Structure and function analysis of the poliovirus cis-acting replication element (CRE)

Abstract

The poliovirus cis-acting replication element (CRE) templates the uridylylation of VPg, the protein primer for genome replication. The CRE is a highly conserved structural RNA element in the enteroviruses and located within the polyprotein-coding region of the genome. We have determined the native structure of the CRE, defined the regions of the structure critical for activity, and investigated the influence of genomic location on function. Our results demonstrate that a 14-nucleotide unpaired terminal loop, presented on a suitably stable stem, is all that is required for function. These conclusions complement the recent analysis of the 14-nucleotide terminal loop in the CRE of human rhinovirus type 14. The CRE can be translocated to the 5' noncoding region of the genome, at least 3.7-kb distant from the native location, without adversely influencing activity, and CRE duplications do not adversely influence replication. We do not have evidence for a specific interaction between the CRE and the RNA-binding 3CD(pro) complex, an essential component of the uridylylation reaction, and the mechanism by which the CRE is coordinated and orientated during the reaction remains unclear. These studies provide a detailed overview of the structural determinants required for CRE function, and will facilitate a better understanding of the requirements for picornavirus replication.

FIGURE 1.

Poliovirus CRE predicted structure and modification. (A) Predicted structure of the native poliovirus type 3 CRE. The designation and location of the stem regions (based on Goodfellow et al. 2000) are indicated together with modifications made to these regions. Nucleotides in bold italics in Stem 3 represent defined mutations, the substitution of two or more of which render the CRE inactive (Goodfellow et al. 2000). The G nucleotide in bold italics in Inv1 (inversion of the Stem 1 region) represents a substitution made to avoid introducing a termination codon to the sequence. The conserved functional A¹A²A³CA motif in the terminal loop of the CRE is highlighted in bold. (B) Schematic diagram of virus genomes and subgenomic replicons used in this study. The noncoding regions are indicated as thin solid horizontal lines. Coding regions are indicated as wide open (unmodified poliovirus sequence) or filled (modified by replacement or mutagenesis) boxes. The region encoding chloramphenicol acetyl transferase expressed by subgenomic replicons is indicated as a solid black filled box labeled CAT. The disrupted native CRE sequence (located within the region encoding the virus 2C protein) is shown with an X. Subgenomic replicon-based cassette vectors (pT7/Rep3/SL3c and pT7/Rep3c) were constructed by the introduction of unique restriction sites flanking a ‘‘stuffer region’’ (indicated by the character ∼), which introduces a frameshift to the encoded polyprotein (see D). Relevant naturally present restriction sites used to introduce sequences into the 5′ NCR are indicated. The panel on the right-hand side defines the role the particular construct was used for in these studies, and whether there is a functional CRE in the native location within the region of the polyprotein encoding 2C. (C) The cassette region of pT7/Rep3c and pT7/Rep3/SL3c. The sequence through the engineered cassette region is shown, together with relevant translation products (indicated using single-letter amino acid codes). Without modification the cassette region is translated in the open reading frame (ORF) highlighted ‘‘0’’, which terminates 19 residues after the carboxyl terminus of the CAT protein. Digestion with Bss HII and Sma I allows replacement of the removed cassette stuffer region (shown in bold text) with complementary oligonucleotides that include the CRE sequence of interest. The oligonucleotides were designed to restore the polyprotein open reading frame to the +1 frame as shown. The YG dipeptide cleaved by the virus 2A^pro protease is shown in double height type, below the filled blocks indicating the short spacer region that flanks the inserted sequence and the start of the 2A^pro protease open reading frame.

FIGURE 1.

Poliovirus CRE predicted structure and modification. (A) Predicted structure of the native poliovirus type 3 CRE. The designation and location of the stem regions (based on Goodfellow et al. 2000) are indicated together with modifications made to these regions. Nucleotides in bold italics in Stem 3 represent defined mutations, the substitution of two or more of which render the CRE inactive (Goodfellow et al. 2000). The G nucleotide in bold italics in Inv1 (inversion of the Stem 1 region) represents a substitution made to avoid introducing a termination codon to the sequence. The conserved functional A¹A²A³CA motif in the terminal loop of the CRE is highlighted in bold. (B) Schematic diagram of virus genomes and subgenomic replicons used in this study. The noncoding regions are indicated as thin solid horizontal lines. Coding regions are indicated as wide open (unmodified poliovirus sequence) or filled (modified by replacement or mutagenesis) boxes. The region encoding chloramphenicol acetyl transferase expressed by subgenomic replicons is indicated as a solid black filled box labeled CAT. The disrupted native CRE sequence (located within the region encoding the virus 2C protein) is shown with an X. Subgenomic replicon-based cassette vectors (pT7/Rep3/SL3c and pT7/Rep3c) were constructed by the introduction of unique restriction sites flanking a ‘‘stuffer region’’ (indicated by the character ∼), which introduces a frameshift to the encoded polyprotein (see D). Relevant naturally present restriction sites used to introduce sequences into the 5′ NCR are indicated. The panel on the right-hand side defines the role the particular construct was used for in these studies, and whether there is a functional CRE in the native location within the region of the polyprotein encoding 2C. (C) The cassette region of pT7/Rep3c and pT7/Rep3/SL3c. The sequence through the engineered cassette region is shown, together with relevant translation products (indicated using single-letter amino acid codes). Without modification the cassette region is translated in the open reading frame (ORF) highlighted ‘‘0’’, which terminates 19 residues after the carboxyl terminus of the CAT protein. Digestion with Bss HII and Sma I allows replacement of the removed cassette stuffer region (shown in bold text) with complementary oligonucleotides that include the CRE sequence of interest. The oligonucleotides were designed to restore the polyprotein open reading frame to the +1 frame as shown. The YG dipeptide cleaved by the virus 2A^pro protease is shown in double height type, below the filled blocks indicating the short spacer region that flanks the inserted sequence and the start of the 2A^pro protease open reading frame.

FIGURE 2.

Structural mapping of the poliovirus CRE. (A) The autoradiograph of a representative mapping acrylamide gel is shown alongside a diagram of the ribonuclease cleavages indicated on the predicted structure of the CRE. T1, V1, and T2 cleavages are indicated with an arrow, black square, or black circle, respectively. The few positions cleaved with both V1 and T2 are indicated with an open square. Lane L is a hydroxidegenerated 1bp ladder produced from the same probe, lanes S are the probe alone incubated in the absence (−) or presence (+) of 100 mM sodium chloride (which is required for V1 ribonuclease activity). (A) The native poliovirus type 3 CRE. (B) The poliovirus type 3 CRE containing two substitutions in the Stem 3 region (see Fig. 1A ▶) of G19A and C22U. (C) The poliovirus type 3 CRE containing three substitutions in the Stem 3 region of G19A, C22U, and U40C. (D) The proposed structure of the native poliovirus type 3 CRE derived from ribonuclease mapping studies.

FIGURE 2.

Structural mapping of the poliovirus CRE. (A) The autoradiograph of a representative mapping acrylamide gel is shown alongside a diagram of the ribonuclease cleavages indicated on the predicted structure of the CRE. T1, V1, and T2 cleavages are indicated with an arrow, black square, or black circle, respectively. The few positions cleaved with both V1 and T2 are indicated with an open square. Lane L is a hydroxidegenerated 1bp ladder produced from the same probe, lanes S are the probe alone incubated in the absence (−) or presence (+) of 100 mM sodium chloride (which is required for V1 ribonuclease activity). (A) The native poliovirus type 3 CRE. (B) The poliovirus type 3 CRE containing two substitutions in the Stem 3 region (see Fig. 1A ▶) of G19A and C22U. (C) The poliovirus type 3 CRE containing three substitutions in the Stem 3 region of G19A, C22U, and U40C. (D) The proposed structure of the native poliovirus type 3 CRE derived from ribonuclease mapping studies.

FIGURE 3.

CAT activity of CRE cassette vector-derived replicons. (A) Reference diagram indicating the cassette and native locations for CRE sequences. Refer to Figure 1B ▶ for further details. (B) Replication activity of CAT-expressing subgenomic replicons containing duplications of the CRE. The name of the replicon cDNA is indicated in the left-hand column next to a phosphorimager-generated image of the ¹⁴C-labeled substrate and monoacetylated CAT products separated by thin-layer chromatography. The proportion of substrate converted to product is indicated (in percent). The columns labeled ‘‘Cassette’’ and ‘‘Native’’ indicate the CRE sequences present in the two positions of the genome; see (A). WT and SL3, respectively, refer to the native CRE sequence or a CRE containing eight nucleotide substitutions designed to disrupt the CRE structure (Goodfellow et al. 2000). WT* indicates native CRE sequences as previously published (Goodfellow et al. 2000). N/A indicates not applicable, where no CRE sequences are present at a particular location. (C) Predicted structures of synthetic CRE sequences used in replication studies. Synth3 to Synth5 are derived from Synth2 by sequential replacement of one to three CG base pairs (highlighted with numerical superscripts in Synth2) with UG pairs. These are shown separated to emphasize the differences, but are predicted to adopt standard Watson-Crick base pairing. The single-point mutation of A¹ to C in the ¹A²A³CA motif, introduced during the construction of Synth2mut7 is also indicated. (D) Replication of CAT-expressing subgenomic replicons containing synthetic CRE sequences. Layout and labeling as in (B), Syn indicates the presence of a synthetic CRE sequence. Each assay shown in (B) or (D) used normalized levels of total cell protein (5–50 μg). Comparison of replication activity between (B) and (D) cannot be made as the control (pT7/Rep3) reaction in (B) was allowed to progress to completion to emphasize differences in the replication of dual CRE containing replicons.

FIGURE 3.

CAT activity of CRE cassette vector-derived replicons. (A) Reference diagram indicating the cassette and native locations for CRE sequences. Refer to Figure 1B ▶ for further details. (B) Replication activity of CAT-expressing subgenomic replicons containing duplications of the CRE. The name of the replicon cDNA is indicated in the left-hand column next to a phosphorimager-generated image of the ¹⁴C-labeled substrate and monoacetylated CAT products separated by thin-layer chromatography. The proportion of substrate converted to product is indicated (in percent). The columns labeled ‘‘Cassette’’ and ‘‘Native’’ indicate the CRE sequences present in the two positions of the genome; see (A). WT and SL3, respectively, refer to the native CRE sequence or a CRE containing eight nucleotide substitutions designed to disrupt the CRE structure (Goodfellow et al. 2000). WT* indicates native CRE sequences as previously published (Goodfellow et al. 2000). N/A indicates not applicable, where no CRE sequences are present at a particular location. (C) Predicted structures of synthetic CRE sequences used in replication studies. Synth3 to Synth5 are derived from Synth2 by sequential replacement of one to three CG base pairs (highlighted with numerical superscripts in Synth2) with UG pairs. These are shown separated to emphasize the differences, but are predicted to adopt standard Watson-Crick base pairing. The single-point mutation of A¹ to C in the ¹A²A³CA motif, introduced during the construction of Synth2mut7 is also indicated. (D) Replication of CAT-expressing subgenomic replicons containing synthetic CRE sequences. Layout and labeling as in (B), Syn indicates the presence of a synthetic CRE sequence. Each assay shown in (B) or (D) used normalized levels of total cell protein (5–50 μg). Comparison of replication activity between (B) and (D) cannot be made as the control (pT7/Rep3) reaction in (B) was allowed to progress to completion to emphasize differences in the replication of dual CRE containing replicons.

FIGURE 4.

The poliovirus type 3-based in vitro uridylylation assay. (A) CRE-mediated uridylylation of VPg requires the presence of 3CD^pro and 3D^pol. The assay was constituted as described in Materials and Methods, with the exclusion of individual components as indicated in the table. Activity was quantified by measuring ³²P-UMP incorporation onto VPg using a Bio-Rad phosphorimager and is shown normalized to the activity observed in the presence of all three proteins. (B) Formation of VPg-pU(pU) requires the CRE template and 3CD^pro. The production of uridylylated VPg is dependent upon the presence of a template containing an unpaired A triplet (CRE, poly[A] or the 3′NCR) and CD^pro, whereas polyadenylated RNA templates (poly[A] and the 3′NCR) yield VPg-poly(U) in the presence or absence of 3CD^pro (indicated above the column labels). The reaction products of the 3D^pol terminal transferase activity are indicated with an asterisk. (C) CRE-templated uridylylation of VPg requires the A triplet in the A¹A²A³CA motif and a base-paired stem. VPg-pU(pU) was quantified by Bio-Rad phosphorimager and normalized to the levels observed with an unmodified CRE template (WT). The mutations present in the CRE RNA template are indicated above the autoradiogram, and relate to the numbering scheme shown in Figure 1A ▶. The G19A/C22U mutations disrupt Stem 3 (Figs. 1A, 2B ▶ ▶; Goodfellow et al. 2000).

FIGURE 5.

Uridylylation of VPg does not require the stem region of the CRE, but is influenced by CRE stability. In both panels the reaction temperature is indicated in the row labeled °C, and in (A) the amount of product is shown normalized to the maximal level observed with the unmodified native CRE (WT). The in vitro uridylylation assay was constituted with RNA templates containing a synthetic stem region and the native CRE terminal loop (Synth2–Synth5). Synth3 to Synth5 differ only in the number of hydrogen bonds stabilizing the base of the stem (see Fig. 3C ▶). (B) In vitro uridylylation of VPg primed with full-length RNA (∼5.5 kb) from subgenomic replicon cDNAs pT7/Rep3, pT7/Rep3/SL3, pT7/Rep3/SL3c^Synth4, and pT7/Rep3/SL3c^Synth5, labeled WT, SL3, Synth4, and Synth5, respectively.

FIGURE 6.

Protein–RNA interactions of the poliovirus CRE. (A) Electrophoretic mobility shift assay (EMSA) of the interaction of the 5′CL or CRE with poliovirus CD^pro. The identity of the input probe is indicated below the autoradiograph, with the position of the free probe indicated with the label ‘‘Probe’’. The lanes labeled (−) contain no added 3CD^pro, the relative concentration of which is indicated with a triangle. The 3CD^pro-containing complex is indicated with an asterisk (*). (B) Specificity of the 3CD^pro interaction with the CRE. Formation of the 3CD^pro/CRE ribonucleoprotein complex (indicated *) was assayed in the presence of no competitor (lane labeled 0) or competing (10× and 100×) molar excess of the native CRE, the 5′CL, 3′ NCR, or the nonfunctional CRE variants, SL3, G19A/C22U, or G19A/C22U/U40C. All lanes contain 3CD^pro, as indicated by the bar below the figure, with the exception of the lane containing probe alone, which is labeled (−).

FIGURE 7.

The poliovirus CRE functions when located in the 5′ NCR of the genome. Complementary oligonucleotides for the CRE were directionally cloned between the Bam HI and Sac I sites located, respectively, in the 5′ NCR and region encoding the amino-terminus of VP4. The CRE sequence was modified by substitution of U15A/A49U (Fig. 1A ▶) to replace the AUG_49–51 to generate pT7/SL3HV^CRE-AUG. The same region of pT7/SL3 was also replaced with oligonucleotides encoding the Synth2 CRE, and a Synth2-derived CRE in which one of the conserved A triplet was deleted (generating pT7/SL3-HV^Synth2 and pT7/SL3-HV^Synth2ΔA, respectively). Normalized levels of RNA generated in vitro, and serial 10-fold dilutions, was transfected into HeLa cells and overlaid with agar. Plaques were visualized after 3 d by staining with crystal violet.