Translating ribosomes inhibit poliovirus negative-strand RNA synthesis

Abstract

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.

FIG. 1

Pathways of poliovirus replication and the dilemma of ribosome-polymerase collisions. (A) The replication of poliovirus RNA in infected cells and in cell-free replication reactions in vitro is fundamentally different. The initial concentration of viral RNA in the cytoplasm of infected cells is very low. In the infected cell, protein synthesis and RNA replication are codependent since protein synthesis requires RNA replication and vice versa. During the course of the infection, this results in the amplification of the input viral RNA and leads to a circular replication pathway. In contrast, a relatively high concentration of input virion RNA is used in the in vitro reactions to achieve maximum rates of protein synthesis. In this case, amplification of the input RNA is not required to achieve maximum rates of protein synthesis and RNA replication in vitro, which leads to a linear replication pathway. (B) Poliovirus RNA is translated before it replicates (30). Translating ribosomes move 5′ to 3′ on the viral mRNA, while the poliovirus polymerase must initiate negative-strand RNA synthesis at the 3′ terminus of the viral RNA and move in a 3′-to-5′ direction. The results of this study indicate that the poliovirus polymerase is not able to dislodge translating ribosomes from viral mRNA. This suggests that the virus has evolved a different mechanism to regulate the switch from translation to RNA replication to avoid this dilemma which would impede the efficient replication of the viral genome.

FIG. 2

Effect of protein synthesis inhibitors on the poliovirus RNA replication cycle. Preinitiation RNA replication complexes were resuspended in reaction mixtures containing fresh HeLa S10 extract, translation initiation factors, and 15 μCi of [α-³²P]CTP (method 3 in Materials and Methods). The individual reactions also contained 2 mM guanidine (lane 1), no additional drug (lane 2), 200 μg of cycloheximide per ml (lane 3), 8 μg of anisomycin per ml (lane 4), 40 μg of diphtheria toxin per ml plus 40 μg of NAD⁺ per ml (lane 5), or 100 μg of puromycin per ml (lane 6). The reactions were incubated at 30°C for 90 min, and the labeled RNA products were characterized by CH₃HgOH-agarose gel electrophoresis and autoradiography.

FIG. 3

Dose-response effect of diphtheria toxin, ricin A chain, and puromycin on viral protein synthesis and RNA replication. Protein synthesis was measured in HeLa S10 translation-RNA replication reactions containing [³⁵S]methionine and diphtheria toxin, ricin A chain, or puromycin at the indicated concentrations. The reactions were incubated at 30°C as indicated in Materials and Methods. The incorporation of [³⁵S]methionine was determined after 30 min (A and B) or 5 h (C) of incubation and was plotted versus the concentration of each drug used. For RNA synthesis, preinitiation RNA replication complexes were resuspended in reaction mixtures containing fresh HeLa S10 extract (method 3; see Materials and Methods). The individual reactions also contained the indicated concentrations of diphtheria toxin, ricin A chain, or puromycin. The reactions were incubated at 30°C for 90 min, and the labeled RNA products were characterized by CH₃HgOH-agarose gel electrophoresis and visualized by autoradiography. The amount of labeled genome-length viral RNA present in each lane was quantitated by using a Molecular Dynamics PhosphorImager and was plotted versus the concentration of each protein synthesis inhibitor.

FIG. 3

Dose-response effect of diphtheria toxin, ricin A chain, and puromycin on viral protein synthesis and RNA replication. Protein synthesis was measured in HeLa S10 translation-RNA replication reactions containing [³⁵S]methionine and diphtheria toxin, ricin A chain, or puromycin at the indicated concentrations. The reactions were incubated at 30°C as indicated in Materials and Methods. The incorporation of [³⁵S]methionine was determined after 30 min (A and B) or 5 h (C) of incubation and was plotted versus the concentration of each drug used. For RNA synthesis, preinitiation RNA replication complexes were resuspended in reaction mixtures containing fresh HeLa S10 extract (method 3; see Materials and Methods). The individual reactions also contained the indicated concentrations of diphtheria toxin, ricin A chain, or puromycin. The reactions were incubated at 30°C for 90 min, and the labeled RNA products were characterized by CH₃HgOH-agarose gel electrophoresis and visualized by autoradiography. The amount of labeled genome-length viral RNA present in each lane was quantitated by using a Molecular Dynamics PhosphorImager and was plotted versus the concentration of each protein synthesis inhibitor.

FIG. 3

Dose-response effect of diphtheria toxin, ricin A chain, and puromycin on viral protein synthesis and RNA replication. Protein synthesis was measured in HeLa S10 translation-RNA replication reactions containing [³⁵S]methionine and diphtheria toxin, ricin A chain, or puromycin at the indicated concentrations. The reactions were incubated at 30°C as indicated in Materials and Methods. The incorporation of [³⁵S]methionine was determined after 30 min (A and B) or 5 h (C) of incubation and was plotted versus the concentration of each drug used. For RNA synthesis, preinitiation RNA replication complexes were resuspended in reaction mixtures containing fresh HeLa S10 extract (method 3; see Materials and Methods). The individual reactions also contained the indicated concentrations of diphtheria toxin, ricin A chain, or puromycin. The reactions were incubated at 30°C for 90 min, and the labeled RNA products were characterized by CH₃HgOH-agarose gel electrophoresis and visualized by autoradiography. The amount of labeled genome-length viral RNA present in each lane was quantitated by using a Molecular Dynamics PhosphorImager and was plotted versus the concentration of each protein synthesis inhibitor.

FIG. 4

Dominant effect of polypeptide chain elongation inhibitors over puromycin. Preinitiation RNA replication complexes were isolated from 50 μl of HeLa S10 translation-replication reactions that contained 2 mM guanidine and were incubated at 30°C for 5 h. The preinitiation replication complexes were resuspended in 50 μl of fresh HeLa S10 translation-replication reactions containing 10 μCi of [α-³²P]CTP as described in Fig. 2 (method 3 in Materials and Methods). The reactions also contained 2 mM guanidine HCl (lane 1); no additional drug (lane 2); 50 μg of puromycin per ml (lane 3); 10 μg of diphtheria toxin per ml plus 40 μg of NAD⁺ per ml (lane 4); 0.2 μg of ricin A chain per ml (lane 5); 200 μg of cycloheximide per ml (lane 6); 8 μg of anisomycin per ml (lane 7); 10 μg of diphtheria toxin, 40 μg of NAD⁺, and 50 μg of puromycin per ml (lane 8); 0.2 μg of ricin A chain and 50 μg of puromycin per ml (lane 9); 200 μg of cycloheximide and 50 μg of puromycin per ml (lane 10); or 8 μg of anisomycin and 50 μg of puromycin per ml (lane 11) as indicated. The reactions were incubated at 30°C for 90 min, and the radiolabeled RNA products were phenol extracted, ethanol precipitated, separated by CH₃HgOH-agarose gel electrophoresis, and visualized by autoradiography.

FIG. 5

Effect of protein synthesis inhibitors on positive-strand RNA synthesis. Normal RNA replication complexes were isolated from 50 μl of HeLa S10 translation-RNA replication reactions that were incubated at 30°C for 5 h in the absence of 2 mM guanidine HCl (method 5 in Materials and Methods). The RNA replication complexes were resuspended in reactions containing HeLa S10 extract and 15 μCi of [α-³²P]CTP. The individual reactions also contained 2 mM guanidine HCl (lanes 2 to 7), 200 μg of cycloheximide per ml (lane 3), 8 μg of anisomycin per ml (lane 4), 20 μg of diphtheria toxin plus 20 μg of NAD⁺ per ml (lane 5), 100 μg of puromycin per ml (lane 6), or 2 μg of ricin A chain per ml (lane 7). The reactions were incubated at 30°C for 90 min, and the labeled RNAs were separated by CH₃HgOH-agarose gel electrophoresis and visualized by autoradiography.

FIG. 6

Two 5′-terminal GMP nucleotides in transcript RNA block positive-strand synthesis. (A) RNA transcripts of poliovirus cDNA clones used in this study contained two nonviral 5′-terminal GMP nucleotides that are required for efficient in vitro transcription by bacteriophage T7 RNA polymerase. The 3′-terminal CGCG sequence is derived from the MluI restriction site in the original plasmid DNA and has no significant inhibitory effect on negative-strand synthesis. RNA2(A)₈₀ is a poliovirus subgenomic transcript RNA that contains a single in-frame deletion of 1,782 nucleotides in the capsid coding region. (B) Preinitiation RNA replication complexes were isolated from HeLa S10 translation-RNA replication reactions containing RNA2(A)₈₀. The RNA2(A)₈₀-preinitiation replication complexes were resuspended in reaction mixtures containing [α-³²P]CTP and were incubated at 37°C for 16 or 32 min (method 4 in Materials and Methods). Labeled RNAs isolated from the 16 min (lane 2) and 32 min (lane 3) reactions were digested with RNase T₁ and then characterized by using a one-dimensional gel electrophoresis procedure as described earlier (6). RNase T₁ digests of [³²P]CMP-labeled negative-strand (lane 1) and positive-strand (lane 4) transcripts were used as markers for the poliovirus strand-specific oligonucleotides.

FIG. 7

Effect of puromycin and cycloheximide on negative-strand RNA synthesis within preinitiation RNA replication complexes. Preinitiation replication complexes were isolated from 50 μl of HeLa S10 translation-replication reactions containing T7-PV1(A)₈₀ RNA (rather than virion RNA) and 2 mM guanidine HCl after incubation at 30°C for 5 h (method 6 in Materials and Methods). The preinitiation complexes were resuspended in 50-μl reaction mixtures containing HeLa S10 extract, translation initiation factors, 50 μCi of [α-³²P]CTP, and 5 μM CTP_final. The individual reactions contained 2 mM guanidine (lane 1), no additional drugs (lane 2), 200 μg of cycloheximide per ml (lane 3), or 100 μg of puromycin per ml (lane 4). The reactions were incubated at 30°C for 60 min (A) or for 90 min (B), and the labeled RNA was phenol extracted and ethanol precipitated. The labeled RNA products were fractionated by CH₃HgOH-agarose gel electrophoresis. The dried gels were subjected to autoradiography for visualization of the labeled RNAs. The amount of labeled negative-strand RNA was quantitated by using a Molecular Dynamics PhosphorImager.