Abstract
Poliovirus RNA replication requires the activities of a viral RNA-dependent RNA polymerase, 3Dpol, in conjunction with several additional viral and likely cellular proteins. The importance of both the 3A and 3B coding regions has been documented previously by genetic tests, and their biochemical activities have been the subject of several recent investigations. In this study, we examined the previously reported stimulation of 3D-catalyzed RNA synthesis by 3AB. We show that 3AB does not stimulate RNA synthesis on templates that are stably base paired to a primer, indicating that 3AB does not stabilize or otherwise activate 3Dpol for chain elongation. Similarly, it does not alter the kinetic parameters or binding affinities of 3D for substrates. In the absence of a primer, or in the presence of a primer that does not form a stable hybrid with the template, 3AB increases the utilization of 3'-hydroxyl termini as sites for chain elongation by 3D, and thereby stimulates RNA synthesis. 3AB may interact with and stabilize these sites and/or may recruit 3Dpol to the site, resulting in stimulation of the initiation of elongation events. We propose that this activity is required for stabilizing weak interactions that occur during nucleotidyl-protein-primed initiation events in the viral RNA replication complex.
Highlights
Proposed some years ago that structural elements in the RNA template may provide a mechanism for self-priming [10]; a more current model involves protein priming [11]
Several polypeptides containing VPg sequences, including 3AB, have been detected in infected cells by immunoprecipitation with anti-VPg antibodies (16 –18). These polypeptides are enriched in membrane fractions containing the viral RNA replication complexes isolated from infected cells
Several laboratories have reported the formation of uridylylated forms of 3B (VPg), both in vivo and in vitro, that could form weakly base paired hybrids with the 3Ј-terminal nucleotides of poliovirus plus and minus strand RNAs; and it is assumed that these nucleotidyl-proteins serve as primers for RNA synthesis
Summary
Materials—Restriction enzymes were obtained from Boehringer Mannheim, Life Technologies, Inc., or New England Biolabs, Inc. The plasmid pT7N224- [29] was digested with EcoRI and Asp718 and the 70-bp fragment was isolated by agarose gel electrophoresis This fragment was inserted into the vector fragment with T4 DNA ligase and used to transform E. coli JM109 cells to obtain pGEM-4Z-PV1-72. In Vitro RNA Transcription and Preparation of Hybrid Substrates— Plasmid pGEM-4Z-PV1-72 was digested with RsaI, and extracted DNA was transcribed with SP6 RNA polymerase (Ambion, Inc.) to yield 36 nt plus strand RNA. PGEM-4Z-PV1-72 was digested with EcoRI, and extracted DNA was transcribed with T7 RNA polymerase (Ambion, Inc.) to yield 88 nt minus strand RNA (complementary to poliovirus plus strand at nt 1–72). Plasmid pGEM-3Z-PV 5522–5627 was digested with HindIII and SspI, and extracted DNA was transcribed with T7 RNA polymerase to produce 118 nt poliovirus plus strand sequences. Precipitates were collected on Whatman GF/C filters and washed with 1 M HCl, 0.1 M
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