Paramecium bursaria chlorella virus (PBCV-1) replicates in certain unicellular eukaryotic exsymbiotic Chorella-like green algae. PBCV-1 contains at least 50 proteins, three of which are glycosylated (Vp 54, the major capsid protein; Vp280 and Vp260). The oligosaccharide portion of these glycoproteins appears to be the major determinate in antibody inhibition of infection by PBCV-1. PBCV-1 has an interesting and apparently unusual method for glycosylating its glycoproteins. These glycoproteins appear to be unique for several reasons: 1) the virus rather than the host dictates glycosylation specificity; 2) at least some of the enzymes involved in the synthesis of the oligosaccharide are virus encoded, and; 3) PBCV-1 glycosylation may occur independently of the host ER and Golgi apparatus and occur in viral assembly centers (VACs) identified by EM immunocytochemistry.

The glycoproteins appear to be unique in that they are assembled independently of the host ER and Golgi apparatus and occur in viral assembly centers (VACs). The glycoproteins appear to be quite homogeneous (they produce sharp bands on SDS-PAGE, even though at least 50% of the weight of Vp260 is carbohydrate and preliminary MS studies of Vp54 (10% carbohydrate) give a sharp peak of mass 53,750). Carbohydrate analysis indicates the presence of Fuc, Ara/Rha, Gal, Glc, Xyl and Man. Preliminary results indicate that the PBCV-1 major capsid protein Vp54 contains either a single oligosaccharide of 32-34 sugars or two identical oligosaccharides of 16-17 sugars with the Xyl:Gal:Glc:Man:Ara/Rha:Fuc ratio of 2:3:3:3:2:3 and, furthermore, that the oligosaccharide may not be linked to the protein via classical N-linkage nor by a typical O-linkage. The working model described above explains the specificity of each antiserum, the different carbohydrate contents of the major capsid proteins from the various antigenic variants, and the mobility differences of the major capsid glycoproteins on SDS-PAGE. The model also predicts that the gene for each enzyme involved in linking the sugars can be independently mutated and that genetic crosses between appropriate serotypes should produce wild-type viruses.

The primary focus of our work begun this year is to rigorously characterize the oligosaccharides of various capsid glycoproteins and discover details of the antigenic determinate. Continuing goals include analysis of the sugar-protein linkage and determination of the glycosyltransferases responsible for synthesis of the oligosaccharides. We also expect to make significant progress detailing the intracellular locales of glycosyltransferase activity.