Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus

Abstract

We report the crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus, a major human pathogen, to 2.8-A resolution. This enzyme is a key target for developing specific antiviral therapy. The structure of the catalytic domain contains 531 residues folded in the characteristic fingers, palm, and thumb subdomains. The fingers subdomain contains a region, the "fingertips," that shares the same fold with reverse transcriptases. Superposition to the available structures of the latter shows that residues from the palm and fingertips are structurally equivalent. In addition, it shows that the hepatitis C virus polymerase was crystallized in a closed fingers conformation, similar to HIV-1 reverse transcriptase in ternary complex with DNA and dTTP [Huang H., Chopra, R., Verdine, G. L. & Harrison, S. C. (1998) Science 282, 1669-1675]. This superposition reveals the majority of the amino acid residues of the hepatitis C virus enzyme that are likely to be implicated in binding to the replicating RNA molecule and to the incoming NTP. It also suggests a rearrangement of the thumb domain as well as a possible concerted movement of thumb and fingertips during translocation of the RNA template-primer in successive polymerization rounds.

Figure 1

(a) Front (Upper) and top (Lower) view of the HCV polymerase in a ribbons representation of the polypeptide chain. α-helices (labeled with capital letters) are shown as red cylinders, β-strands (labeled with numbers) as blue arrows, and connecting loops as yellow tubes. The front view has the palm subdomain at the base of the cleft, which is closed at either side by the fingers and thumb and at the back by loops Λ1 and Λ2. (b) The fingertips. Shown is the mixed barrel, composed of β-strands and an α-helix, that lies at the tip of the fingers and aligns structurally between RTs and RdRps. The left panel shows the ribbon diagram of this domain in the HCV polymerase along with a topology diagram. Two long loops (Λ1 and Λ2) emanate from the barrel to close the back of the enzyme. The right panel depicts the corresponding domain of HIV-1 RT in the same orientation. The topology diagram shows that there is a difference in connectivity in the left-most (N-terminal) strands. It is clear that Λ1 is absent but that Λ2 has its counterpart in RT. Broken lines indicate regions in which the polypeptide chain makes excursions into neighboring regions of the fingers to come back and complete the fold of the barrel.

Figure 2

Structural alignment of RdRps and RTs. The HCV sequence is displayed in blue letters, with the poliovirus RdRp sequence above and the sequence of RT from Moloney leukemia virus and HIV-1 below. α-helices (red) and β-strands (blue) are indicated (with the corresponding label) as solid symbols above the sequences for HCV and as boxes surrounding the corresponding sequences for poliovirus and HIV-1. Conserved residues are indicated with a dark background (blue for HCV, black for the others). White letters denote strict conservation, which for HCV means conserved also in the related GB viruses (32). Yellow letters denote conservation to a lesser extent, which in the case of NS5B means conservation in all HCV strains and at least one of the GB viruses. In the case of poliovirus, we have not included residues that were disordered in the structure. The number of the N-terminal amino acid is shown next to each aligned segment. Green marks in the sequences indicate places in which there are insertions with respect to the HCV RdRp. The known polymerase “motifs,” A through E (28), are indicated by black boxes that comprise all four sequences. Blue and green stars underneath the HIV-1 RT sequences denote residues that contact the dNTP molecule and DNA, respectively. Underlines show regions of NS5B that would be implicated in such contacts in NS5B, as inferred from the superposition of the two structures. Orange, green, and magenta underlines mark regions that surround the NTP site, the template strand, and the primer strand, respectively. In the thumb, a cyan underline marks residues that would contact bases in the minor groove. In this domain, the underlined regions would contact the nucleic acid after a structural rearrangement as explained in the text.

Figure 3

The molecular surface of the HCV RdRp, front view (Left) and back view (Right). (Upper) Electrostatic potential calculated on the molecular surface. Positive and negative charges are indicated in blue and red, respectively. The positively charged groove along which RNA would bind during viral replication is apparent in the front view. The region of the thumb at the very C terminus of the catalytic domain of NS5B is also positively charged. In the back view, the lower ridge of the “bridge” formed by loops Λ1 and Λ2 is seen to have a strong positive charge. This area corresponds to the “tunnel” where NTP would access to the polymerizing complex. The red patch underneath the tunnel corresponds to the strictly conserved aspartic acids that are known to coordinate the catalytic Mg²⁺ ions. (Lower) The molecular surface is colored according to sequence conservation from an alignment that included one representative from each of the know HCV serotypes (1 through 6) and all three of the related GB viruses (A, B, and C) (32). The scoring ranges from 0 (white, no conservation) to 1 (dark orange, strict conservation). Note that both the RNA binding groove and the putative NTP tunnel are highly conserved. Note also the conserved ridges lining the acidic patch mentioned above and the conservation of the basic region at the back of the thumb.

Figure 4

Interactions of the HCV RdRp with nucleic acid as inferred from the comparison with the structure of HIV-1 RT in a ternary complex with DNA and dTTP (26). (a) The template–primer duplex and dTTP molecule from the structure of the RT ternary complex superposed on a surface representation of the HCV polymerase colored according to electrostatic potential (as in Fig. 3 Upper). The view is a rotation of 90 degrees with respect to the front view of Figs. 1a and 3, looking from the thumb domain, which was removed for clarity. Some of the amino acids seen in the contacts are labeled. The transformation that superposes the palm and fingertips of the two enzymes has been applied to the nucleic acid coordinates for this figure. Five nucleotides of the template strand are shown, two of them base paired to the 3′ bases of the primer strand and a third to the incoming nucleotide, the triphosphate moiety of which points toward the tunnel mentioned in Fig. 3 and described in the text. (b) Proposed movement of the thumb on binding of RNA. The HCV polymerase is shown in a ribbon representation colored according to the different subdomains: fingers, red; palm, yellow; and thumb, blue. A rotation of ≈10 degrees (indicated by the black arrow in the left panel) about the connection between thumb and palm brings helix P in our structure into coincidence with helix H in HIV-1 RT, which tracks the minor groove of the A form DNA duplex. The resulting position of helix P in the complex is shown in the left panel, in a top view of the molecule where the template and primer backbone are displayed as green and magenta ribbons, respectively. The NTP molecule in the active site is colored cyan. The “flap” is a β-hairpin (strands 17 and 18), which has to move as well, as shown by the blue arrow in the left panel of the figure. (c) Diagram of the template–primer duplex in the catalytic site indicating the amino acid residues seen to contact the nucleic acid and nucleotide molecules after superposition to HIV-1 RT. These residues are very likely involved in binding RNA and nucleotide during polymerization. Residues behind an arc, around the triphosphate moiety of the dNTP molecule, are those lining the tunnel through which the NTP molecules are likely to access the active site of the enzyme. Residues indicated in red are those that come into contact after applying the rearrangement on the thumb shown in b.