A Complex Network of Interactions between S282 and G283 of Hepatitis C Virus Nonstructural Protein 5B and the Template Strand Affects Susceptibility to Sofosbuvir and Ribavirin

Abstract

The hepatitis C virus (HCV) RNA-dependent RNA-polymerase NS5B is essentially required for viral replication and serves as a prominent drug target. Sofosbuvir is a prodrug of a nucleotide analog that interacts selectively with NS5B and has been approved for HCV treatment in combination with ribavirin. Although the emergence of resistance to sofosbuvir is rarely seen in the clinic, the S282T mutation was shown to decrease susceptibility to this drug. S282T was also shown to confer hypersusceptibility to ribavirin, which is of potential clinical benefit. Here we devised a biochemical approach to elucidate the underlying mechanisms. Recent crystallographic data revealed a hydrogen bond between S282 and the 2'-hydroxyl of the bound nucleotide, while the adjacent G283 forms a hydrogen bond with the 2'-hydroxyl of the residue of the template that base pairs with the nucleotide substrate. We show that DNA-like modifications of the template that disrupt hydrogen bonding with G283 cause enzyme pausing with natural nucleotides. However, the specifically introduced DNA residue of the template reestablishes binding and incorporation of sofosbuvir in the context of S282T. Moreover, the DNA-like modifications of the template prevent the incorporation of ribavirin in the context of the wild-type enzyme, whereas the S282T mutant enables the binding and incorporation of ribavirin under the same conditions. Together, these findings provide strong evidence to show that susceptibility to sofosbuvir and ribavirin depends crucially on a network of interdependent hydrogen bonds that involve the adjacent residues S282 and G283 and their interactions with the incoming nucleotide and complementary template residue, respectively.

FIG 1

Hydrogen bonding network at the active site of NS5B. The network involves three components of a ternary complex of NS5B, primer/template, and bound nucleotide. The nucleotide (UDP) is shown in green, and the complementary residue of the template is shown in beige. The 2′-hydroxyl of the template forms a hydrogen bond with backbone oxygen of G283 (purple), and the covalently linked S282 (red) forms a hydrogen bond with the 2′-hydroxyl of the bound nucleotide. Active site residues D318 and D319 (blue) and the diphosphate moiety of the bound nucleotide interact with the catalytic metal ions. The figure was generated with SwissPdbViewer software (PDB 4WTA).

FIG 2

NS5B-mediated RNA synthesis and effects of DNA-like modifications on the template. (A) Experimental design for RNA synthesis with a short model template. The modification (X) in the template and its complementary residue Y of the newly synthesized RNA are indicated in red. The reaction is initiated with a dinucleotide primer and yields a 20mer full-length product after exposure to all four NTPs. (B) Structures of modifications at template position +14: dT, dG, dU (South sugar pucker conformation), and U, 2′F-U (80 to 90% North sugar pucker conformation). (C) RNA synthesis with modified templates as described in panel B with RNA length markers at positions +15, +16, +17, and +20 are shown on the left. Enzyme pausing is seen at position +13.

FIG 3

Incorporation efficiency on modified templates. (A) Schematic of NS5B elongation on T20-G16 templates modified at position +16 (red). The addition of GTP and ATP to the reaction mixture leads to a 15mer RNA product, and the subsequent addition of UTP and increasing concentrations of CTP yields a 20mer full-length product. (B) WT NS5B-mediated RNA synthesis performed with increasing concentrations of CTP in the presence of RNA templates containing G, dG, and 2′-O-Me-G at position +16. (C) Graphical representation of the data in panel B.

FIG 4

RNA synthesis performed in the presence of increasing concentrations of 2′C-Methyl-CTP. (A) Schematic of chain termination by 2′-C-Me-CTP on T20-G16 modified at position +16 to G, dG, and 2′-F-G (red). (B and C) RNA synthesis was performed in the presence of increasing concentrations of 2′-C-Me-CTP with WT NS5B (B) and the S282T mutant enzyme (C).

FIG 5

RNA synthesis performed in the presence of increasing concentrations of sofosbuvir-TP. (A) Schematic of chain termination by 2′-C-Me-2′-F-UTP on T20-A16 modified at position +16 to A or dA (red). (B and C) RNA synthesis performed in the presence of increasing concentrations of 2′-C-Me-2′-F-UTP with WT NS5B and the S282T mutant on the natural template T20-AU16 (B) and the modified template T20-dA16 (C).

FIG 6

RNA synthesis performed in the presence of increasing concentrations of ribavirin-TP. (A) Chemical structure of ribavirin-5′-triphosphate. (B) A scheme depicts the incorporation of ribavirin (R), indicated in red, on T20-C16 templates modified at position +16. Note that ribavirin can get incorporated opposite C and U. (C) RNA synthesis performed by WT NS5B and S282T with increasing concentrations of ribavirin on T20-C16 and T20-dC16. (D) Graphical representation of the data shown in panel C.

FIG 7

Model of interdependent hydrogen bonding affects susceptibility to 2′C-methylated compounds and ribavirin. (A) S282T prevents binding of 2′-C-methyl modified nucleotides. (B) The loss of a hydrogen bond between G283 and a DNA template increases the flexibility at S282T and facilitates binding the inhibitor. (C) The loss of a hydrogen bond between G283 and a DNA template prevents binding of ribavirin (R) that shows per se weak base pairing. (D) S282T can partially compensate for this deficiency and facilitates the binding and/or incorporation of ribavirin.