Unlinkase activity remains unchanged during poliovirus infection.

<p>HeLa cells were infected with wild type poliovirus and cytoplasmic extracts were collected at 0 (lanes 3 and 9), 2 (lanes 4 and 10), 4 (lanes 5 and 11), or 6 (lanes 6 and 12) hours post-infection. 1 µg (lanes 1–6) or 5 µg (lanes 7–12) of total protein from these extracts was incubated with full-length ³⁵S-methionine radiolabeled W1-VPg 31 virion RNA substrate. No difference in activity was observed between the mock lanes (lanes 1, 2, 7, and 8) and the different time points collected during the infection.</p

Optimal assay conditions for detecting unlinkase activity using full-length poliovirus virion RNA ³⁵S-methionine labeled substrate.

<p>A protein chromatography fraction enriched for unlinkase activity was incubated with full-length ³⁵S-methionine radiolabeled W1-VPg 31 virion RNA substrate with either (A) increasing amounts of protein (0.01, 0.02, 0.04, 0.1, 0.2, 0.4, and 0.6 µg/µl) from an enriched source of unlinkase activity (Fraction SA) at 30°C for 30 minutes or (B) increasing incubation time (0, 1, 2, 5, 10, 15, 20, and 30 minutes) with 0.4 µg/µl of protein from a partially-purified fraction of unlinkase activity (Fraction SA) to determine optimal assay conditions for the full-length substrate. (C) ³⁵S-methionine radiolabeled W1-VPg 31 virion RNA substrate was mock-incubated (lane 1), incubated with 0.8 µg/µl RSW (lane 2), 0.4 µg/µl of protein from a partially-purified fraction of unlinkase activity (Fraction SA) (lane 3), or one unit of RNase A (lane 4) to differentiate between non-specific nuclease activity and authentic unlinkase activity.</p

VPg-RNA covalent linkage and VPg sequences of wild type and mutated W1-VPg31 polioviruses.

<p>Schematic of the VPg-RNA covalent linkage between the single tyrosine residue in poliovirus VPg and the first uridine at the 5′ terminus of the genomic RNA (A). Image was generated using ChemDraw software. The arrow indicates where unlinkase cleaves the bond. (B) VPg sequences of wild type poliovirus and W1-VPg31 (wild type poliovirus mutated to encode two methionines in the VPg coding region).</p

Oligonucleotides used for cloning of S1-VPgR1.

<p>Oligonucleotides used for cloning of S1-VPgR1.</p

Unlinkase activity on a poliovirus versus a rhinovirus substrate.

<p>(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.</p

Unlinkase activity in different cytoplasmic extracts from human cell lines.

<p>Increasing amounts of total protein (0.1 µg/µl, lanes 1, 4, 7, 10, and 13; 0.2 µg/µl, lanes 2, 5, 8, 11, and 14; 0.4 µg/µl, lanes 3, 6, 9, 12, and 15) from cytoplasmic extracts from different cell lines were incubated with ³⁵S-methionine radiolabeled W1-VPg 31 virion RNA substrate. HeLa (lanes 1–3), SK-OV-3 (lanes 4–6), NGP (lanes 7–9), and K562 (lanes 13–15) are all human cell lines and have differing levels of unlinkase activity; RRL (rabbit reticulocyte lysate) (lanes 10–12), contrary to previous publications, did not exhibit unlinkase activity in this assay (lanes 10–12).</p