Cytomegalovirus UL97 kinase catalytic domain mutations that confer multidrug resistance

Abstract

Human cytomegalovirus UL97 kinase mutations that commonly confer ganciclovir resistance cluster in different parts of the gene than those conferring resistance to maribavir, an experimental UL97 kinase inhibitor. The drug resistance, growth, and autophosphorylation phenotypes of several unusual UL97 mutations in the kinase catalytic domain were characterized. Mutations V466G and P521L, described in clinical specimens from ganciclovir-treated subjects, conferred a UL97 kinase knockout phenotype with no autophosphorylation, a severe growth defect, and high-level ganciclovir, cyclopropavir, and maribavir resistance, similar to mutations at the catalytic lysine residue K355. Mutations F342S and V356G, observed after propagation under cyclopropavir in vitro, showed much less growth attenuation and moderate- to high-level resistance to all three drugs while maintaining UL97 autophosphorylation competence and normal cytopathic effect in cell culture, a novel phenotype. F342S is located in the ATP-binding P-loop and is homologous to a c-Abl kinase mutation conferring resistance to imatinib. UL97 mutants with relatively preserved growth fitness and multidrug resistance are of greater concern in antiviral therapy than the severely growth-impaired UL97 knockout mutants. Current diagnostic genotyping assays are unlikely to detect F342S and V356G, and the frequency of their appearance in clinical specimens remains undefined.

Fig 1

Growth curves of UL97 mutants. Growth was assayed by accumulation of supernatant SEAP activity at days 4 through 8 postinoculation in HEL fibroblast (A) and HFF (B) cultures. Inoculum was calibrated to a multiplicity of infection of 0.01 to 0.02 by assay of SEAP activity at 1 day. Data points are means and standard deviations of 4 replicates. Line labels show strain serial numbers and UL97 mutations. RLU, relative light units.

Fig 2

In vitro autophosphorylation assay. In vitro kinase assays used [γ-³²P]ATP and immunoprecipitated pUL97 from control and mutant UL97 strains. (A) Phosphorimages of radiolabeled proteins. (B) Corresponding HA-antibody immunodetectable pUL97 in the same blot as the phosphorimage. The HA-tagged full-length pUL97 corresponds to the upper band in the phosphorimage lanes. The lower, non-HA-tagged radioactive band likely represents matrix protein pp65 that coimmunoprecipitated with and was phosphorylated by pUL97. The position of the 82-kDa molecular mass marker is shown. Lanes: 1, K355M mutant; 2, V466G mutant; 3, P521L mutant; 4, F342S mutant; 5, V356G mutant; 6, wild type; 7, wild type + 5 μM MBV.

Fig 3

P-loop amino acid sequence alignments of UL97 and c-Abl kinases. Beginning and ending residue numbers and amino acid sequences are shown for the UL97 kinase (NCBI gi: 4493302) and c-Abl kinase (NCBI gi: 177943). Apart from the 3 conserved glycine residues, identical (|) and similar (:) residues are shown, as well as the positional homology of the F342S UL97 mutation and the Y253F/H c-Abl imatinib resistance mutations.