Our long-range goal is to characterize a family of large (>330 kb) dsDNA, plaque-forming viruses, which infect unicellular, eukaryotic chiorella-like green algae. The type chiorella virus, PBCV-1, encodes 375 proteins and 10 tRNAs. Many PBCV-1 encoded enzymes are the smallest representatives of their functional class. Consequently some of these enzymes are the subject of intense physical and biochemical investigation. The chlorella viruses have other unusual features including: i) They encode multiple DNA methyltransferases and DNA restriction endonucleases. ii) They are the first viruses with three types of introns in their genome. iii) Phylogenetic analyses suggest these viruses have a long evolutionary history, indicating that virus-specific pathways may closely resemble ancestral forms. This proposal focuses on two features that are unique to these viruses. First, unlike other glycoprotein containing viruses, PBCV-1 encodes most of the machinery to glycosylate its major capsid protein. Furthermore, accumulating experimental evidence indicates that glycosylation is cytoplasmic and does not involve the ER or Golgi. We propose to continue investigating three aspects of this glycosylation process: i) To identify and characterize the PBCV-1 genes involved in glycosylation. ii) To determine the intracellular location of protein glycosylation. iii) To determine the structure of the complex glycan. These experiments will provide a solid foundation to begin investigating the biosynthesis of the oligosaccharide. Second, PBCV-1 encodes a 94 amino acid protein, named Kcv, that forms a K+ selective channel in frog oocytes. The Kcv protein is the smallest K+ channel protein identified to date and phylogenetic analyses indicate that Kcv may be a primitive form of more complex ion channel proteins. We propose to initiate studies on the role of Kcv in PBCV-1 replication.
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