Figure 1.
(A) Schematic representation of a poliovirus-luciferase replicon. (B and C) Secondary structure of the PLuc cloverleaf and the two tandem cloverleaves in dCL-PLuc with wild-type sequences in CL1 and four G-C pairs in “stem a” of the downstream cloverleaf G/C-CL (highlighted in blue). (D) RNA replication in a cell-free system. RNA transcripts either Pluc (lanes 1, 2+5), N50-Pluc (50 non-polio nucleotides at the 5′-end of the viral RNA) (lanes 3+6), Pluc-GC (lane 4), or dCL-Pluc (lane 7) were used to program HeLa cell S10 extract. After 4 hours of incubation at 30°C, translation levels were measured as luciferase activity (arbitrary units [AU]), and pre-initiation complexes were isolated by centrifugation in the presence of 2 mM guanidinium hydrochloride (Gdn). RNA synthesis was initiated by addition of NTP and monitored by [α32P]UTP incorporation for 2 hr. The RNA synthesized was analyzed using native agarose gels. (E) Replication of poliovirus replicon in intact HeLa S3 cells. RNA transcripts (PLuc or dCL-PLuc) were transfected into HeLa S3 cells, and luciferase activity [AU] corresponding to 2.5×105 cells was measured every hour for 8h. The cells were incubated either in the presence (dashed lines) or absence (solid lines) of 2 mM Gdn. Each measurement was carried out in triplicate; standard deviations are indicated by vertical bars.
Figure 2.
Efficient replication requires a full-length cloverleaf structure at the most 5′-end of the virus genome.
(A) Schematic representation of the secondary structure of the 5′- ends of plus9, plus20, plus27, and StemA-disr RNAs. A SacI site (in lower case letters) was introduced as a linker between the partial cloverleaf 5′-ends (in red) and a downstream G/C-CL cloverleaf. (B) RNA replication in a cell-free system. RNA transcripts of either PLuc-GC (lane 1), dCL-PLuc (lane 2), plus9 (lane 3), plus20 (lane 4), plus27 (lane 5), or StemA-disr (lane 6) were used to program a cell extract. (C) Replication of replicons bearing partial cloverleaf structure at the 5′-end. RNA transcripts (dCL-PLuc, plus9 or StemA-disr) were transfected into HeLa S3 cells, and luciferase activity [AU] was measured every hour for 8h. Incubations were carried out in the presence (+Gdn) or absence of 2 mM Gdn. The graphs are representative of three independent experiments.
Figure 3.
Elements within StemB or StemD required for RNA replication.
(A) Representation of the secondary structure of tandem cloverleaf replicons with either StemB or StemD mutations (highlighted in red) in the 5′-end cloverleaf. (B and C) Luciferase expression in replicon RNA-transfected HeLa S3 cells. RNA transcripts containing mutations either within StemB or StemD were transfected into HeLa S3 cells, and luciferase activity [AU] was determined every hour for 8h. Control experiment included the addition of 2 mM Gdn. The graphs are representative of three independent experiments. (D and E) RNA replication in a cell-free system. Replicon RNA transcripts with mutations in either StemB or StemD were used to program a cell extract. (F) VPg-uridylylation in a cell-free replication system. RNA transcripts corresponding to tandem cloverleaf replicons containing mutations in either StemB or StemD were employed to program cell-free replications systems. VPg-pU(pU) formation was monitored by incubating the extracts with [α32P]UTP for 1 hour. The radiolabeled RNA was immuno-precipitated using anti-VPg antibodies, separated on a Tris-Tricine SDS-Page gel and visualized by using autoradiography.
Figure 4.
Replication of Double Cloverleaf replicons bearing mutations within StemA.
(A) Schematic representation of the secondary structure of Tandem cloverleaf structures replicons with either StemA mutations (highlighted in red) in the 5′-end cloverleaf. (B) Summary of replication rates of StemA mutants in a cell-free system and in HeLa S3 cells. (C) RNA replication in a cell-free system. RNA transcripts containing StemA mutations were used to program a cell extract. RNA products were analyzed on native agarose gels and detected by autoradiography. (D) Replication of virus RNA containing mutations within the StemA. Luciferase activity [AU] corresponding to 2.5×105 transfected HeLa-cells was measured every hour for 8h. The graphs are representative of three independent experiments.
Figure 5.
Analyzing the evolution of poliovirus carrying lesions within Stem A.
(A) One-step growth curve of poliovirus carrying either one cloverleaf (WT-polio type1) or tandem cloverleaf structures (dCL-polio type 1). HeLa S3 cells were infected at an MOI of 10 with either WT-polio type 1 or dCL-polio type 1 viruses. At indicated time-points viruses were harvested and their titers were determined according to standard plaque assays. The graphs are the mean of triplicate samples. Standard deviations are indicated by error bars. (B) Schematic representation of the cloverleaf StemA-8 and StemA-10 mutations (mutations highlighted in blue) and the changes in sequence observed in revertants, stemA-R8 or stemA-R10 (highlighted in red), with increased replication capacity. Multiple plaques from viruses with higher replication capacity were sequenced and they all contained the mutations highlighted in the figure.
Figure 6.
An integrated model for enterovirus replication.
Negative-strand synthesis is initiated by circularization of the positive-strand genome via a protein-protein bridge through the interaction of the ternary complex at the 5′-end (3CD and PCBP bound to the cloverleaf structure) and PABP bound to the 3′-poly(A)tail (I. + II.). CRE-mediated VPg-pUpU acts as primer of the reaction and the polymerase 3D synthesizes the new negative-strand (III.), resulting in a double-stranded intermediate (RF) (IV.). The positive-negative duplex RNA intermediate unwinds, so that the cloverleaf structure at the 5′-end of the positive-strand can form. 3CD and PCBP bind to the cloverleaf to form a ternary complex, which, in turn, will initiate positive-strand synthesis on the 3′-end of the negative-strand (V.). The primer, VPg-pUpU, is recruited and binds to the 3′-terminal AA of the negative strand, and the new positive-strand is synthesized by the polymerase, 3D (VI.).