Generation of a panel of mutants that are resistant to standard of care therapies in a clinically relevant strain of human cytomegalovirus for drug resistance profiling

Infection with human cytomegalovirus (HCMV) can result in a significant disease burden within the immunosuppressed and immunocompromised patient populations. Current standard of care (SOC) relies on direct-acting antivirals which target a limited group of Viral Proteins including the viral polymerase (UL54), terminase (UL56), and protein kinase (UL97). Incomplete inhibition of virally encoded proteins result in a selective pressure towards the generation of "breakthrough" drug resistant variants. One limitation in evaluating novel antivirals is the difficulty in profiling their Antiviral activity against variants resistant to current SOC interventions, as these resistant variants have arisen in different genetic backgrounds with distinct replication kinetics and yields. To limit strain variation we generated a targeted mutant panel of viruses in a Bacterial artificial chromosome (BAC) derived clinically relevant laboratory strain of HCMV, TB40e, that expresses the fluorescent proteins mCherry upon viral entry and eGFP at times after viral DNA replication. This unique construct allows for the monitoring of viral entry and viral DNA replication independently. This panel consists of WT and seven mutant viruses harboring mutations that confer resistance to ganciclovir, maribavir, cidofovir, and letermovir. In addition, we characterized a host-targeted Sirtuin 2 deacetylase (SIRT2) inhibitor, FLS-359, against the SOC resistant variants. We observed that mutant viruses demonstrated increased EC₅₀ concentrations for SOC inhibition, and that host directed FLS-359 demonstrated broad-spectrum Antiviral activity against known SOC drug-resistant mutants. This panel represents a much-needed comparatively innovative platform for screening the efficacy of new direct-acting antivirals and host-directed antivirals against HCMV variants refractive to therapeutic interventions.