Comparison of Cytomegalovirus Terminase Gene Mutations Selected after Exposure to Three Distinct Inhibitor Compounds

Abstract

Letermovir, GW275175X (a benzimidazole), and tomeglovir (Bay38-4766) are chemically unrelated human cytomegalovirus (CMV) terminase complex inhibitors that have been tested in human subjects. UL56 gene mutations are the dominant pathway of letermovir resistance, while UL89 and UL56 mutations are known to confer benzimidazole resistance. This study compares the mutations elicited by the three inhibitors during CMV propagation. GW275175X consistently selected for UL89 D344E and sometimes selected for UL89 C347S, UL89 R351H, or UL56 Q204R. Tomeglovir consistently selected for UL89 V362M and sometimes selected for UL89 N329S, T350M, H389N, or N405D or UL56 L208M, E407D, H637Q, or V639M. Adding to known and novel UL56 mutations, letermovir occasionally selected for UL89 N320H, D344E, or M359I. Recombinant phenotyping confirmed that UL89 D344E conferred 9-fold resistance (an increased 50% effective concentration [EC]) for GW275175X and increased the letermovir and tomeglovir ECs by 1.7- to 2.1-fold for the baseline virus and the UL56 Q204R, E237D, F261L, and M329T mutants. UL89 N320H and M359I conferred <2-fold letermovir resistance but 7-fold tomeglovir resistance; the N320H mutant was also 4-fold resistant to GW275175X. UL89 N329S conferred tomeglovir and letermovir cross-resistance. UL89 T350M conferred resistance to all three inhibitors. UL89 C347S conferred 27-fold GW275175X resistance. UL89 V362M and H389N conferred 98-fold and 29-fold tomeglovir resistance, respectively, without conferring cross-resistance. Thus, characteristic UL89 mutations confer substantial resistance to GW275175X and tomeglovir and are an uncommon accessory pathway of letermovir resistance. Instances of moderate cross-resistance and the proximity of the selected UL89 and UL56 mutations suggest targeting of a similar terminase functional locus involving UL56 and UL89 interaction.

A third component of the human cytomegalovirus terminase complex is involved in letermovir resistance

“…Unlike some previous work, this strain has an intact UL54 DNA polymerase gene without an exonuclease defect designed to accelerate the evolution of drug resistance (Chou, 2015, 2017). Other BD1-derived baseline strains T4190 and T4272 contain a silent Frt motif upstream of UL56 and UL89 exon 2 respectively (Chou, 2015, 2017), for insertion and removal of a Kan selection marker during BAC mutagenesis and were used as the parental strain for comparisons of growth and drug susceptibility with mutant strains.…”

“…Mutant recombinant viruses were constructed as described below for phenotypic assays of specific mutations and combinations of interest. Drug susceptibility and growth assays were performed in retinal epithelial cells transduced to over-express platelet-derived growth factor receptor α (ARPEp), which facilitate assay standardization as described (Chou, 2017; Chou et al, 2017). …”

“…Mutagenesis of BAC clones to introduce specific amino acid substitutions in UL51 and UL56 was performed as previously described (Chou, 2015, 2017). New UL56 mutations were introduced by recombination of a transfer vector containing a selectable Kan resistance marker and subsequent removal by Flp recombinase, while the one UL51 mutation studied was introduced by the markerless en passant procedure (Tischer et al, 2010) into BAC clones containing either wild type UL56 and UL89 sequences, or pre-existing UL56 or UL89 mutations.…”

“…BAC cloned viral DNA was transfected into HFF or ARPEp to yield cell-free CMV stocks, which were sequenced throughout the mutagenized gene for the presence of the intended mutation(s). Phenotypic assays for letermovir susceptibility were performed as recently detailed (Chou, 2017), using SEAP activity in culture supernatants as a measure of viral growth. The drug concentration required to reduce supernatant SEAP activity by 50% (EC50) at 6 to 7 days was determined by assaying growth under no drug and at 5 two-fold increasing concentrations centered on the estimated EC50 value.…”

“…Notable examples are amino acid substitution V236M, which is the first one observed in a treated human subject (Lischka et al, 2016), and various substitutions at the C325 residue that confer absolute letermovir resistance with minor impact on viral growth fitness. Additional in vitro selection experiments with letermovir showed the occasional emergence of UL89 mutations that confer cross-resistance to older terminase inhibitors (Chou, 2017). UL89 is homologous to the bacteriophase terminase large subunit and encodes nuclease activity essential to the cleavage of replicated viral DNA into unit length genomes, in addition to other incompletely understood functions (Bogner, 2010; Neuber et al, 2017).…”