Evasion of a Human Cytomegalovirus Entry Inhibitor with Potent Cysteine Reactivity Is Concomitant with the Utilization of a Heparan Sulfate Proteoglycan-Independent Route of Entry

The dependence of viruses on the host cell to complete their replicative cycle renders cellular functions potential targets for novel antivirals. We screened a panel of broadly acting cellular ion channel inhibitors for activity against human cytomegalovirus (HCMV) and identified the voltage-gated chloride ion channel inhibitor 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) as a potent inhibitor of HCMV replication. Time-of-addition studies demonstrated that DIDS inhibited entry via direct interaction with the virion that impeded binding to the plasma membrane. Synthesis and analysis of pharmacological variants of DIDS suggested that intrinsic cysteine, and not lysine, reactivity was important for activity against HCMV. Although sequencing of DIDS-resistant HCMV revealed enrichment of a mutation within UL100 (encoding glycoprotein M) and a specific truncation of glycoprotein RL13, these did not explain the DIDS resistance phenotype. Specifically, only the introduction of the RL13 mutant partially phenocopied the DIDS resistance phenotype. Serendipitously, the entry of DIDS-resistant HCMV also became independent of heparan sulfate proteoglycans (HSPGs), suggesting that evasion of DIDS lowered dependence on an initial interaction with HSPGs. Intriguingly, the DIDS-resistant virus demonstrated increased sensitivity to antibody neutralization, which mapped, in part, to the presence of the gM mutation. Taken together the data characterize the antiviral activity of a novel HCMV inhibitor that drives HCMV infection to occur independently of HSPGs and the generation of increased sensitivity to humoral immunity. The data also demonstrate that compounds with cysteine reactivity have the potential to act as antiviral compounds against HCMV via direct engagement of virions. IMPORTANCE Human cytomegalovirus (HCMV) is major pathogen of nonimmunocompetent individuals that remains in need of new therapeutic options. Here, we identify a potent antiviral compound (4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid [DIDS]), its mechanism of action, and the chemical properties required for its activity. In doing so, the data argue that cysteine-reactive compounds could have the capacity to be developed for anti-HCMV activity. Importantly, the data show that entry of DIDS-resistant virus became independent of heparan sulfate proteoglycans (HSPGs) but, concomitantly, became more sensitive to neutralizing antibody responses. This serendipitous observation suggests that retention of an interaction with HSPGs during the entry process in vivo may be evolutionarily advantageous through better evasion of humoral responses directed against HCMV virions.