NIH Research Festival
There have been relatively few examples of viral O-GlcNAc proteins relative to the large numbers discovered in animals and plants. Here, we describe the presence of a novel O-GlcNAc protein in vaccinia virus (VACV) infectious particles. VACV, the prototypic member of the Poxviridae family, comprised the live-virus vaccine that eradicated smallpox and harbors a linear, double-stranded DNA genome encoding approximately 200 proteins. Early studies from our laboratory demonstrated the presence of a 40-kDa protein that contains N-acetylglucosamine in purified virions. The small size of the pronase-digestion product and the absence of other sugars suggested one or few glucosamines. In the present study, extracts of purified virions were enzymatically labeled utilizing the mutant Œ≤-1,4-galactosyltransferase (Gal-T1 (Y289L)) to specifically transfer azido-modified galactose (GalNAz) from UDP-GalNAz to O-GlcNAc residues. Following copper catalyzed azide-alkyne cycloaddition, the candidate GlcNAc proteins were detected by SDS-polyacrylamide gel electrophoresis and identified by mass spectrometry. Then using strain-promoted cycloaddition chemistry to attach a polyethylene glycol mass tag of 10 kDa on to the GlcNAc protein, a significant shift in the electrophoretic mobility of the VACV A4 protein was documented by Western blotting. The presence of O-GlcNAc and one major modification site in A4 was confirmed by mass spectrometry and specific antibody. Expression of A4 is essential for virus replication and is required for morphogenesis of mature virions. Further studies to determine the serine/threonine residues modified and to determine the role of GlcNAc in the function of A4 are in progress.
Scientific Focus Area: Virology
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