Hepatitis C virus NS5B polymerase exhibits distinct nucleotide requirements for initiation and elongation

Abstract

The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3'-end, we examined de novo initiation and primer extension in a system devoid of self-priming and terminal nucleotide transferase activities. Products predominantly of template size and its multiples were detected. High concentrations of nucleoside triphosphates (K(app)(m) approximately 100-400 mum) corresponding to the first three incorporated nucleotides were found to be required for efficient de novo RNA synthesis. In the presence of initiating di- or trinucleotides, however, the amount of NTP needed to achieve maximal activity dropped 10(3)- to 10(4)-fold, revealing a much reduced nucleotide requirement for elongation (K(app)(m) approximately 0.03-0.09 microm). Accordingly, single round extension from an exogenous primer following preincubation of the enzyme with template and primer could also be supported by <0.1 microm levels of NTP. De novo synthesis at high NTP concentrations was shown to be preferred over primer extension. On a dideoxycytidine-blocked synthetic RNA template derived from the 3'-end of the HCV(-)UTR, the addition of the corresponding initiating trinucleotide also dramatically reduced the NTP levels needed to achieve efficient RNA synthesis. Thus, distinct nucleotide requirements exist for initiation and elongation steps catalyzed by the HCV NS5B polymerase.

FIGURE 1.

De novo initiation of RNA synthesis by HCV NS5B on a synthetic heteropolymer RNA template. A, sequence of DCoH75, the 75-mer RNA template used in most of this study. DCoH75ddC represents an otherwise identical template with the 3′-terminal cytidine replaced by a ddC moiety. B, NS5B-catalyzed RNA products directed from DCoH75 (lanes 1–5) or DCoH75ddC (lanes 6–10) template were analyzed by TBE-urea denaturing PAGE with all four ribonucleotides present in the reaction (lanes 1 and 6), without ATP (lanes 2 and 7), without UTP (lanes 3 and 8), without GTP (lanes 4 and 9), or with only CTP present (lanes 5 and 10). C, 3′-end labeling of DCoH75 (lane 3) or DCoH75ddC (lane 2) template was performed with T4 RNA ligase in the presence of [³²P]pCp. RNA Decade Marker (Ambion) was labeled by T4 polynucleotide kinase in the presence of [γ-³²P]ATP (lane 1). D, RNase T1 treatment of NS5B reaction products using DCoH75 (lanes 2 and 3) or DCoH75ddC (lanes 4 and 5) template as generated in B was performed under digest conditions that cleaved single-stranded but not double-stranded RNA. Digested products were then analyzed by denaturing PAGE as shown. Nt, nucleoide.

FIGURE 2.

Nucleotide requirements for de novo RNA synthesis. A, NS5B activity on DCoH75ddC template was monitored by [α-³²P]CTP incorporation for 1 h at room temperature. In addition to radiolabeled CTP (at 0.1 μm), reaction mixtures contained either a 500 μm concentration each of ATP, GTP, and UTP or the indicated nucleotide(s) at 5 μm with the remaining NTP at 500 μm. B, NS5B activity was assayed for 2 h at room temperature in separate mixtures containing one of the four alternative [α-³³P]NTPs at a concentration of 20 nm, with the other three unlabeled nucleotides at 500 μm each. The maximal activity obtained for each template was expressed as 100%.

FIGURE 3.

Effect of dinucleotide addition on NS5B activity. NS5B activity on DCoH75ddC template was assayed by [α-³²P]CTP incorporation as in Fig. 2A, in the presence of a 5 μm concentration each of GTP and ATP, 200 μm UTP, with or without a 1 mm concentration of the indicated dinucleotide.

FIGURE 4.

Reduction in NS5B nucleotide requirements in the presence of di- and trinucleotides. A, RdRp reactions were carried out for 1 h at room temperature in the presence or absence of 0.5 mm GA dinucleotide with a 500 μm concentration each of GTP and UTP, [α-³²P]CTP (0.1 μm), and various concentrations of ATP as shown. B, titration of UTP was performed in the presence of 0.5 mm GAU trinucleotide with a 0.1 μm concentration each of GTP and ATP and [α-³²P]CTP (20 nm). Curve fitting was carried out with a hyperbolic equation for one-site binding in Prism (GraphPad).

FIGURE 5.

Effect of primer addition on NS5B activity. An 11-mer primer complementing the 3′ terminus of the DCoH75ddC template was used to probe primer extension activity. A, RdRp activity was measured by [α-³³P]CTP incorporation with or without primer in the presence of a 500 μm concentration each of GTP, ATP, and UTP at 30 °C for 2 h. Where indicated, enzyme, template, and primer (each at 0.1 μm final concentration) were preincubated at room temperature for 18 h prior to the addition of nucleotides to start the reaction. B, primer was 5′-end-labeled with T4 polynucleotide kinase and [γ-³²P]ATP. A 500 μm concentration each of GTP, ATP, and UTP and 1 μm CTP were present in the RdRp reaction with radiolabeled primer, template, and enzyme (at 0.1 μm each) at 30 °C for 2 h, with (lane 1) or without (lane 2) preincubation of enzyme, template, and primer at room temperature for 18 h. Primer extension activity was evaluated by comparing the amount of ³²P-labeled primer incorporated into the template-length product with that of total [³²P]CTP incorporation in a preincubated reaction performed as in A.

FIGURE 6.

Effect of heparin trap on NS5B activity in the presence of a primer. A, a time course was performed on NS5B activity in the presence of an 11-mer primer (enzyme, template, and primer each at 0.1 μm final concentration) by following [α-³³P]CTP incorporation at 15 s, 30 s, 1 min, 5 min, 20 min, 60 min, and 120 min in the presence of a 500 μm concentration each of GTP, ATP, and UTP. RdRp reactions were initiated by the addition of nucleotides in the presence or absence of heparin (0.2 mg/ml final concentration). Where indicated, enzyme, template, and primer were preincubated at room temperature for 22 h prior to the reaction. Curve fitting of data were performed in Prism (GraphPad) using a burst linear model and linear regression, respectively, for preincubated and nonpreincubated reactions. B, titration of GTP under single-round elongation conditions was performed at room temperature for 10 min in the presence of 0.2 mg/ml heparin with preincubated enzyme, template, and primer (room temperature/22-h preincubation, 0.1 μm each final concentration), 500 μm concentration each of ATP and UTP, [α-³³P]CTP (20 nm), and various concentrations of GTP, as shown. For determination, see “Experimental Procedures.”

FIGURE 7.

Nucleotide requirements for RNA synthesis directed by HCV(–)UTR67ddC template. A, sequence of HCV(–)UTR67ddC. B, NS5B activity was assayed by [α-³³P]UTP (20 nm) incorporation with 0.1 μm NS5B-Δ21 and 0.1 μm HCV(–)UTR67ddC template at 30 °C for 1.5 h in the presence of the indicated levels of the other nucleotides. Initiator GC or GCC was added as indicated at 0.5 mm.