Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg

Abstract

The first step in the replication of the plus-stranded poliovirus RNA is the synthesis of a complementary minus strand. This process is initiated by the covalent attachment of UMP to the terminal protein VPg, yielding VPgpU and VPgpUpU. We have previously shown that these products can be made in vitro in a reaction that requires only synthetic VPg, UTP, poly(A), purified poliovirus RNA polymerase 3D(pol), and Mg(2+) (A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998). Since such a poly(A)-dependent process cannot confer sufficient specificity to poliovirus RNA replication, we have developed a new assay to search for a viral RNA template in conjunction with viral or cellular factors that could provide this function. We have now discovered a small RNA hairpin in the coding region of protein 2C as the site in PV1(M) RNA that is used as the primary template for the in vitro uridylylation of VPg. This hairpin has recently been described in poliovirus RNA as being an essential structure for the initiation of minus strand RNA synthesis (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000). The uridylylation reaction either with transcripts of cre(2C) RNA or with full-length PV1(M) RNA as the template is strongly stimulated by the addition of purified viral protein 3CD(pro). Deletion of the cre(2C) RNA sequences from minigenomes eliminates their ability to serve as template in the reaction. A similar signal in the coding region of VP1 in HRV14 RNA (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the poliovirus cre(2C) can be functionally exchanged in the assay. The mechanism by which the VPgpUpU precursor, made specifically on the cre(2C) template, might be transferred to the site where it serves as primer for poliovirus RNA synthesis, remains to be determined.

FIG. 1

(A) Structure of poliovirus genomic RNA. The single-stranded RNA genome of poliovirus is shown with the terminal protein VPg (3B) at the 5′ end of the NTR (single line) and the 3′NTR (single line) with the poly(A) tail. The 5′NTR consists of a small cloverleaf and a large IRES element. The location of the cre(2C) element and of SLII of the IRES are indicated. The attachment site of the 5′-terminal UMP of the RNA to the tyrosine of VPg is shown enlarged. The polyprotein contains structural (P1) and nonstructural (P2 and P3) domains. The boxed region containing vertical lines represents the polyprotein with the proteinase cleavage sites. Processing of the P3 domain by proteinases 3C and 3CD^pro is shown enlarged. (B) Predicted secondary structures of the HRV14 cre(VP1) (35, 36) and the PV1(M) cre(2C) RNAs (19). Conserved sequences are shown in boldface. (C) Conserved cre sequences in the coding regions of picornaviral RNAs (, –36).

FIG. 2

Uridylylation of VPg in vitro on full-length or truncated PV1(M) transcript RNA templates. The synthesis of VPgpU(pU) was measured as described in Materials and Methods, except that the UTP concentration was 0.1 μM and the poly(A) template was replaced with 1.5 μg of PV1(M) transcript RNA, either full length or truncated. Where indicated, 3CD^pro was added to the reaction mixtures. (A) Stimulation by 3CD^pro. (B) Full-length and truncated PV1(M) transcript RNAs as templates.

FIG. 3

Polioviral cis-replicating RNAs as templates in the in vitro uridylylation of VPg. Synthesis of VPgpU(pU) was measured as described in Materials and Methods, except that poly(A) was replaced by a transcript or tRNA (0.5 μg) template. Where indicated, the reactions contained 3CD^pro. The top of the figure shows the autoradiography of the reaction products, and the bottom portion displays the quantitation of the data. (A) Comparison of cre(2C) RNA with 3′NTR-poly(A), −cloverleaf and +cloverleaf RNAs, and tRNA. (B) Comparison of cre(2C) RNA with the IRES SLII RNA.

FIG. 4

Uridylylation of VPg on minigenomic RNA templates. Synthesis of VPgpU(pU) was measured as described in Materials and Methods with minigenomic transcript RNAs (1.0 μg) as templates. Where indicated, 3CD^pro was added to the reaction mixtures. Minigenome RNAs (shown below) contained the cre(2C) element either in the forward (F) direction (I) or in the reverse (R) direction (III). The cre(2C) was deleted from RNA II. (A) The cre(2C) element is required for optimal template activity. (B) The reaction requires the plus strand sequence of the cre(2C) element. The top of the figure shows the autoradiography of the reaction products, and below it the quantitation of the data are displayed.

FIG. 5

Comparison of poly(A) and cre(2C) RNA as templates in the in vitro uridylylation of VPg. Synthesis of VPgpU(pU) was measured as described in Materials and Methods with either Mg²⁺ or Mn²⁺, and the template was varied, as indicated. Some samples contained 3CD^pro, as indicated. The top of the figure shows the autoradiography of the reaction products, and the bottom portion displays the quantitation of the data.

FIG. 6

Uridylylation of VPg in vitro on a cre(2C) template. The synthesis of VPgpU(pU) was measured as described in Materials and Methods, containing 0.5 μg of cre(2C) transcript RNA. (A) Optimal concentrations of 3CD^pro required for stimulation. The amounts of 3CD^pro added were varied, as shown in the figure. (B) 3CD^pro stimulates the 3D^pol catalyzed reaction. Sample 1 contained no 3CD^pro; in sample 2 3D^pol was omitted but 3CD^pro was added, and sample 3 contained 3CD^pro heated for 3 min at 70°C. (C) VPg specificity of the reaction. Where indicated, wt VPg was either omitted (lane 4) or replaced with a synthetic mutant VPg peptide (2 μg) that contained either the Y3F (lane 2) or the R17E (lane 3) amino acid change. All samples contained 3CD^pro. The top of the figure shows the autoradiography of the reaction products and the bottom portion displays the quantitation of the data.

FIG. 6

Uridylylation of VPg in vitro on a cre(2C) template. The synthesis of VPgpU(pU) was measured as described in Materials and Methods, containing 0.5 μg of cre(2C) transcript RNA. (A) Optimal concentrations of 3CD^pro required for stimulation. The amounts of 3CD^pro added were varied, as shown in the figure. (B) 3CD^pro stimulates the 3D^pol catalyzed reaction. Sample 1 contained no 3CD^pro; in sample 2 3D^pol was omitted but 3CD^pro was added, and sample 3 contained 3CD^pro heated for 3 min at 70°C. (C) VPg specificity of the reaction. Where indicated, wt VPg was either omitted (lane 4) or replaced with a synthetic mutant VPg peptide (2 μg) that contained either the Y3F (lane 2) or the R17E (lane 3) amino acid change. All samples contained 3CD^pro. The top of the figure shows the autoradiography of the reaction products and the bottom portion displays the quantitation of the data.

FIG. 7

Effect of translation reactions of wt and mutant 3C^pro(R84S/I86A) PV1(M) RNA on the VPg-uridylylation reaction. The synthesis of VPgpU(pU) was measured as described in Materials and Methods, except that the template was varied, as indicated. Translation reaction mixtures (2 μl) were added, as shown in the figure. PV1(M) wt or mutant 3C^pro(R84S/I86A) RNAs, derived from two independently isolated pT7PVM[3C^pro(R84S/I86A)] cDNAs, were translated in HeLa cell extracts (40) for either 0 or 6 h at 34°C, and an aliquot of each translation mixture was heated 3 min at 70°C. The following template RNAs were used: samples 1 to 6, cre(2C) RNA (0.5 μg); samples 7 to 11, PV1(M) transcript RNA (1.5 μg); samples 12 to 14, poly(A) (0.5 μg). The top of the figure shows the autoradiography of the reaction products, and the bottom portion displays the quantitation of the data.

FIG. 8

PV1(M) cre(2C) RNA is functionally exchangeable with the cre(VP1) RNA of HRV14 in the in vitro assay. The synthesis of VPgpU(pU) was measured as described in Materials and Methods, except that the template and VPg were varied, as indicated. The template was either PV1 cre(2C) RNA or HRV14 cre(VP1) RNA. Where indicated, PV1(M) VPg was replaced by HRV14 VPg (2 μg). Samples 2, 4, 6, and 8 contained 3CD^pro. The top of the figure shows the autoradiography of the reaction products; below it the quantitation of the data is displayed. The amino acid sequences of PV1(M) and HRV14 VPg are shown below. Amino acid differences between PV1(M) VPg and that of HRV14 are indicated with boldface letters.

FIG. 9

Proposed model for poliovirus RNA minus-strand RNA synthesis compared to that of hepatitis B virus and adenovirus. Poliovirus minus-strand synthesis might resemble that used by hepatitis B virus (60) and adenovirus (25). Both DNA and RNA synthesis takes place in three consecutive steps: protein priming, jumping back, and elongation. Poliovirus 3D^pol complexed with VPg and 3CD^pro binds to the cre(2C) RNA (19). RNA polymerase 3D^pol catalyzes the covalent linkage of UMP to VPg using the first two A's of the conserved AAACA sequence of the cre(2C) RNA as a template. The complex of 3D^pol and VPgpUpU is released and translocated to the 3′ end of the poly(A) tail. The precursor VPgpUpU then serves as primer for minus-strand RNA synthesis. Abbreviations: RT, reverse transcriptase; pTP, preterminal protein; DP, DNA polymerase.