title: In vitro translational analysis of genomic, defective, and satellite RNAs of Cryphonectria hypovirus 3-GH2.
abstract: Cryphonectria hypovirus 3-GH2 (CHV3-GH2) is a member of the fungal virus family Hypoviridae that differs from previously characterized members in having a single large open reading frame with the potential to encode a protein of 326 kDa from its 9.8-kb genome. The N-terminal portion of the ORF contains sequence motifs that are somewhat similar to papain-like proteinases identified in other hypoviruses. Translation of the ORF is predicted to release autocatalytically a 32.5-kDa protein. A defective RNA, predicted to encode a 91.6-kDa protein representing most of the N-terminal proteinase fused to the entire putative helicase domain, and two satellite RNAs, predicted to encode very small proteins, also are associated with CHV3-GH2 infected fungal cultures. We performed in vitro translation experiments to examine expression of these RNAs. Translation of three RT-PCR clones representing different lengths of the amino-terminal portion of the ORF of the genomic RNA resulted in autocatalytic release of the predicted 32.5-kDa protein. Site-directed mutagenesis was used to map the processing site between Gly(297) and Thr(298). In vitro translation of multiple independent cDNA clones of CHV3-GH2-defective RNA 2 resulted in protein products of approximately 92 kDa, predicted to be the full-length translation product, 32 kDa, predicted to represent the N-terminal proteinase, and 60 kDa, predicted to represent the C-terminal two-thirds of the full-length product. In vitro translation of cDNA clones representing satellite RNA 4 resulted in products of slightly less than 10 kDa, consistent with the predicted 9.4 kDa product.
authors.affiliation: Department of Plant Pathology, Cook College, Foran Hall, Rutgers University, 59 Dudley Rd., New Brunswick, New Jersey 08901-8520, USA.
authors.forename: W
authors.initials: W
authors.lastname: Yuan
authors.forename: B I
authors.initials: BI
authors.lastname: Hillman
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003673:Defective Viruses / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D005658:Fungi / Q000821:virology*; D016679:Genome, Viral; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012328:RNA Viruses / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D019244:RNA, Satellite / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D014158:Transcription, Genetic; D014197:Trees / Q000382:microbiology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D019244:RNA, Satellite; D012367:RNA, Viral; D014764:Viral Proteins