Selection of 3'-template bases and initiating nucleotides by hepatitis C virus NS5B RNA-dependent RNA polymerase

Abstract

De novo RNA synthesis by hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase has been investigated using short RNA templates. Various templates including those derived from the HCV genome were evaluated by examining the early steps of de novo RNA synthesis. NS5B was shown to be able to produce an initiation dinucleotide product from templates as short as 4-mer and from the 3'-terminal sequences of both plus and minus strands of the HCV RNA genome. GMP, GDP, and guanosine were able to act as an initiating nucleotide in de novo RNA synthesis, indicating that the triphosphate moiety is not absolutely required by an initiating nucleotide. Significant amounts of the initiation product accumulated in de novo synthesis, and elongation from the dinucleotide was observed when large amounts of dinucleotide were available. This result suggests that NS5B, a template, and incoming nucleotides are able to form an initiation complex that aborts frequently by releasing the dinucleotide product before transition to an elongation complex. The transition is rate limiting. Furthermore, we discovered that the secondary structure of a template was not essential for de novo initiation and that 3'-terminal bases of a template conferred specificity in selection of an initiation site. Initiation can occur at the +1, +2, or +3 position numbered from the 3' end of a template depending on base composition. Pyrimidine bases at any of the three positions are able to serve as an initiation site, while purine bases at the +2 and +3 positions do not support initiation. This result implies that HCV possesses an intrinsic ability to ensure that de novo synthesis is initiated from the +1 position and to maintain the integrity of the 3' end of its genome. This assay system should be an important tool for investigating the detailed mechanism of de novo initiation by HCV NS5B as well as other viral RNA polymerases.

FIG. 1.

Formation of initiation products in NS5B de novo RNA synthesis using oligoribonucleotide 5′-A₈GC-3′ as a template. Each reaction contained 20 μM RNA template, 2.5 μM NS5B, 100 μM [α-³³P]CTP, and 1 mM concentration of one of the following initiating nucleotides or nucleosides unless specified: lane 1, GTP (no enzyme); lane 2, GTP (no template); lane 3, no initiating nucleotide; lane 4, CTP; lane 5, UTP; lane 6, ATP; lane 7, GTP; lane 8, 100 μM [γ-³³P]GTP (initiating nucleotide) to replace [α-³³P]CTP, and 1 mM CTP; lane 9, GDP; lane 10, GMP; lane 11, guanosine; lanes 12 to 15, A₈GC^3′d as an template and GMP (lane 12), guanosine (lane 13), N-2 methyl guanosine (lane 14), and inosine (lane 15) as an initiating nucleotide; and lane 16, GTP as an initiating nucleotide with NS5B D318A/D319A double mutant. The reaction products were resolved on a 25% polyacrylamide-7 M urea-TBE gel and were analyzed by a PhosphorImager.

FIG. 2.

Formation of initiation products in NS5B de novo RNA synthesis using A₈GC (lane 1) or HCV(+) templates (lanes 2 to 5). Each reaction contained 2.5 μM NS5B, 100 μM [α-³³P]CTP, 20 μM specified RNA template, and 1 mM concentration of a nucleotide as follows: lane 1, A₈GC and GTP; lane 2, HCV(+)GC and GTP; lane 3, HCV(+)GU and ATP; lane 4, HCV(+)GG and CTP; and lane 5, HCV(+)GA and UTP. The reaction products were resolved on a 25% polyacrylamide-7 M urea-TBE gel and were subjected to PhosphorImager analysis.

FIG. 3.

Elongation from initiation products in NS5B de novo RNA synthesis. Each reaction contained 2.5 μM NS5B, 100 μM [α-³³P]CTP, 2 mM UTP, 20 μM RNA template, and 1 mM concentration of an initiating nucleotide as indicated. Lane 1, HCV(+)GC template and GTP; lane 2, HCV(+)GU template and ATP; lane 3, HCV(+)GG template and CTP; lane 4, HCV(+)GA template and UTP; and lane 5, A₈GC template and GTP. The reaction products were resolved on a 25% polyacrylamide-7 M urea-TBE gel and were analyzed using a PhosphorImager. Asterisk denotes gel artifact.

FIG. 4.

Elongation in NS5B de novo synthesis with HCV(+)GU as a template and ApC as a primer. Each reaction contained 2.5 μM NS5B, 100 μM [α-³³P]UTP, 20 μM HCV(+)GU template, and one of the following concentrations of ApC: lane 1, none; lane 2, 50 μM; lane 3, 100 μM; lane 4, 500 μM; and lane 5, 1 mM. The reaction products were resolved on a 25% polyacrylamide-7 M urea-TBE gel and were subjected to PhosphorImager analysis.

FIG. 5.

Minimal length requirement of an RNA template in NS5B de novo synthesis. Each reaction contained 2.5 μM NS5B, 100 μM [α-³³P]CTP, 1 mM GTP, and 1 mM concentrations of the RNA template and nucleotide as follows: lane 1, template UUUGC only; lane 2, UUUGC and ATP; lane 3, template UUGC only; and lane 4, UUGC and ATP. The reaction products were resolved on a 25% polyacrylamide-7 M urea-TBE gel and were analyzed using a PhosphorImager. Asterisk denotes gel artifact.

FIG. 6.

De novo initiation by NS5B using a template derived from the HCV minus strand genome. Each reaction contained 5 μM NS5B, 100 μM [α-³³P]CTP, 1 mM GTP, and 20 μM concentration of the HCV(−) template (lane 1). For comparison, the same reaction was run with A₈GC as a template (lane 2). The reaction products were resolved on a 25% polyacrylamide-7 M urea-TBE gel and were analyzed using a PhosphorImager.

FIG. 7.

Effect of Mn²⁺ on NS5B de novo RNA synthesis activity. Each reaction contained 5 μM NS5B, 100 μM [α-³³P]CTP, 1 mM GTP, 1 mM UTP, 5 mM concentration of a metal cofactor, and 20 μM concentration of an RNA template as listed. Lane 1, Mn²⁺ and the HCV(−) template; lane 2, Mg²⁺ and the HCV(−) template; lane 3, Mn²⁺ and A₈GC^3′d template; and lane 4, Mg²⁺ and A₈GC^3′d template. The reaction products were resolved on a 25% polyacrylamide-7 M urea-TBE gel and were analyzed using a PhosphorImager. Asterisk denotes gel artifact.

FIG. 8.

Selective initiation from +1, +2, and +3 positions of an RNA template by NS5B. Each reaction contained 2.5 μM NS5B, 20 μM concentration of an RNA template (template A₈GCC for panel A and template A₈GCCC for panel B), and 100 μM and 1 mM concentrations, respectively, of the following [α-³³P] nucleotide and unlabeled nucleotide(s): for panel A, lane 1, [α-³³P]CTP and GTP; lane 2, [α-³³P]CTP and GMP; lane 3, [α-³³P]CTP, GTP and GMP; lane 4, [γ-³³P]GTP; and lane 5, [γ-³³P]GTP and CTP; and for panel B, lane 1, [α-³³P]CTP and GTP; lane 2, [α-³³P]CTP and GMP; lane 3, [α-³³P]CTP, GTP, and GMP; lane 4, [γ-³³P]GTP, and lane 5, [γ-³³P]GTP and CTP. The reaction products were resolved on a 25% polyacrylamide-7 M urea-TBE gel and were subjected to PhosphorImager analysis. Asterisk denotes gel artifact.

FIG. 9.

Selective initiation by NS5B from the +2 or +3 position of the HCV(+) templates. Each reaction contained 2.5 μM NS5B, 100 μM [α-³³P]UTP, 1 mM CTP, and 20 μM concentration of one of the following RNA templates: lane 1, HCV(+)GC; lane 2, HCV(+)GU; lane 3, HCV(+)GG; and lane 4, HCV(+)GA. The reaction products were resolved on a 25% polyacrylamide-7 M urea-TBE gel and were analyzed using a PhosphorImager.

FIG. 10.

Selective initiation using HCV(+)GUA (A) or HCV(+)GUAG (B) as a template. Each reaction contained 2.5 μM NS5B and a 20 μM concentration of the specified RNA template and the following nucleotides: for panel A, lane 1, 100 μM [α-³³P]CTP, 1 mM ATP, and 1 mM UTP; lane 2, 100 μM [α-³³P]UTP and 1 mM ATP (no enzyme control); lane 3, 100 μM [α-³³P]UTP and 1 mM ATP; and lane 4, 100 μM [α-³³P]CTP and 1 mM ATP; and for panel B, lane 1, 100 μM [α-³³P]CTP and 1 mM UTP; lane 2, 100 μM [α-³³P]CTP, 1 mM UTP, and 1 mM ATP; lane 3, 100 μM [α-³³P]CTP and 1 mM ATP; and lane 4, 100 μM [α-³³P]UTP and 1 mM ATP. The reaction products were resolved on a 25% polyacrylamide-7 M urea-TBE gel and were analyzed using a PhosphorImager.