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.
Highlights
The presence of protein-nucleic acid covalent bonds is not unusual in viral pathogens
There is evidence for a tyrosyl-RNA phosphodiesterase activity in eukaryotic cells. Existence of this enzyme is demonstrated by the efficient removal of the picornavirus protein VPg, which is attached to the genomic RNA via an O4-(59-Uridylyl)tyrosine bond, from the 59 end of the viral genome (Figure 1) [17,18]
We report that unlinkase activity can be detected using this assay and confirm previous reports that the fulllength virion RNA is a much more efficient substrate than a VPgnonanucleotide substrate. We demonstrate that this unique activity remains unchanged over the course of a poliovirus infection in HeLa cells and that it is unaffected by the presence of exogenous VPg or anti-VPg antibodies
Summary
The presence of protein-nucleic acid covalent bonds is not unusual in viral pathogens. The occurrence of protein-nucleic acid covalent bonds in uninfected cells is slightly more novel These linkages result from the failure of a topoisomerase to dissociate properly from target DNA [for review see [12]]. In such instances additional enzymes, termed tyrosyl-DNA phosphodiesterases, are required to resolve the phosphodiester bond formed with the nucleic acid. There is evidence for a tyrosyl-RNA phosphodiesterase activity in eukaryotic cells Existence of this enzyme is demonstrated by the efficient removal of the picornavirus protein VPg, which is attached to the genomic RNA via an O4-(59-Uridylyl)tyrosine bond, from the 59 end of the viral genome (Figure 1) [17,18]. This activity has been given several names in the literature, including unlinking enzyme [18], VPg unlinkase [19], and uridylylpolynucleotide-(59 P-.O)tyrosine phosphodiesterase (Y-pUpN PDE) [20], in this manuscript we will refer to the enzymatic activity as ‘‘unlinkase.’’
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