the viral and cellular membranes. Here, we describe the structural characterization of the extracellular region of the subgroup A
avian sarcoma and leukosis viruses envelope
glycoproteins, SUATM129 produced in chicken DF-1 cells. We developed a simple, automated method for acquiring high resolution mass spectrometry data using electron capture dissociation conditions that preferentially cleave the
disulfide bond more readily than the
peptide backbone
amide bonds that enabled the identification of
disulfide-linked
peptides. Seven of nine
disulfide bonds were definitively assigned; the remaining two bonds were assigned to an adjacent pair of
cysteine residues. The first
cysteine of surface and the last
cysteine of the transmembrane form a
disulfide bond linking the heterodimer. The
surface glycoprotein contains a free
cysteine at residue 38 previously reported to be critical for virus entry. Eleven of 13 possible SUATM129 N-linked glycosylation sites were modified with
carbohydrate. This study demonstrates the utility of this simple yet powerful method for assigning
disulfide bonds in a complex
glycoprotein.