Abstract
The antimalarial agents artemisinins inhibit cytomegalovirus (CMV) in vitro and in vivo, but their target(s) has been elusive. Using a biotin-labeled artemisinin, we identified the intermediate filament protein vimentin as an artemisinin target, validated by detailed biochemical and biological assays. We provide insights into the dynamic and unique modulation of vimentin, depending on the stage of human CMV (HCMV) replication. In vitro, HCMV entry and viral progeny are reduced in vimentin-deficient fibroblasts, compared with control cells. Similarly, mouse CMV (MCMV) replication in vimentin knockout mice is significantly reduced compared with controls in vivo, confirming the requirement of vimentin for establishment of infection. Early after HCMV infection of human foreskin fibroblasts vimentin level is stable, but as infection proceeds, vimentin is destabilized, concurrent with its phosphorylation and virus-induced calpain activity. Intriguingly, in vimentin-overexpressing cells, HCMV infection is reduced compared with control cells. Binding of artesunate, an artemisinin monomer, to vimentin prevents virus-induced vimentin degradation, decreasing vimentin phosphorylation at Ser-55 and Ser-83 and resisting calpain digestion. In vimentin-deficient fibroblasts, the anti-HCMV activity of artesunate is reduced compared with controls. In summary, an intact and stable vimentin network is important for the initiation of HCMV replication but hinders its completion. Artesunate binding to vimentin early during infection stabilizes it and antagonizes subsequent HCMV-mediated vimentin destabilization, thus suppressing HCMV replication. Our target discovery should enable the identification of vimentin-binding sites and compound moieties for binding.
Highlights
Repurposing of the antimalarial agents artemisinins for treatment of human cytomegalovirus (HCMV) attracted interest, fueled by clinical experience and safety data from malaria therapy [1,2,3,4,5]
Artesunate and an inactive metabolite, deoxyartemisinin, that lacks anti-human CMV (HCMV) activity were tested at concentrations ranging from 0.93 to 33.3 mM along with at least two zero concentrations
Deoxyartemisinin did not show any interaction with vimentin (Fig. 1, G and H), indicating that the endoperoxide bridge in the artemisinin pharmacophore is required for binding to vimentin
Summary
Trioxane C10 primary alcohol [1], derived from dihydroartemisinin (DHA), the active metabolite of all monomeric artemisinins, was coupled with carboxylic acid [2] to produce biotinlabeled trioxane (552 kDa) (Fig. 1A). Artesunate-mediated changes in vimentin were HCMV-specific, because in HSV1-infected HFFs artesunate did not modify vimentin level and HSV1-encoded ICP8 was not reduced (Fig. 2E). Artemisinins were reported to reverse HCMV-mediated cell cycle changes and reduce the levels of CDKs. In addition, in confluent HFFs (at the time of infection), artesunate inhibited HCMV, but in subconfluent cells, its antiHCMV activity was lost [12]. We investigated the correlation between cell cycle and vimentin changes in infected HFFs. HCMV inhibition by artesunate was associated with reduced Ser-83 phosphorylation and vimentin stabilization in contact-inhibited cells. Artesunate-mediated changes in CDKs and PLK1 were observed only in infected cells, and not in noninfected cells (Fig. 7D), suggesting that the cell cycle in noninfected cells overcomes the effect of artesunate or that viral proteins. The anti-HCMV activity of artesunate is mediated through vimentin, and the cell cycle drives changes in vimentin. At the best cell cycle stage (early G1), artesunate will bind to vimentin and compete with HCMV to prevent its degradation, winning the battle on HCMV replication
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