Stimulation of poliovirus synthesis in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CDpro

Abstract

The plus-strand RNA genome of poliovirus serves three distinct functions in the life cycle of the virus. The RNA is translated and then replicated, and finally the progeny RNAs are encapsidated. These processes can be faithfully reproduced in a HeLa cell-free in vitro translation-RNA replication system that produces viable poliovirus. We have previously observed a stimulation of virus synthesis when an mRNA, encoding protein 3CD(pro), is added to the translation-RNA replication reactions of poliovirus RNA. Our aim in these experiments was to further define the factors that affect the stimulatory activity of 3CD(pro) in virus synthesis. We observed that purified 3CD(pro) protein also enhances virus synthesis by about 100-fold but has no effect on the translation of the polyprotein. Optimal stimulation is observed only when 3CD(pro) is present early in the incubation period. The stimulation, however, is abolished by a mutation either in the RNA binding domain of 3CD(pro), 3C(pro)R84S/I86A, or by each of two groups of complementary mutations R455A/R456A and D339A/S341A/D349A at interface I in the 3D(pol) domain of 3CD(pro). Surprisingly, virus synthesis is strongly inhibited by the addition of both 3C(pro) and 3CD(pro) at the beginning of incubation. We also examined the effect of other viral or cellular proteins on virus synthesis in the in vitro system. No enhancement of virus synthesis occurred with viral proteins 3BC, 3ABC, 3BCD, 3D(pol), and 3C(pro) or with cellular protein PCBP2. These results suggest that 3CD(pro) has to be present in the reaction at the time the replication complexes are assembled and that both the 3C(pro) and 3D(pol) domains of the protein are required for its activity that stimulates virus production.

FIG. 1.

Genomic structure of poliovirus and processing of the P3 domain of the polyprotein. The single-stranded RNA genome of poliovirus is shown with the terminal protein VPg attached to the 5′ end of the RNA and the poly(A) tail at the 3′ end. The 5′ NTR and the 3′ NTR are shown with single lines. The polyprotein (open box) contains structural (P1) and nonstructural (P2, P3) domains that are cleaved into individual polypeptides. Processing of the P3 domain by proteinase 3C^pro/3CD^pro is shown enlarged.

FIG. 2.

Stimulation of virus production in the in vitro translation-RNA replication system by 3CD^pro. In vitro translation-RNA replication reactions and plaque assays were carried out as described in Materials and Methods. Where indicated, 3CD^pro mRNA or purified 3CD^pro(3C^proH40A) protein was added to the translation reactions. (A) Effect of 3CD^pro mRNA concentration. The amount of 3CD^pro mRNA added to the translation reactions at t = 0 h is indicated in the figure. (B) Effect of purified 3CD^pro protein concentration. The amount of purified 3CD^pro(3C^proH40A) protein added to the translation reactions at t = 0 h was varied as indicated in the figure. From three different experiments, the average value for the stimulation of virus synthesis by 3CD^pro was 130-fold. (C) Comparison of the stimulatory activities of 3CD^pro mRNA and purified 3CD^pro(3C^proH40A) protein. Virus production was measured with optimal concentrations of either 3CD^pro mRNA (1.4 μg/ml) or 3CD^pro(3C^proH40A) protein (5.5 nM) added to the translation-RNA replication reactions.

FIG. 3.

3CD^pro has to be added early to the translation-RNA replication reactions to exert optimal stimulatory activity. In vitro translation-RNA replication reactions and plaque assays were carried out as described in Materials and Methods. Purified 3CD^pro(3C^proH40A) protein was added to the reactions at the indicated times. (A) Effect of time of 3CD^pro addition. The time at which 3CD^pro(H40A) protein (5.5 nM) was added to the translation reactions was varied as indicated. (B) Addition of purified 3CD^pro(3C^proH40A) protein to preinintiation replication complexes. Preinitiation replication complexes (PIRC) were made as described in Materials and Methods. The complexes were resuspended in either the absence (column 1) or presence (column 2) of purified 3CD^pro(3C^proH40A) (5.5 nM). In column 3, 3CD^pro(3C^proH40A) (5.5 nM) was added at t = 0 h before the formation of the PIRC.

FIG. 4.

Mutations both in the 3C^pro and 3D^pol domains of 3CD^pro affect the stimulation of virus synthesis. In vitro translation-RNA replication reactions and plaque assays were carried out as described in Materials and Methods. (A) Either wild-type or mutant 3C^pro(R84S/I86A) 3CD^pro mRNAs (1.4 μg/ml) were added to the reactions at t = 0 h as indicated. From three different experiments, the average value for the stimulation of virus synthesis by 3CD^pro(3C^proR84S/I86A) was 1.3-fold. (B) CD^pro(3C^proH40A), 3CD^pro(3C^proH40G; 3D^polR455A/R456A), or 3CD^pro(3C^proH40G; 3D^polD339A/S341A/D349A) purified proteins (5.5 nM) were added to the reactions at t = 0 h as indicated. From two different experiments, the average value for the stimulation of virus synthesis by 3CD^pro(3C^proH40G/3D^polR455A/R456A) protein was 1.0-fold and by 3CD^pro(3C^proH40G; 3D^polD339A/S341A/D349A) it was 1.4-fold.

FIG. 5.

Effect of 3ABC, 3BC, 3BCD, 3C^pro, and PCBP2 on virus production in the translation-RNA replication system. In vitro translation-RNA replication reactions and plaque assays were carried out as described in Materials and Methods. Purified viral protein was added (t = 0 h) to the translation-RNA replication reactions (40 ng/ml and 400 ng/ml). The effect of each protein on virus synthesis was tested at least twice, and the data shown are an average of the two experiments. (A) The effect of 3ABC, 3BC, 3BCD, or 3C^pro on virus production. Column 2, 3CD^pro (3C^proH40A), 5.5 nM; columns 3 and 4, 3ABC(3C^proH40A), 2.5 nM and 12.5 nM, respectively; columns 5 and 6, 3C^pro(C147G), 2 nM and 20 nM, respectively; columns 7 and 8, 3BC(3C^proC147G), 1.8 nM and 18 nM, respectively; columns 9 and10, 3BCD(3C^proC147G), 0.54 nM and 5.4 nM, respectively. (B) Effect of adding PCBP2 with or without 3CD^pro on virus production. Purified PCBP2 and/or purified 3CD^pro(3C^proH40A) was added as indicated in the figure.

FIG. 6.

Effect of adding 3CD^pro together with 3C^pro and/or 3D^pol on virus production in translation-RNA replication reactions. In vitro translation-RNA replication reactions and plaque assays were carried out as described in Materials and Methods. Purified 3CD^pro(3C^proH40A), 3D^pol, or 3C^pro(C147G), each at 400 ng/ml, was added to the reactions at t = 0 h as indicated in the figure. The molar concentration of 3CD^pro(3C^proH40A) was 5.5 nM, that of 3C^pro(C147G) was 20 nM, and that of 3D^pol was 7.7 nM. The average value obtained from three different experiments for the effect of 3C^pro on virus synthesis was 1.0-fold; the inhibitory effect of 3C^pro and 3CD^pro added together was 60-fold.

FIG. 7.

In vitro translation of vRNA is not affected by the addition of 3CD^pro or 3C^pro. In vitro translation reactions of viral RNA were incubated for 12 h at 34°C, and the products were analyzed (see Materials and Methods). Purified proteins (400 ng/ml) or mRNA (1.4 μg/ml) was added to the reactions at t = 0 h. Lane 1, vRNA; lane 2, 5.5 nM 3CD^pro(3C^proH40A) protein; lane 3, 3CD^pro(3C^proR84S/I86A) mRNA; lane 4, 5.5 nM 3CD^pro(3C^proH40G; 3D^polR455A/R456A) protein; lane 5, 20 nM 3C^pro(C147G) protein; lane 6, 5.5 nM 3CD^pro(3C^proH40A) and 20 nM 3C^pro(C147G) proteins.