Inhibition of Human Cytomegalovirus Replication by Artemisinins: Effects Mediated through Cell Cycle Modulation

Abstract

dArtemisinin-derived monomers and dimers inhibit human cytomegalovirus (CMV) replication in human foreskin fibroblasts (HFFs). The monomer artesunate (AS) inhibits CMV at micromolar concentrations, while dimers inhibit CMV replication at nanomolar concentrations, without increased toxicity in HFFs. We report on the variable anti-CMV activity of AS compared to the consistent and reproducible CMV inhibition by dimer 606 and ganciclovir (GCV). Investigation of this phenomenon revealed that the anti-CMV activity of AS correlated with HFFs synchronized to the G 0 /G 1 stage of the cell cycle. In contact-inhibited serum-starved HFFs or cells arrested at early/late G 1 with specific checkpoint regulators, AS and dimer 606 efficiently inhibited CMV replication. However, in cycling HFFs, in which CMV replication was productive, virus inhibition by AS was significantly reduced, but inhibition by dimer 606 and GCV was maintained. Cell cycle analysis in noninfected HFFs revealed that AS induced early G 1 arrest, while dimer 606 partially blocked cell cycle progression. In infected HFFs, AS and dimer 606 prevented the progression of cell cycle toward the G 1 /S checkpoint. AS reduced the expression of cyclin-dependent kinases (CDK) 2, 4, and 6 in noninfected cycling HFFs, while the effect of dimer 606 on these CDKs was moderate. Neither compound affected CDK expression in noninfected contact-inhibited HFFs. In CMV-infected cells, AS activity correlated with reduced CDK2 levels. CMV inhibition by AS and dimer 606 also correlated with hypophosphorylation (activity) of the retinoblastoma protein (pRb). AS activity was strongly associated with pRb hypophosphorylation, while its reduced anti-CMV activity was marked by pRb phosphorylation. Roscovitine, a CDK2 inhibitor, antagonized the anti-CMV activities of AS and dimer 606. These data suggest that cell cycle modulation through CDKs and pRb might play a role in the anti-CMV activities of artemisinins. Proteins involved in this modulation may be identified and targeted for CMV inhibition.A rtemisinins, drugs of choice for malaria therapy, inhibit human cytomegalovirus (CMV) replication (1-4). Artesunate (AS) and the parent compound artemisinin inhibit CMV replication in vitro and in vivo. Variable responses to AS in case reports of CMV-infected patients have been attributed to the dosing regimen, duration of therapy, or tissue penetration of AS (5-8). In our effort to improve and uncover the anti-CMV activities of artemisinins, we reported on in vitro highly selective inhibition of CMV replication with artemisinin-derived dimers, significantly more than with their monomeric counterparts, without increasing toxicity in human foreskin fibroblasts (HFFs) (3, 9). Although similar effects on CMV replication were observed between monomers and dimers (timing of CMV inhibition, effects on DNA replication, and virus yield), dimers inhibited CMV at nanomolar concentrations and had a high slope of the dose-response curve, a measure of cooperativity in binding of multiple ligands to linked bi...

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Treatment of CMV infection after allogeneic hematopoietic stem cell transplantation

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