5' cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis.

A cloverleaf structure at the 5' terminus of poliovirus RNA binds viral and cellular proteins. To examine the role of the cloverleaf in poliovirus replication, we determined how cloverleaf mutations affected the stability, translation and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Mutations within the cloverleaf destabilized viral RNA in these reactions. Adding a 5' 7-methyl guanosine cap fully restored the stability of the mutant RNAs and had no effect on their translation. These results indicate that the 5' cloverleaf normally protects uncapped poliovirus RNA from rapid degradation by cellular nucleases. Preinitiation RNA replication complexes formed with the capped mutant RNAs were used to measure negative-strand synthesis. Although the mutant RNAs were stable and functional mRNAs, they were not active templates for negative-strand RNA synthesis. Therefore, the 5' cloverleaf is a multifunctional cis-acting replication element required for the initiation of negative-strand RNA synthesis. We propose a replication model in which the 5' and 3' ends of viral RNA interact to form a circular ribonucleoprotein complex that regulates the stability, translation and replication of poliovirus RNA.

Translating ribosomes inhibit poliovirus negative-strand RNA synthesis.

Poliovirus has a single-stranded RNA genome of positive polarity that serves two essential functions at the start of the viral replication cycle in infected cells. First, it is translated to synthesize viral proteins and, second, it is copied by the viral polymerase to synthesize negative-strand RNA. We investigated these two reactions by using HeLa S10 in vitro translation-RNA replication reactions. Preinitiation RNA replication complexes were isolated from these reactions and then used to measure the sequential synthesis of negative- and positive-strand RNAs in the presence of different protein synthesis inhibitors. Puromycin was found to stimulate RNA replication overall. In contrast, RNA replication was inhibited by diphtheria toxin, cycloheximide, anisomycin, and ricin A chain. Dose-response experiments showed that precisely the same concentration of a specific drug was required to inhibit protein synthesis and to either stimulate or inhibit RNA replication. This suggested that the ability of these drugs to affect RNA replication was linked to their ability to alter the normal clearance of translating ribosomes from the input viral RNA. Consistent with this idea was the finding that the protein synthesis inhibitors had no measurable effect on positive-strand synthesis in normal RNA replication complexes. In marked contrast, negative-strand synthesis was stimulated by puromycin and was inhibited by cycloheximide. Puromycin causes polypeptide chain termination and induces the dissociation of polyribosomes from mRNA. Cycloheximide and other inhibitors of polypeptide chain elongation "freeze" ribosomes on mRNA and prevent the normal clearance of ribosomes from viral RNA templates. Therefore, it appears that the poliovirus polymerase was not able to dislodge translating ribosomes from viral RNA templates and mediate the switch from translation to negative-strand synthesis. Instead, the initiation of negative-strand synthesis appears to be coordinately regulated with the natural clearance of translating ribosomes to avoid the dilemma of ribosome-polymerase collisions.

Synchronous replication of poliovirus RNA: initiation of negative-strand RNA synthesis requires the guanidine-inhibited activity of protein 2C.

We report that protein 2C, the putative nucleoside triphosphatase/helicase protein of poliovirus, is required for the initiation of negative-strand RNA synthesis. Preinitiation RNA replication complexes formed upon the translation of poliovirion RNA in HeLa S10 extracts containing 2 mM guanidine HCI, a reversible inhibitor of viral protein 2C. Upon incubation in reactions lacking guanidine, preinitiation RNA replication complexes synchronously initiated and elongated negative-strand RNA molecules, followed by the synchronous initiation and elongation of positive-strand RNA molecules. The immediate and exclusive synthesis of negative-strand RNA upon the removal of guanidine demonstrates that guanidine specifically blocks the initiation of negative-strand RNA synthesis. Readdition of guanidine HCl to reactions synchronously elongating nascent negative-strand RNA molecules did not prevent their continued elongation and completion. In fact, readdition of guanidine HCl to reactions containing preinitiation complexes elongating nascent negative-strand RNA molecules had no effect on subsequent positive-strand RNA synthesis initiation or elongation. Thus, the guanidine-inhibited function of viral protein 2C was not required for the elongation of negative-strand RNA molecules, the initiation of positive-strand RNA molecules, or the elongation of positive-strand RNA molecules. The guanidine-inhibited function of viral protein 2C is required only immediately before or during the initiation of negative-strand RNA synthesis. We suggest that guanidine may block an irreversible structural maturation of protein 2C and/or RNA replication complexes necessary for the initiation of RNA replication.

Poliovirus RNA recombination in cell-free extracts.

Poliovirus RNA has been shown to undergo homologous genetic recombination at a high frequency in infected human cells. Recently it has become possible to mimic the entire intracellular replicative cycle of poliovirus replication in cytoplasmic extracts prepared from HeLa cells, resulting in the generation of infectious poliovirions. The mechanism of poliovirus RNA recombination has been shown previously to be coupled to RNA replication, presumably by template switching during the replication of parental RNAs. Experiments were designed to test whether recombinant poliovirus RNA molecules are produced in a cell-free environment. Recombinant molecules generated bear marker sequences that can be detected physically by reverse transcription and PCR. We report here successful detection of poliovirus RNA recombination in a cell-free replication system. The frequency measured for cell-free RNA recombination between two polymorphic marker loci 656 nt apart was between 10(-2) and 10(-3) recombinants/genome, a frequency comparable to or slightly higher than that measured for RNA recombination in infected cells.

Poliovirus RNA polymerase mutation 3D-M394T results in a temperature-sensitive defect in RNA synthesis.

Mutant ts10 is an RNA-negative temperature-sensitive mutant of Mahoney type 1 poliovirus. Mutant ts10 3D pol was purified from infected cells and was shown to be rapidly heat-inactivated at 45 degrees when compared to wild-type polymerase. Sequencing of mutant ts10 genomic RNA revealed a U to C transition at nt 7167 resulting in an amino acid change of methionine 394 of 3D pol to threonine. The 3D-M394T mutation was engineered into a wild-type infectious clone of poliovirus type 1. The resultant mutant virus, 3D-105, had a temperature-sensitive phenotype in plaque assays. The translation and replication of wild-type, ts10, and 3D-105 virion RNAs were all characterized in HeLa S10 translation-RNA replication reactions in vitro. The optimum temperatures for the replication of the wild-type and mutant viral RNAs in the HeLa S10 translation-replication reactions were 37 and 34 degrees, respectively. To characterize the temperature-sensitive defect in the replication of the mutant RNA, we used preinitiation RNA replication complexes which were formed in HeLa S10 in vitro reactions containing guanidine HCl. Negative-strand RNA synthesis in 3D-M394T mutant preinitiation replication complexes was normal at 34 degrees but was rapidly and irreversibly inhibited at 39.5 degrees. To differentiate between the initiation and elongation steps in RNA replication, we compared the elongation rates in mutant and wild-type replication complexes at 39.5 degrees. The results showed that the elongation rates for nascent negative strands in both the mutant and wild-type replication complexes were identical. Therefore, the results indicate that the heat-sensitive step in negative-strand synthesis exhibited by the 3D-M394T replication complexes is in the initiation of RNA synthesis and not in the elongation of nascent chains.

Complete replication of poliovirus in vitro: preinitiation RNA replication complexes require soluble cellular factors for the synthesis of VPg-linked RNA.

Translation of poliovirion RNA in HeLa S10 extracts resulted in the formation of RNA replication complexes which catalyzed the asymmetric replication of poliovirus RNA. Synthesis of poliovirus RNA was detected in unfractionated HeLa S10 translation reactions and in RNA replication complexes isolated from HeLa S10 translation reactions by pulse-labeling with [32P]CTP. The RNA replication complexes formed in vitro contained replicative-intermediate RNA and were enriched in viral protein 3CD and the membrane-associated viral proteins 2C, 2BC, and 3AB. Genome-length poliovirus RNA covalently linked to VPg was synthesized in large amounts by the replication complexes. RNA replication was highly asymmetric, with predominantly positive-polarity RNA products. Both anti-VPg antibody and guanidine HCl inhibited RNA replication and virus formation in the HeLa S10 translation reactions without affecting viral protein synthesis. The inhibition of RNA synthesis by guanidine was reversible. The reversible nature of guanidine inhibition was used to demonstrate the formation of preinitiation RNA replication complexes in reaction mixes containing 2 mM guanidine HCl. Preinitiation complexes sedimented upon centrifugation at 15,000 x g and initiated RNA replication upon their resuspension in reaction mixes lacking guanidine. Initiation of RNA synthesis by preinitiation complexes did not require active protein synthesis or the addition of soluble viral proteins. Initiation of RNA synthesis by preinitiation complexes, however, was absolutely dependent on soluble HeLa cytoplasmic factors. Preinitiation complexes also catalyzed the formation of infectious virus in reaction mixes containing exogenously added capsid proteins. The titer of infectious virus produced in such trans-encapsidation reactions reached 4 x 10(7) PFU/ml. The HeLa S10 translation-RNA replication reactions represent an efficient in vitro system for authentic poliovirus replication, including protein synthesis, polyprotein processing, RNA replication, and virus assembly.

Coupled translation and replication of poliovirus RNA in vitro: synthesis of functional 3D polymerase and infectious virus.

Poliovirus RNA polymerase and infectious virus particles were synthesized by translation of virion RNA in vitro in HeLa S10 extracts. The in vitro translation reactions were optimized for the synthesis of the viral proteins found in infected cells and in particular the synthesis of the viral polymerase 3Dpol. There was a linear increase in the amount of labeled protein synthesized during the first 6 h of the reaction. The appearance of 3Dpol in the translation products was delayed because of the additional time required for the proteolytic processing of precursor proteins. 3Dpol was first observed at 1 h in polyacrylamide gels, with significant amounts being detected at 6 h and later. Initial attempts to assay for polymerase activity directly in the translation reaction were not successful. Polymerase activity, however, was easily detected by adding a small amount (3 microliters) of translation products to a standard polymerase assay containing poliovirion RNA. Full-length minus-strand RNA was synthesized in the presence of an oligo(U) primer. In the absence of oligo(U), product RNA about twice the size of virion RNA was synthesized in these reactions. RNA stability studies and plaque assays indicated that a significant fraction of the input virion RNA in the translation reactions was very stable and remained intact for 20 h or more. Plaque assays indicated that infectious virus was synthesized in the in vitro translation reactions. Under optimal conditions, the titer of infectious virus produced in the in vitro translation reactions was greater than 100,000 PFU/ml. Virus was first detected at 6 h and increased to maximum levels by 12 h. Overall, the kinetics of poliovirus replication (protein synthesis, polymerase activity, and virus production) observed in the HeLa S10-initiation factor in vitro translation reactions were similar to those observed in infected cells.

Replication of poliovirus RNA and subgenomic RNA transcripts in transfected cells.

Full-length and subgenomic poliovirus RNAs were transcribed in vitro and transfected into HeLa cells to study viral RNA replication in vivo. RNAs with deletion mutations were analyzed for the ability to replicate in either the absence or the presence of helper RNA by using a cotransfection procedure and Northern (RNA) blot analysis. An advantage of this approach was that viral RNA replication and genetic complementation could be characterized without first isolating conditional-lethal mutants. A subgenomic RNA with a large in-frame deletion in the capsid coding region (P1) replicated more efficiently than full-length viral RNA transcripts. In cotransfection experiments, both the full-length and subgenomic RNAs replicated at slightly reduced levels and appeared to interfere with each other's replication. In contrast, a subgenomic RNA with a similarly sized out-of-frame deletion in P1 did not replicate in transfected cells, either alone or in the presence of helper RNA. Similar results were observed with an RNA transcript containing a large in-frame deletion spanning the P1, P2, and P3 coding regions. A mutant RNA with an in-frame deletion in the P1-2A coding sequence was self-replicating but at a significantly reduced level. The replication of this RNA was fully complemented after cotransfection with a helper RNA that provided 2A in trans. A P1-2A-2B in-frame deletion, however, totally blocked RNA replication and was not complemented. Control experiments showed that all of the expected viral proteins were both synthesized and processed when the RNA transcripts were translated in vitro. Thus, our results indicated that 2A was a trans-acting protein and that 2B and perhaps other viral proteins were cis acting during poliovirus RNA replication in vivo. Our data support a model for poliovirus RNA replication which directly links the translation of a molecule of plus-strand RNA with the formation of a replication complex for minus-strand RNA synthesis.

Determination of the poliovirus RNA polymerase error frequency at eight sites in the viral genome.

The poliovirus RNA polymerase error frequency was measured in vivo at eight sites in the poliovirus genome. The frequency at which specific G residues in poliovirion RNA changed to another base during one round of viral RNA replication was determined. Poliovirion RNA uniformly labeled with 32Pi was hybridized to a synthetic DNA oligonucleotide that was complementary to a sequence in the viral genome that contained a single internal G residue. The nonhybridized viral RNA was digested with RNase T1, and the protected RNA oligonucleotide was purified by gel electrophoresis. The base substitution frequency at the internal G residue was measured by finding the fraction of this RNA oligonucleotide that was resistant to RNase T1 digestion. A mean value of 2.0 x 10(-3) +/- 1.2 x 10(-3) was obtained at two sites. A modification of the above procedure involved the use of 5'-end-labeled RNA oligonucleotides. The mean value of the error frequency determined at eight sites in the viral genome by using this technique was 4.1 x 10(-3) +/- 0.6 x 10(-3). Sequencing two of the RNase T1-resistant RNA oligonucleotides confirmed that the internal G was changed to a C, A, or U residue in most of these oligonucleotides. Thus, our results indicated that the polymerase had a high error frequency in vivo and that there was no significant variation in the values determined at the specific sites examined in this study.

Interference with vaccinia virus growth caused by insertion of the coding sequence for poliovirus protease 2A.

Attempts were made to express noninfectious derivatives of full-length type 1 (Mahoney) and type 2 (Lansing) poliovirus cDNAs in live recombinant vaccinia viruses for vaccine purposes. Vaccinia virus (VV) would not tolerate insertions of polio cDNA containing the coding sequence for the polio protease 2A. However, polio cDNA with the 2A gene deleted either in vivo or in vitro could be inserted into VV and stably maintained. Genetic evidence indicated that expression of the polio 2A gene in trans from transfected plasmid DNA was deleterious to vaccinia virus within the same cell. The 2A product presumably interferes with VV growth by modifying the host translational machinery such that translation of host and vaccinia capped mRNAs is inhibited. Polio cDNA containing a mutated 2A gene whose product is no longer active in host protein shutoff could be inserted into VV. However, inserts containing the intact mutated 2A gene did not synthesize detectable poliovirus protein, although they did produce polio-specific RNA. Expression of polio-specific protein was detected from a VV-polio recombinant containing cDNA encoding the capsid proteins plus an incomplete 2A gene. These results have implications regarding possible vaccine construction, and suggest a mechanism for interference between polio and vaccinia viruses in mixed infection.

Self-catalyzed linkage of poliovirus terminal protein VPg to poliovirus RNA.

The poliovirus terminal protein, VPg, was covalently linked to poliovirus RNA in a reaction that required synthetic VPg, Mg2+, and a replication intermediate synthesized in vitro. The VPg linkage reaction did not require the viral polymerase, host factor, or ribonucleoside triphosphates and was specific for template-linked minus-strand RNA synthesized on poliovirion RNA. The covalent nature of the bond between VPg and the RNA was demonstrated by the isolation of VPg-pUp from VPg-linked RNA. A model is proposed in which the tyrosine residue in VPg forms a phosphodiester bond with the 5'UMP in minus-strand RNA in a self-catalyzed transesterification reaction. It appears that either the RNA, VPg, or a combination of both forms the catalytic center for this reaction.

A conserved base pair within helix P4 of the Tetrahymena ribozyme helps to form the tertiary structure required for self-splicing.

Site-specific mutagenesis of the self-splicing Tetrahymena intron has been used to investigate the function of C109-G212, a conserved base pair in the P4 stem of group I introns. Mutation of C109 to G affects splicing only slightly, whereas mutation of G212 to A or C reduces the rate of splicing substantially (500-fold reduction in kcat/Km under standard in vitro splicing conditions for the G212C mutant). Splicing activity of the compensatory double mutant (C109G:G212C) is intermediate between those of the two single mutants. Thus, the stability of the P4 stem as well as the identity of the base at position 212 are important for self-splicing. Single and double mutants containing the G212C substitution have a decreased temperature optimum for self-splicing and are partially Mg2+ suppressible, both indicative of structural destabilization. Chemical structure mapping indicates that the mutations do not redirect the global folding of the RNA, but affect the structure locally and at one other site (A183) that is distant in the secondary structure. We propose that, in addition to its pairing in P4, G212 is involved in a base triplet or an alternate base pair that contributes to the catalytically active tertiary structure of the ribozyme.

A point mutation in the poliovirus polymerase gene determines a complementable temperature-sensitive defect of RNA replication.

We have previously described the isolation of a RNA- temperature-sensitive (ts) mutant of poliovirus type 1, ts035, after chemical mutagenesis by 5-fluorouracil. The ts defect of ts035 correlated with defective RNA replication, since the two characters corevert in the case of spontaneous revertants. The alteration of a trans-acting replication function of ts035 was suggested by significant rescue following mixed infection with another ts mutant, ts221, or with wild-type virus. Protein synthesis appeared normal at 39 degrees (nonpermissive temperature) in shift-up experiments and no defect of RNA elongation was evidenced when the activity of replication complexes or purified polymerase was measured at 39 degrees. These results provide circumstantial evidence that the initiation of ts035 RNA synthesis at 39 degrees is impaired. Molecular cloning of the ts035 genome allowed us to construct a recombinant virus with the same ts phenotype as ts035, by the transfer of a fragment of the mutant polymerase gene into the wild-type genome. Two mutations were present in this region of the ts035 genome but the determination of nucleotide sequences in the case of ts035 revertants indicated that only the substitution from A to G at nucleotide 7256 was necessary for the ts phenotype. This mutation replaces Asn 426 by an Asp in polypeptide 3D, the viral polymerase.

Measurement of poliovirus RNA polymerase binding to poliovirion and nonviral RNAs using a filter-binding assay.

The binding of the purified poliovirus RNA-dependent RNA polymerase to viral and nonviral RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. Optimal conditions for the binding of purified polymerase (fraction 5-PAS) to 32P-labeled poliovirion RNA were determined. The binding of purified polymerase to 32P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) much much greater than poly(U) greater than poly(C) greater than poly(A). In competitive binding studies, the polymerase bound with equal efficiency to virion RNA and to a subgenomic transcript which contained the 3' end of the genome. The polymerase bound to 18S ribosomal RNA and to globin mRNA equally well, but with a five-fold lower affinity than to virus-specific RNAs. The results suggest that the polymerase exhibits sequence specificity in binding and that polymerase binding sites in poliovirus RNA may contain (G- and/or U)-rich sequences.

Direct measurement of the poliovirus RNA polymerase error frequency in vitro.

The fidelity of RNA replication by the poliovirus-RNA-dependent RNA polymerase was examined by copying homopolymeric RNA templates in vitro. The poliovirus RNA polymerase was extensively purified and used to copy poly(A), poly(C), or poly(I) templates with equimolar concentrations of noncomplementary and complementary ribonucleotides. The error frequency was expressed as the amount of a noncomplementary nucleotide incorporated divided by the total amount of complementary and noncomplementary nucleotide incorporated. The polymerase error frequencies were very high and ranged from 7 x 10(-4) to 5.4 x 10(-3), depending on the specific reaction conditions. There were no significant differences among the error frequencies obtained with different noncomplementary nucleotide substrates on a given template or between the values determined on two different templates for a specific noncomplementary substrate. The activity of the polymerase on poly(U) and poly(G) was too low to measure error frequencies on these templates. A fivefold increase in the error frequency was observed when the reaction conditions were changed from 3.0 mM Mg2+ (pH 7.0) to 7.0 mM Mg2+ (pH 8.0). This increase in the error frequency correlates with an eightfold increase in the elongation rate that was observed under the same conditions in a previous study.

Characterization of product RNAs synthesized in vitro by poliovirus RNA polymerase purified by chromatography on hydroxylapatite or poly(U) Sepharose.

The size of the product RNA synthesized by the poliovirus RNA polymerase and host factor was significantly affected by the type of column chromatography used to purify the polymerase. Dimer length product RNA was synthesized by the polymerase purified by chromatography on hydroxylapatite. This contrasted with the monomer length product RNA synthesized by the polymerase purified by chromatography on poly(U) Sepharose. The poly(U) Sepharose-purified polymerase was shown to contain oligo(U) that functioned as a primer. The addition of host factor to reactions containing the poly(U) Sepharose-purified polymerase significantly increased the synthesis of monomer length product RNA, in agreement with previous studies. This product RNA, however, did not immunoprecipitate with anti-VPg antibody and thus was not linked to VPg or a VPg-related protein. Thus, it was concluded that the synthesis of monomer length product RNA by the poly(U) Sepharose-purified polymerase and host factor was caused by oligo(U) priming rather than VPg priming.

Anti-VPg antibody precipitation of product RNA synthesized in vitro by the poliovirus polymerase and host factor is mediated by VPg on the poliovirion RNA template.

Antibody to the poliovirus genome-linked protein, VPg, specifically immunoprecipitated the product RNA synthesized in vitro by the poliovirus RNA polymerase and HeLa cell host factor when VPg-linked poliovirion RNA was used as a template. The largest product RNA that was immunoprecipitated was twice the size of the template RNA. The complete denaturation of the product RNA with CH3HgOH had no effect on the immunoprecipitation reaction. In contrast, CH3HgOH denaturation prevented the immunoprecipitation of the oligo(U)-primed product RNA. Immunoprecipitation of the product RNA synthesized in the host-factor-dependent reaction was prevented if VPg was removed from the template RNA by pretreatment with proteinase K or if an RNA template without VPg was used in the reaction. The results support our previous evidence that a covalent linkage exists between the labeled negative-strand product RNA and the VPg-linked template RNA and suggest that the purified polymerase and host factor initiated RNA synthesis in vitro in the absence of VPg or a VPg-precursor protein.

Poliovirus RNA-dependent RNA polymerase and host cell protein synthesize product RNA twice the size of poliovirion RNA in vitro.

The poliovirus RNA-dependent RNA polymerase required an oligouridylate primer or a HeLa cell protein (host factor) to initiate RNA synthesis on poliovirion RNA in vitro. The polymerase synthesized template-sized product RNA in the oligouridylate-primed reaction. In the host factor-dependent reaction, the largest product RNA synthesized by the polymerase was twice the size of the template RNA. About half of the product RNA recovered from this reaction was shown to exist in the form of a snapback sequence. Time-course reactions and pulse-chase experiments showed that the product RNA was only slightly larger than the template RNA at early reaction times and that with time it increased in size to form the dimer-sized product RNA. Inhibition of the elongation reaction by adding only [alpha-32P]UTP and ATP resulted in the formation of template-sized product RNA. The dimer-sized product RNA was unaffected by phenol extraction or proteinase K treatment but was converted to template-sized molecules by S1 nuclease. Dimer-sized poliovirus RNA that was sensitive to S1 nuclease was also isolated from poliovirus-infected cells. The results from this study indicate that the labeled negative-strand product RNA synthesized in vitro was covalently linked to the positive-strand template RNA. Thus, in vitro, the primer-dependent poliovirus RNA polymerase may initiate RNA synthesis in the presence of the host factor by using the 3' end of the template RNA as a primer.

Poliovirus RNA-dependent RNA polymerase synthesizes full-length copies of poliovirion RNA, cellular mRNA, and several plant virus RNAs in vitro.

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.