3'-Terminal sequence in poliovirus negative-strand templates is the primary cis-acting element required for VPgpUpU-primed positive-strand initiation

Abstract

The 5' cloverleaf in poliovirus RNA has a direct role in regulating the stability, translation, and replication of viral RNA. In this study, we investigated the role of stem a in the 5' cloverleaf in regulating the stability and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Our results showed that disrupting the duplex structure of stem a destabilized viral RNA and inhibited efficient negative-strand synthesis. Surprisingly, the duplex structure of stem a was not required for positive-strand synthesis. In contrast, altering the primary sequence at the 5'-terminal end of stem a had little or no effect on negative-strand synthesis but dramatically reduced positive-strand initiation and the formation of infectious virus. The inhibition of positive-strand synthesis observed in these reactions was most likely a consequence of nucleotide alterations in the conserved sequence at the 3' ends of negative-strand RNA templates. Previous studies suggested that VPgpUpU synthesized on the cre(2C) hairpin was required for positive-strand synthesis. Therefore, these results are consistent with a model in which preformed VPgpUpU serves as the primer for positive-strand initiation on the 3'AAUUUUGUC5' sequence at the 3' ends of negative-strand templates. Our results suggest that this sequence is the primary cis-acting element that is required for efficient VPgpUpU-primed positive-strand initiation.

FIG. 1.

Stem a mutations in the 5′ cloverleaf of poliovirus RNA and predicted secondary structure of the 5′ cloverleaf formed by poliovirus wild-type RNA and the stem a mutants used in this study. The nucleotides that were altered or deleted are underlined. (A) The duplex structure of stem a was disrupted in mut1, mut6, and mut9. (B) By making compensatory nucleotide changes in both strands of stem a, the duplex structure was restored while the primary sequence was changed in mut2, mut7, and mut10. (C) The 5′-terminal nucleotides were sequentially deleted in mut3, mut4, and mut8.

FIG. 2.

Schematic of poliovirus RNAs utilized in this study. (A) Diagram of the nonreplicating helper RNA, PV1ΔGUA₃, which encodes all of the viral proteins. (B) Diagram of F3 template RNA. F3 RNA contains a frameshift mutation, as indicated in the diagram, and does not encode any viral proteins. This RNA contains two 5′-terminal nonviral G nucleotides and was used in assays for negative-strand RNA synthesis. (C) Diagram of Rz-F3 template RNA. This RNA contains a self-cleaving 5′-terminal hammerhead ribozyme that results in the formation of viral transcripts with an authentic 5′ end. Rz-F3 RNA supports the synthesis of both negative- and positive-strand product RNAs.

FIG. 3.

Effects of stem a mutations on viral RNA stability. HeLa S10 translation-replication reaction mixtures (150 μl) containing equimolar amounts of helper RNA and the indicated ³²P-labeled viral RNA were incubated for 4 h at 34°C, and 20-μl samples of the reaction mixture were removed at the indicated time points. The ³²P-labeled viral RNA remaining at each time point was determined by gel electrophoresis (A, C, and E) or by precipitation in trichloroacetic acid (B, D, and F) as described in Materials and Methods.

FIG. 4.

Effect of adding a 5′ cap on the stability of F3mut1 RNA. HeLa S10 translation-replication reaction mixtures (150 μl) containing equimolar amounts of helper RNA and ³²P-labeled F3 RNA, F3mut1 RNA, or F3mut1 RNA with a 5′ cap (cap F3mut1) were incubated for 4 h at 34°C, and 20-μl samples of the reaction mixture were removed at the indicated time points. The ³²P-labeled viral RNA remaining at each time point was determined by gel electrophoresis as described in Materials and Methods.

FIG. 5.

Effect of disrupting the duplex structure of stem a on negative-strand RNA synthesis. PIRCs were isolated from HeLa S10 translation-replication reactions containing the nonreplicating helper RNA and the indicated template RNAs and were resuspended in reaction mixtures containing [³²P]CTP and incubated at 37°C for 8 min. The ³²P-labeled product RNAs synthesized in these reactions were characterized by denaturing agarose gel electrophoresis and quantitated using a PhosphorImager as described in Materials and Methods. Labeled RNA synthesized in the reactions containing the mutant RNAs (lanes 2 to 4) was expressed as a percentage of the labeled RNA synthesized with F3 RNA (lane 1).

FIG. 6.

Effect of altering the 5′-terminal sequence on negative-strand RNA synthesis. PIRCs isolated from HeLa S10 reactions containing helper RNA and the indicated template RNA were resuspended in reaction mixtures containing [³²P]CTP and incubated at 37°C for 8 min. The ³²P-labeled product RNAs synthesized in these reactions were characterized by gel electrophoresis and quantitated as described for Fig. 5.

FIG. 7.

Effect of the sequential deletion of the 5′-terminal nucleotides on negative-strand RNA synthesis. PIRCs isolated from HeLa S10 reactions containing helper RNA and the indicated template RNA were resuspended in reaction mixtures containing [³²P]CTP and incubated at 37°C for 8 min. The ³²P-labeled product RNAs synthesized in these reactions were characterized by gel electrophoresis and were quantitated as described for Fig. 5.

FIG. 8.

Effect of disrupting the duplex structure of stem a on positive-strand RNA synthesis. HeLa S10 translation-replication reaction mixtures that contained 2 mM guanidine HCl and helper RNA were incubated at 34°C for 1 h. The indicated template RNA was added and allowed to remain in the reaction for 1 h. PIRCs were isolated from these reactions, resuspended in reaction mixtures containing [³²P]CTP, and incubated for 1 h at 37°C. The resulting ³²P-labeled RNA products were then analyzed by gel electrophoresis and quantitated using a PhosphorImager. The ratios of positive/negative-strand synthesis shown were calculated for the wild-type and mutant RNAs as described in Materials and Methods. The predicted secondary structures for the 5′ end of the positive strand and the 3′ end of the negative strand are shown for each RNA. The altered nucleotides are underlined.

FIG. 9.

Effect of changing the primary sequence of stem a on positive-strand RNA synthesis. PIRCs were isolated from HeLa S10 reactions containing helper RNA and the indicated template RNA and were resuspended in reaction mixtures containing [³²P]CTP and incubated for 1 h at 37°C. The resulting ³²P-labeled product RNAs were analyzed by gel electrophoresis and quantitated as described for Fig. 8. Results for separate experiments are shown in panels A and B. The ratios of positive/negative-strand synthesis shown were calculated for the wild-type and mutant RNAs as described in Materials and Methods. The predicted secondary structures for the 5′ end of the positive strand and the 3′ end of the negative strand are shown for mutant RNAs. The altered nucleotides are underlined.

FIG. 10.

Effect of sequential deletion of 5′-terminal nucleotides on positive-strand RNA synthesis. (A) HeLa S10 translation-replication reaction mixtures that contained 2 mM guanidine HCl, helper RNA, and the indicated template RNA were incubated at 34°C for 4 h. (B) HeLa S10 translation-replication reaction mixtures that contained 2 mM guanidine HCl and helper RNA were incubated at 34°C for 1 h. The indicated template RNA was added and incubated for 1 h. PIRCs were isolated from the reactions shown in panels A and B, resuspended in reaction mixtures containing [³²P]CTP, and incubated for 1 h at 37°C. The resulting ³²P-labeled product RNA was analyzed by gel electrophoresis and quantitated as described for Fig. 8. The ratios of positive/negative-strand synthesis shown were calculated for the wild-type and mutant RNAs as explained in Materials and Methods. The predicted secondary structures for the 5′ end of the positive strand and the 3′ end of the negative strand are shown for mutant RNAs. The deleted nucleotides are underlined.

FIG. 11.

Plaque morphology of virus produced in HeLa S10 reactions. Monolayers of BSC40 cells were infected with the virus produced in the HeLa S10 translation replication reactions containing the indicated RNAs. The cells were overlaid with Eagle's minimum essential medium containing 1% methyl cellulose and incubated at 37°C for 2 days. Plaques were visualized by staining the monolayers with 0.02% crystal violet. Representative wells for the virus produced in the reactions containing Rz-PV1 RNA (Wild-type) and each of the indicated mutant RNAs are shown.

FIG. 12.

Model for the VPgpUpU-primed initiation of positive-strand RNA synthesis at the 3′ end of negative-strand RNA templates. (A) The poliovirus polymerase, 3D^pol, utilizes preformed VPgpUpU as a primer to initiate positive-strand RNA synthesis at the 3′ ends of poliovirus negative-strand RNA templates. VPgpUpU pairs with the two complementary A nucleotides at the 3′-terminal end of a negative-strand RNA template to facilitate the efficient initiation of positive-strand synthesis. (B) mut3 and mut4 RNAs contain either a 5′-terminal U or UU deletion, respectively (Fig. 1). Therefore, mut3 and mut4 negative-strand RNAs would contain a 3′-terminal A or AA deletion. As shown in panel B, VPgpUpU is able to function as a primer for positive-strand initiation on both of the mutant negative-strand templates. In this case, however, initiation would be inefficient (depicted by light-gray arrows) due to the absence of either one or both of the 3′-terminal A nucleotides. This mechanism would restore the wild-type sequence at the 5′ ends of nascent positive strands and would explain why only wild-type progeny virus was recovered from reactions containing either mut3 or mut4 input RNAs. For clarity, the model was simplified to focus on the roles of 3D^pol, VPgpUpU, and the 3′ termini of negative-strand templates during positive-strand initiation. Not depicted in this model are the cellular membranes, the cellular proteins, and the precursor forms of VPg, 3D^pol, and the other viral proteins that are most likely part of functional RNA replication complexes. In addition, this model is not meant to imply that other sequences in negative-strand RNA, including the 5′ end, might play a role in positive-strand initiation in some host cells (16).