Accelerated evolution of maribavir resistance in a cytomegalovirus exonuclease domain II mutant

Abstract

A human cytomegalovirus (CMV) UL54 pol exonuclease domain II mutation, D413A, found in a clinical specimen, conferred ganciclovir (GCV) and cidofovir resistance but not foscarnet resistance when incorporated into laboratory strain T2294. After several passages without drug, mutation was observed in five of eight plaques of T2294, and its plating efficiency under foscarnet was increased approximately 30-fold over that of a control strain. When T2294 was serially passed under maribavir (MBV), phenotypic changes and viral UL97 mutations were detected by passage 5, much earlier than previously reported for other CMV strains. By passage 15, mutations included two cases of H411Y, one each of H411L and H411N, three of T409M, five of V353A, and one of L397R. Five instances of codon 409 or 411 mutations evolved into double mutations including V353A. Marker transfer experiments showed H411N/Y/L to confer 9- to 70-fold-increased MBV resistance and combinations of H411L/Y and V353A to confer >150-fold-increased MBV resistance, but no GCV resistance. These findings are consistent with defective exonuclease activity of the pol D413A mutant T2294, leading to the accelerated evolution of UL97 mutations under MBV. This recapitulated the known resistance mutations V353A, L397R, and T409M; suggested their relative frequency; and identified new ones at codon 411. These UL97 mutations predict an MBV binding region overlapping the kinase ATP binding site and located upstream of known GCV resistance mutations. The existence of viable pol D413A mutants may facilitate the selection of additional drug resistance mutations in vivo and the study of these mutations in vitro.

FIG. 1.

Effect of MBV on CMV CPE. HEL fibroblasts 6 days after infection at a multiplicity of infection of 0.2 were photographed at an original magnification of ×20 with phase contrast. (A) Control strain T2233 without MBV, showing normal (type 1) CPE. (B) Strain T2233 under 0.3 μM MBV, showing UL97-inhibited (type 2) CPE characterized by refractile nuclear inclusions increasing with advancing CPE. (C) Strain T2923 (H411L mutation) showing mixed type 1 and type 2 CPE under 10 μM MBV. (D) Strain T2903 (V353A and H411L mutations), showing advanced type 1 CPE with apoptotic cells but no nuclear inclusions in the presence of 20 μM MBV.

FIG. 2.

Sequence alignments of UL97 and kinases of known structure. Alignments of UL97 amino acid residues of kinase domains I to VIb (12) with the corresponding residues of yeast GCN2 kinase (PDB 1zyc) (24) (A) and human SR protein kinase 1 (PDB 1wbp) (23) (B) are shown. These alignments were used to calculate the UL97 structure models shown in Fig. 3. Arrows indicate UL97 residues 353, 397, 409, and 411, which are involved in MBV resistance.

FIG. 3.

Structure models of UL97 kinase ATP and MBV binding sites. (A) Corresponding residues of the UL97 sequence are labeled on the experimentally determined structure of yeast GCN2 kinase complexed to ATP (PDB 1zyd) (24). Residues identical in the two kinases are shown in red on the peptide backbone. G, conserved glycine residues in kinase domain I. (B) UL97 kinase ATP binding site structure model, based on the yeast GCN2 kinase structure (PDB 1zyc) (24) and the sequence alignment shown in Fig. 2A, as constructed by CPH Models 2.0. (C) MBV docked to the same UL97 structure model as above, guided by residues 353, 409, 411, and 460. (D) MBV docked to an alternative UL97 structure model based on the human SRPK1 structure (PDB 1wbp) (23) and the sequence alignment shown in Fig. 2B, as constructed by MODELLER v9.1. In the UL97 structure models, the ATP or MBV molecule was docked using ArgusLab v4 and structure displayed as a ribbon cartoon of protein backbone. Conserved kinase domains I and VIb and residues affecting MBV sensitivity are labeled.