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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 in vitro 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 [EC50]) for GW275175X and increased the letermovir and tomeglovir EC50s 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.

SUBMITTER: Chou S

PROVIDER: S-EPMC5655092 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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

Chou Sunwen S

Antimicrobial agents and chemotherapy 20171024 11

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 <i>in vitro</i> CMV propagation. GW275175X consistently selected for UL89 D344E and som ...[more]

PMID: 28827420

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Project description:Human cytomegalovirus (HCMV) genome encapsidation requires several essential viral proteins, among them pUL56, pUL89, and the recently described pUL51, which constitute the viral terminase. To gain insight into terminase complex assembly, we investigated interactions between the individual subunits. For analysis in the viral context, HCMV bacterial artificial chromosomes carrying deletions in the open reading frames encoding the terminase proteins were used. These experiments were complemented by transient-transfection assays with plasmids expressing the terminase components. We found that if one terminase protein was missing, the levels of the other terminase proteins were markedly diminished, which could be overcome by proteasome inhibition or providing the missing subunit in trans These data imply that sequestration of the individual subunits within the terminase complex protects them from proteasomal turnover. The finding that efficient interactions among the terminase proteins occurred only when all three were present together is reminiscent of a folding-upon-binding principle leading to cooperative stability. Furthermore, whereas pUL56 was translocated into the nucleus on its own, correct nuclear localization of pUL51 and pUL89 again required all three terminase constituents. Altogether, these features point to a model of the HCMV terminase as a multiprotein complex in which the three players regulate each other concerning stability, subcellular localization, and assembly into the functional tripartite holoenzyme.IMPORTANCE HCMV is a major risk factor in immunocompromised individuals, and congenital CMV infection is the leading viral cause for long-term sequelae, including deafness and mental retardation. The current treatment of CMV disease is based on drugs sharing the same mechanism, namely, inhibiting viral DNA replication, and often results in adverse side effects and the appearance of resistant virus strains. Recently, the HCMV terminase has emerged as an auspicious target for novel antiviral drugs. A new drug candidate inhibiting the HCMV terminase, Letermovir, displayed excellent potency in clinical trials; however, its precise mode of action is not understood yet. Here, we describe the mutual dependence of the HCMV terminase constituents for their assembly into a functional terminase complex. Besides providing new basic insights into terminase formation, these results will be valuable when studying the mechanism of action for drugs targeting the HCMV terminase and developing additional substances interfering with viral genome encapsidation.

Project description:The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease. IMPORTANCE:Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus drug development. We identified and characterized an inhibitor of pUL89 endonuclease activity that also inhibits human cytomegalovirus replication in cell culture. pUL89 endonuclease, therefore, should be explored as a potential target for antiviral development against human cytomegalovirus.

Dataset Information

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

Publications

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

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