Poliovirus RNA-Dependent RNA Polymerase Synthesizes Full-Length Copies of Poliovirion RNA, Cellular mRNA, and Several Plant Virus RNAs In Vitro

Abstract

The poliovirus RNA-dependent RNA polymerase was active on synthetic homopolymeric RNA templates as well as on every natural RNA tested. The polymerase copied polyadenylate· oligouridylate [oligo(U)], polycytidylate · oligoinosinate, and polyinosinate· oligocytidylate templates to about the same extent. The observed activity on polyuridylate· oligoadenylate was about fourfold less. Full-length copies of both poliovirion RNA and a wide variety of other polyadenylated RNAs were synthesized by the polymerase in the presence of oligo(U). Polymerase elongation rates on poliovirion RNA and a heterologous RNA (squash mosaic virus RNA) were about the same. Changes in the Mg 2+ concentration affected the elongation rates on both RNAs to the same extent. With two non-polyadenylated RNAs (tobacco mosaic virus RNA and brome mosaic virus RNA3), the results were different. The purified polymerase synthesized a subgenomic-sized product RNA on brome mosaic virus RNA3 in the presence of oligo(U). This product RNA appeared to initiate on oligo(U) hybridized to an internal oligoadenylate sequence in brome mosaic virus RNA3. No oligo(U)-primed product was synthesized on tobacco mosaic virus RNA. When partially purified polymerase was used in place of the completely purified enzyme, some oligo(U)-independent activity was observed on the brome mosaic virus and tobacco mosaic virus RNAs. The size of the product RNA from these reactions suggested that at least some of the product RNA was full-sized and covalently linked to the template RNA. Thus, the polymerase was found to copy many different types of RNA and to make full-length copies of the RNAs tested.

ATP is required for initiation of poliovirus RNA synthesis in vitro: demonstration of tyrosine-phosphate linkage between in vitro-synthesized RNA and genome-linked protein

“…Template specificity of poliovirus replicase. Previous results have shown that purified poliovirus replicase can copy a variety of poly(A)-containing RNAs in the presence of host factor or oligo(U) (5,16,44). However, with host factor, the efficiency of copying of poliovirion RNA was two-to threefold more than that of other poly(A)-containing RNAs (5, P-Ser I / P-Thr *~('?…”

Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus.

“…Several laboratories investigating viral RdRps in vitro obtained dimer-sized RNA products. In the case of the poliovirus RdRp 3DPol protein, this phenomenon has been described and discussed by a number of different authors (Tuschall et al, 1982;Young et al, 1985;Hey et al, 1986Hey et al, , 1987Lubinski et al, 1986;Plotch et al, 1989;Neufeld et al, 1991;Cho et al, 1993). Interestingly, in the case of polioviruses and EMCVs, dimer-sized genomic RNA molecules have also been detected in vivo (Senkevich et al, 1980;Young et al, 1985), which fuels speculations that these molecules may represent intermediates in the RNA replication process.…”

Partial Purification and Characterization of Cucumber necrosis virus and Tomato bushy stunt virus RNA-Dependent RNA Polymerases: Similarities and Differences in Template Usage between Tombusvirus and Carmovirus RNA-Dependent RNA Polymerases

“…RNA templates with alternative structures may determine whether the RdRp can initiate de novo at the initiator sequence (promoter) or it may use the 3Ј end of the template as primer. The 3Ј-terminal extension reaction ("self-priming") on the template RNA is not unique to the tombusvirus RdRp and is also generated in many in vitro RdRp systems, including TCV (Song and Simon, 1995a;Nagy et al, 1998;Nagy and Simon, 1998a,b), Poliovirus (Tuschall et al, 1982;Lubinski et al, 1986Lubinski et al, , 1987Hey et al, 1986), and Hepatitis C virus (Behrens et al, 1996).…”