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Engineered picornavirus VPg-RNA substrates: analysis of a tyrosyl-RNA phosphodiesterase activity.

Rozovics JM, Virgen-Slane R, Semler BL - PLoS ONE (2011)

Bottom Line: 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.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.Furthermore, we have determined that unlinkase recognizes and cleaves a human rhinovirus-poliovirus chimeric substrate with the same efficiency as the poliovirus substrate.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, School of Medicine, University of California Irvine, Irvine, California, United States of America.

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.

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Unlinkase activity on a poliovirus versus a rhinovirus substrate.(A) VPg sequences of HRV14 and S1-VPgR1 (wild type poliovirus mutated to encode an HRV14 VPg that encodes two methionine residues). (B) Single-cycle growth assay of wild type poliovirus (closed circles) and S1-VPgR1 (closed squares). 35S-methionine radiolabeled W1-VPg 31 or S1-VPgR1 substrate that was treated with RNase T1 (C) or untreated (D) was incubated with increasing amounts of total protein from a partially-purified fraction of unlinkase activity (Fraction CA) ((C) 0, 5, 10, and 20 µg and (D) 0, 0.5, 1, 2, 5 and 10 µg) to demonstrate that unlinkase activity recognizes each substrate equally.
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pone-0016559-g007: Unlinkase activity on a poliovirus versus a rhinovirus substrate.(A) VPg sequences of HRV14 and S1-VPgR1 (wild type poliovirus mutated to encode an HRV14 VPg that encodes two methionine residues). (B) Single-cycle growth assay of wild type poliovirus (closed circles) and S1-VPgR1 (closed squares). 35S-methionine radiolabeled W1-VPg 31 or S1-VPgR1 substrate that was treated with RNase T1 (C) or untreated (D) was incubated with increasing amounts of total protein from a partially-purified fraction of unlinkase activity (Fraction CA) ((C) 0, 5, 10, and 20 µg and (D) 0, 0.5, 1, 2, 5 and 10 µg) to demonstrate that unlinkase activity recognizes each substrate equally.

Mentions: To determine if unlinkase activity can recognize and cleave a human rhinovirus substrate, we developed a chimeric virus consisting of a poliovirus genome that encodes a human rhinovirus 14 (HRV 14) VPg; the rhinovirus VPg was also mutated to encode two methionine residues for radiolabeling. We developed this chimeric virus, termed S1-VPgR1 (Figure 7A), due to the difficulty of generating sufficient amounts of purified virion RNA from human rhinovirus-infected HeLa cells. Growth kinetics determined that this virus chimera has a somewhat slower growth phenotype at four hours post infection when compared to wild type poliovirus in a single-cycle growth assay, but this difference is eliminated by six hours post infection, when virions are harvested for virus RNA purification (Figure 7B). S1-VPgR1 was used to infect HeLa cells in the presence of 35S-methionine, and virion RNA was purified. T1-treated virion RNA (Figure 7C) or full-length virion RNA (Figure 7D) from both W1-VPg31 and S1-VPgR1 radiolabeled substrates was incubated with an unlinkase source to determine if cleavage efficiency differed between the two viruses. RNase T1-generated substrates from both viruses (Figure 7C) were incubated with 0, 5, 10 or 20 µg of total protein from an enriched unlinkase activity source (Fraction CA), and no significant difference in cleavage efficiency between the two substrates was observed (Figure 7C compare lanes 4 and 8). Full-length substrates from both viruses (Figure 7D) were incubated with 0.5, 1, 2, 5, or 10 µg of total protein from the same enriched unlinkase activity source, and again no significant differences in cleavage between the two substrates were observed (Figure 7D compare lanes 6 and 12). No difference was observed for unlinkase cleavage of either substrate, leading to the conclusion that this enzyme can recognize and cleave a human rhinovirus substrate.


Engineered picornavirus VPg-RNA substrates: analysis of a tyrosyl-RNA phosphodiesterase activity.

Rozovics JM, Virgen-Slane R, Semler BL - PLoS ONE (2011)

Unlinkase activity on a poliovirus versus a rhinovirus substrate.(A) VPg sequences of HRV14 and S1-VPgR1 (wild type poliovirus mutated to encode an HRV14 VPg that encodes two methionine residues). (B) Single-cycle growth assay of wild type poliovirus (closed circles) and S1-VPgR1 (closed squares). 35S-methionine radiolabeled W1-VPg 31 or S1-VPgR1 substrate that was treated with RNase T1 (C) or untreated (D) was incubated with increasing amounts of total protein from a partially-purified fraction of unlinkase activity (Fraction CA) ((C) 0, 5, 10, and 20 µg and (D) 0, 0.5, 1, 2, 5 and 10 µg) to demonstrate that unlinkase activity recognizes each substrate equally.
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pone-0016559-g007: Unlinkase activity on a poliovirus versus a rhinovirus substrate.(A) VPg sequences of HRV14 and S1-VPgR1 (wild type poliovirus mutated to encode an HRV14 VPg that encodes two methionine residues). (B) Single-cycle growth assay of wild type poliovirus (closed circles) and S1-VPgR1 (closed squares). 35S-methionine radiolabeled W1-VPg 31 or S1-VPgR1 substrate that was treated with RNase T1 (C) or untreated (D) was incubated with increasing amounts of total protein from a partially-purified fraction of unlinkase activity (Fraction CA) ((C) 0, 5, 10, and 20 µg and (D) 0, 0.5, 1, 2, 5 and 10 µg) to demonstrate that unlinkase activity recognizes each substrate equally.
Mentions: To determine if unlinkase activity can recognize and cleave a human rhinovirus substrate, we developed a chimeric virus consisting of a poliovirus genome that encodes a human rhinovirus 14 (HRV 14) VPg; the rhinovirus VPg was also mutated to encode two methionine residues for radiolabeling. We developed this chimeric virus, termed S1-VPgR1 (Figure 7A), due to the difficulty of generating sufficient amounts of purified virion RNA from human rhinovirus-infected HeLa cells. Growth kinetics determined that this virus chimera has a somewhat slower growth phenotype at four hours post infection when compared to wild type poliovirus in a single-cycle growth assay, but this difference is eliminated by six hours post infection, when virions are harvested for virus RNA purification (Figure 7B). S1-VPgR1 was used to infect HeLa cells in the presence of 35S-methionine, and virion RNA was purified. T1-treated virion RNA (Figure 7C) or full-length virion RNA (Figure 7D) from both W1-VPg31 and S1-VPgR1 radiolabeled substrates was incubated with an unlinkase source to determine if cleavage efficiency differed between the two viruses. RNase T1-generated substrates from both viruses (Figure 7C) were incubated with 0, 5, 10 or 20 µg of total protein from an enriched unlinkase activity source (Fraction CA), and no significant difference in cleavage efficiency between the two substrates was observed (Figure 7C compare lanes 4 and 8). Full-length substrates from both viruses (Figure 7D) were incubated with 0.5, 1, 2, 5, or 10 µg of total protein from the same enriched unlinkase activity source, and again no significant differences in cleavage between the two substrates were observed (Figure 7D compare lanes 6 and 12). No difference was observed for unlinkase cleavage of either substrate, leading to the conclusion that this enzyme can recognize and cleave a human rhinovirus substrate.

Bottom Line: 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.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.Furthermore, we have determined that unlinkase recognizes and cleaves a human rhinovirus-poliovirus chimeric substrate with the same efficiency as the poliovirus substrate.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, School of Medicine, University of California Irvine, Irvine, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus