title: Engineered picornavirus VPg-RNA substrates: analysis of a tyrosyl-RNA phosphodiesterase activity.
abstract: Using poliovirus, the prototypic member of Picornaviridae, we have further characterized a host cell enzymatic activity found in uninfected cells, termed "unlinkase," that recognizes and cleaves the unique 5' tyrosyl-RNA phosphodiester bond found at the 5' end of picornavirus virion RNAs. This bond connects VPg, a viral-encoded protein primer essential for RNA replication, to the viral RNA; it is cleaved from virion RNA prior to its engaging in protein synthesis as mRNA. Due to VPg retention on nascent RNA strands and replication templates, but not on viral mRNA, we hypothesize that picornaviruses utilize unlinkase activity as a means of controlling the ratio of viral RNAs that are translated versus those that either serve as RNA replication templates or are encapsidated. To test our hypothesis and further characterize this enzyme, we have developed a novel assay to detect unlinkase activity. We demonstrate that unlinkase activity can be detected using this assay, that this unique activity remains unchanged over the course of a poliovirus infection in HeLa cells, and that unlinkase activity is unaffected by the presence of exogenous VPg or anti-VPg antibodies. Furthermore, we have determined that unlinkase recognizes and cleaves a human rhinovirus-poliovirus chimeric substrate with the same efficiency as the poliovirus substrate.
authors.affiliation: Department of Microbiology and Molecular Genetics, School of Medicine, University of California Irvine, Irvine, California, United States of America.
authors.forename: Janet M
authors.initials: JM
authors.lastname: Rozovics
authors.forename: Richard
authors.initials: R
authors.lastname: Virgen-Slane
authors.forename: Bert L
authors.initials: BL
authors.lastname: Semler
mesh_terms: D000595:Amino Acid Sequence; D002457:Cell Extracts; D057075:Enzyme Assays; D005818:Genetic Engineering; D016679:Genome, Viral / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D017955:Poliovirus / Q000235:genetics* / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D012229:Rhinovirus / Q000378:metabolism; D006163:Ribonuclease T1 / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism; D014771:Virion / Q000378:metabolism
chemical_list: D002457:Cell Extracts; D050505:Mutant Proteins; D012367:RNA, Viral; C520979:VPg protein, poliovirus; D014764:Viral Proteins; D014443:Tyrosine; D006163:Ribonuclease T1; D010727:Phosphoric Diester Hydrolases