A multi-step process of viral adaptation to a mutagenic nucleoside analogue by modulation of transition types leads to extinction-escape

Abstract

Resistance of viruses to mutagenic agents is an important problem for the development of lethal mutagenesis as an antiviral strategy. Previous studies with RNA viruses have documented that resistance to the mutagenic nucleoside analogue ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is mediated by amino acid substitutions in the viral polymerase that either increase the general template copying fidelity of the enzyme or decrease the incorporation of ribavirin into RNA. Here we describe experiments that show that replication of the important picornavirus pathogen foot-and-mouth disease virus (FMDV) in the presence of increasing concentrations of ribavirin results in the sequential incorporation of three amino acid substitutions (M296I, P44S and P169S) in the viral polymerase (3D). The main biological effect of these substitutions is to attenuate the consequences of the mutagenic activity of ribavirin -by avoiding the biased repertoire of transition mutations produced by this purine analogue-and to maintain the replicative fitness of the virus which is able to escape extinction by ribavirin. This is achieved through alteration of the pairing behavior of ribavirin-triphosphate (RTP), as evidenced by in vitro polymerization assays with purified mutant 3Ds. Comparison of the three-dimensional structure of wild type and mutant polymerases suggests that the amino acid substitutions alter the position of the template RNA in the entry channel of the enzyme, thereby affecting nucleotide recognition. The results provide evidence of a new mechanism of resistance to a mutagenic nucleoside analogue which allows the virus to maintain a balance among mutation types introduced into progeny genomes during replication under strong mutagenic pressure.

Figure 1. Passage history of FMDV in the presence of increasing cencentrations of ribavirin.

Biological clone C-S8c1of FMDV was subjected to up to 460 serial passages in BHK-21 cells , . Al passage 213, biological clone MARLS was selected by its resistance to neutralization by monoclonal antibody (MAb) SD6 , and the population derived from the clone was subjected to serial passages either in the absence (white circles) or presence (grey circles) of increasing concentrations of ribavirin (R) . In this scheme biological clones (virus derived from a single plaque developed on a BHK-21 cell monolayer) are indicated as black squares, and uncloned populations as circles; “p” indicates passage number. The concentrations of R included in the culture medium are indicated below the corresponding passages. The procedures involved in the isolation of the initial FMDV C-S8c1 clone and in infections of BHK-21 cells have been described in our previous studies , , , and are detailed in Materials and Methods.

Figure 2. Progeny production in BHK-21 cells infected with viruses encoding mutant polymerases.

Kinetics of progeny production of infectious virus in BHK-21 infected cells infected at a MOI of 0.5 PFU/cell by the indicated FMDVs. Data have been divided in two separate graphs for clarity. Results are the average of three determinations and standard deviations are given. Procedures are described in Materials and Methods.

Figure 3. Effect of ribavirin on progeny infectivity and viral RNA in serial infections with FMDV Wt or FMDV 3D(SSI).

(A) BHK-21 cells (2×10⁶) were infected with wild type FMDV (Wt) (progeny of infectious clone pMT28 [57], [70]) at a multiplicity of infection of 0.3 PFU/cell. In successive passages, the same number of cells was infected with 1∶10 of the volume of the supernatant from the previous passage in the absence (−R) or in the presence of 5000 µM R (+R). The discontinuous line indicates the limit of detection of viral infectivity. (B) Same as (A), using mutant FMDV 3D(SSI). (C) and (D) FMDV RNA levels in the supernatants of BHK-21 cells infected with FMDV Wt or FMDV 3D(SSI) in the absence (−R) or presence (+R) of 5000 µM R. The discontinuous line indicates the limit of detection of viral RNA. (E) and (F) Specific infectivity (PFUs/RNA molecules) calculated from the data in panels (A) to (D). The specific infectivity from passage 7 of FMDV Wt in the presence of R was not calculated due to undetectable virus titer (<5 PFU/ml). Procedures are described in Materials and Methods.

Figure 4. Incorporation of nucleotides into sym/sub-AC by mutant FMDV polymerases.

(A) Kinetics of incorporation of GMP into sym/sub-AC (sequence shown at the top) by the indicated FMDV polymerases. The reactions were initiated by addition of 50 µM GTP after the formation of 3D-RNA(n+1) complex, as described in Materials and Methods. At different time points the reaction was quenched by addition of EDTA. (B) Same as (A), except that the reaction was started by addition of 50 µM RTP after the formation of the 3D-RNA (n+1) complex. (C) Percentage of primer elongated to position +2 (12 mer or larger RNAs synthesized), calculated from the densitometric analysis of the electrophoreses shown in A. The results are the average of three independent experiments, and standard deviations are given. (D) Same as (C) for the incorporation of RMP at position +2 (12 mer) calculated from the densitometric analysis of the electrophoreses shown in (B). Procedures are detailed in Materials and Methods.

Figure 5. Incorporation of nucleotides into sym/sub-AC by mutant FMDV polymerases.

Kinetics of incorporation of AMP into sym/sub-AU (sequence shown at the top) by the indicated polymerases. The reactions were initiated by addition of 50 µM ATP after the formation of 3D-RNA (n+1) complex. At different time points the reaction was quenched by addition of EDTA. (B) Same as (A), except that the reaction was started by addition of 50 µM RTP after the formation of 3D-RNA (n+1) after formation of the 3D-RNA (n+1) complex. (C) Percentage of primer elongated (12 mer or larger RNAs synthesized) calculated from the densitometric analysis of the electrophoreses shown in (A). The results are the average of three independent experiments, and standard deviations are given. (D) Same as (C) for the incorporation of RMP at position +2 (12 mer) calculated from the densitometric analysis of the electrophoreses shown in (B). Procedures are detailed in Materials and Methods.

Figure 6. FMDV 3D residues around the substituted sites.

Stereoviews of σ_A-weighted |F_o|-|F_c| electron density maps at 2.5 Å resolution (contoured at 3 σ) around the mutated amino acids (A) S44, (B) S169 and (C) I296. The substituted residues and surrounding amino acids were omitted from the phasing model. The model is placed inside in ball and stick representation and colored in atom type code. The names of the mutated residues are labeled.

Figure 7. The structure and interactions of the FMDV 3D active site in two different complexes.

(A) 3D(SSI)-RNA template/primer and (B) 3D(wild type)-RNA-ribavirin-triphosphate (RTP) (PDB: 2E9R). The polymerase residues in the active site are shown in grey with the loop β9-α11 highlighted in green. The first base pairs of the template/primer RNA are shown in yellow with the incoming RTP molecule in orange (in B). When the RTP molecule is located at the active site of the wild type 3D, the β9-α11 loop changes its conformation to accommodate the nucleoside analogue into the cavity, and the ribavirin pseudo-base appears hydrogen bonded to residues S298 and G299 within the loop. The side chains of residues Asp245 of motif A and Asn307 of motif B have also changed their rotamer conformations to facilitate the interactions with the ribose moiety of the mutagenic nucleotide. Substitution M296I seems to prevent the mentioned conformational changes in the loop β9-α11 as well as the side chain rearrangements in residues Asp245 and Asn307 required to interact with ribavirin.

Figure 8. Structure and interactions in the template channel of the FMDV 3D polymerase.

(A) the 3D(SSI)RNA mutant complex and (B) the 3DWt-RNA complex (PDB 1WNE). The molecular surface of the polymerase is shown in grey with the acidic residues of the active site in red and the RNA depicted as a ribbon in yellow. Only the 5′ overhang moiety and the first base pair in the active site is shown for clarity. Residues of the β2-α2 loop (containing S44), the amino acid interacting with β2-α2 loop and, those contacting the RNA template are shown as sticks in atom type colour. The left side insets in A and B show close-ups of the interactions involving the loop β2-α2 (top) and template nucleotides A3 and U4 (bottom).

Figure 9. Ribbon diagram of the structure of FMDV 3D polymerase, SSI mutant, in complex with the RNA template-primer.

The polymerase is depicted in blue and the RNA in yellow. The substituted amino acids, S44, S169 and I296, are shown as red balls and explicitly labelled.