De novo synthesis of negative-strand RNA by Dengue virus RNA-dependent RNA polymerase in vitro: nucleotide, primer, and template parameters

Abstract

By using a purified dengue virus RNA-dependent RNA polymerase and a subgenomic 770-nucleotide RNA template, it was shown previously that the ratio of the de novo synthesis product to hairpin product formed was inversely proportional to increments of assay temperatures (20 to 40 degrees C). In this study, the components of the de novo preinitiation complex are defined as ATP, a high concentration of GTP (500 micro M), the polymerase, and the template RNA. Even when the 3'-terminal sequence of template RNA was mutated from -GGUUCU-3' to -GGUUUU-3', a high GTP concentration was required for de novo initiation, suggesting that high GTP concentration plays a conformational role. Furthermore, utilization of synthetic primers by the polymerase indicated that AGAA is the optimal primer whereas AG, AGA, and AGAACC were inefficient primers. Moreover, mutational analysis of the highly conserved 3'-terminal dinucleotide CU of the template RNA indicated that change of the 3'-terminal nucleotide from U to C reduced the efficiency about fivefold. The order of preference for the 3'-terminal nucleotide, from highest to lowest, is U, A - G, and C. However, change of the penultimate nucleotide from C to U did not affect the template activity. A model consistent with these results is that the active site of the polymerase switches from a "closed" form, catalyzing de novo initiation through synthesis of short primers, to an "open" form for elongation of a double-stranded template-primer.

FIG. 1.

Components of the de novo preinitiation complex. The protocol for determining the components for the de novo preinitiation complex is outlined in each of the panels on the right. The conditions under which de novo initiation occurs in preference to 3′-end elongation (1×/2× ratio, ≥2) define the components for formation of the de novo preinitiation complex. It was previously established that one of the determinants for preferential de novo initiation is incubation at a low temperature (1). In experiments A to E, if components in an initial preincubation mixture are favorable for de novo initiation, then subsequent additions of missing components should not affect the preferential formation of the de novo product. RdRP assays were performed, and the products were fractionated on formaldehyde-agarose gels and subjected to autoradiography. The template-sized and hairpin products were cut out and quantitated by scintillation counting. The 1×/2× ratio on the y axis is plotted against the temperature of the RdRP assay on the x axis.

FIG. 2.

Preincubation of either ATP or GTP alone with NS5 and template is not sufficient for de novo initiation. (A) RdRP assays were carried out at 30°C with limiting components before the addition of heparin, and then the complete system was reconstituted after its addition. At 30°C, if all components were added at the same time, de novo initiation and 3′-end elongation occurred on the template RNA, and equal amounts of 1× and 2× products were formed (1). (B) The reaction products were analyzed by formaldehyde-agarose gel electrophoresis and detected by autoradiography.

FIG. 3.

Analysis of NTP requirement for de novo initiation or 3′-end elongation. For RdRPs, the initiating nucleotide is required at high concentrations for de novo synthesis whereas lower concentrations are sufficient for elongation (see the text). Standard RdRp assays in which all components were held at a fixed concentration except for the NTP that was varied (UTP [A], ATP [B], or GTP[C]) were performed at 30°C. Products were analyzed by formaldehyde-agarose gel electrophoresis followed by autoradiography. The 1× and 2× products were cut out and quantified by either scintillation counting (A and B) or density analysis using the imagej program, a free software product available at the National Institutes of Health website (http://rsb.info.nih.gov/ij/). ND, not done.

FIG. 3.

Analysis of NTP requirement for de novo initiation or 3′-end elongation. For RdRPs, the initiating nucleotide is required at high concentrations for de novo synthesis whereas lower concentrations are sufficient for elongation (see the text). Standard RdRp assays in which all components were held at a fixed concentration except for the NTP that was varied (UTP [A], ATP [B], or GTP[C]) were performed at 30°C. Products were analyzed by formaldehyde-agarose gel electrophoresis followed by autoradiography. The 1× and 2× products were cut out and quantified by either scintillation counting (A and B) or density analysis using the imagej program, a free software product available at the National Institutes of Health website (http://rsb.info.nih.gov/ij/). ND, not done.

FIG. 3.

Analysis of NTP requirement for de novo initiation or 3′-end elongation. For RdRPs, the initiating nucleotide is required at high concentrations for de novo synthesis whereas lower concentrations are sufficient for elongation (see the text). Standard RdRp assays in which all components were held at a fixed concentration except for the NTP that was varied (UTP [A], ATP [B], or GTP[C]) were performed at 30°C. Products were analyzed by formaldehyde-agarose gel electrophoresis followed by autoradiography. The 1× and 2× products were cut out and quantified by either scintillation counting (A and B) or density analysis using the imagej program, a free software product available at the National Institutes of Health website (http://rsb.info.nih.gov/ij/). ND, not done.

FIG. 4.

Effects of varying amounts of ATP and GTP on RdRP activity. RdRP assays were carried out as described in Materials and Methods except that the concentrations of ATP (A and B) or GTP (C) were varied. The amounts of the hairpin product produced by 3′ elongation and the template-sized product produced by de novo synthesis of RNA were determined by counting the radioactivity by use of a scintillation counter. Data are presented as double-reciprocal plots (1/V versus 1/[S]) generated with Microsoft Excel. The plot for GTP utilization in de novo synthesis showed a complex kinetics and therefore could not be accurately determined (see the text).

FIG. 5.

Analysis of primer utilization by the DEN2 polymerase reveals that the optimal primer for DEN2 polymerase is AGAA. (A) The oligoribonucleotide primer AG, AGA, AGAA, or AGAACC (40 μM) (lanes 1 to 4) was simply mixed with the subgenomic RNA template (0.1 μg) in a standard RdRP assay. The temperature of incubation of the RdRP assays was kept at 35°C, at which predominantly a hairpin product was formed in the absence of any primer (lane 5). (B) The concentration of AGAA was varied while all other components were held at constant levels. Products were analyzed by formaldehyde-agarose gel electrophoresis and autoradiography as described in Materials and Methods. The products were quantified by densitometric analysis as described in the legend to Fig. 3.

FIG. 6.

High GTP concentration requirement for de novo initiation on a mutant RNA template. The positive-strand RNA template containing the 3′-end sequence GGUUCU-3′ in the wild-type RNA was mutated to GGUUUU-3′ by PCR as described in Materials and Methods. RdRP assays were performed at 30°C by using fixed amounts of all components except GTP (A) or ATP (B). Products were analyzed by formaldehyde-agarose gel electrophoresis followed by autoradiography. The intensities of bands were quantified by using the imagej program as described in the Fig. 3 legend. ND, not done.

FIG. 7.

Mutational analysis of the 3′-terminal sequences of the template RNA. The wild-type sequence, UUCU-3′, was mutated to UUUU-3′ (see Fig. 6), UCCU-3′, UUCA-3′, UUCG-3′, or UUCC-3′ as described in Materials and Methods. The mutant template RNAs in three different concentrations (0.02, 0.1, and 1.0 μg) were used in the RdRP assays, and the products were analyzed by formaldehyde-agarose gel electrophoresis and autoradiography. The radioactivities for the 1× and 2× products were quantified by liquid scintillation counting. The sum of the radioactivity, representing total RNA synthesis, was determined by using 0.1 μg each of wild-type and mutant RNA templates. The activity of each mutant relative to that of wild-type RNA, taken as 100%, is given below the gel.