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Artemisinins target the intermediate filament protein vimentin for human cytomegalovirus inhibition

2020; Elsevier BV; Volume: 295; Issue: 44 Linguagem: Inglês

10.1074/jbc.ra120.014116

1083-351X

Sujayita Roy, Arun Kapoor, Fei Zhu, Rupkatha Mukhopadhyay, Ayan Kumar Ghosh, Hyun Lee, Jennifer R. Mazzone, Gary H. Posner, Ravit Arav‐Boger,

Glycosylation and Glycoproteins Research

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. 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. Repurposing of the antimalarial agents artemisinins for treatment of human cytomegalovirus (HCMV) attracted interest, fueled by clinical experience and safety data from malaria therapy (1Arav-Boger R. He R. Chiou C.J. Liu J. Woodard L. Rosenthal A. Jones-Brando L. Forman M. Posner G.H. Artemisinin-derived dimers have greatly improved anti-cytomegalovirus activity compared to artemisinin monomers.PLoS One. 2010; 5 (20442781): e1037010.1371/journal.pone.0010370Crossref PubMed Scopus (47) Google Scholar, 2He R. Mott B.T. Rosenthal A.S. Genna D.T. Posner G.H. Arav-Boger R. An artemisinin-derived dimer has highly potent anti-cytomegalovirus (CMV) and anti-cancer activities.PLoS One. 2011; 6 (21904628): e2433410.1371/journal.pone.0024334Crossref PubMed Scopus (56) Google Scholar, 3Efferth T. Marschall M. Wang X. Huong S.M. Hauber I. Olbrich A. Kronschnabl M. Stamminger T. Huang E.S. Antiviral activity of artesunate towards wild-type, recombinant, and ganciclovir-resistant human cytomegaloviruses.J. Mol. Med. (Berl.). 2002; 80 (11976732): 233-24210.1007/s00109-001-0300-8Crossref PubMed Scopus (148) Google Scholar, 4Adjuik M. Babiker A. Garner P. Olliaro P. Taylor W. 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We and others have reported that artemisinin-derived monomers (artemisinin, artesunate, artemether, artemisone) inhibit HCMV at micromolar concentrations, whereas the dimeric versions are inhibitory at nanomolar concentrations (1Arav-Boger R. He R. Chiou C.J. Liu J. Woodard L. Rosenthal A. Jones-Brando L. Forman M. Posner G.H. Artemisinin-derived dimers have greatly improved anti-cytomegalovirus activity compared to artemisinin monomers.PLoS One. 2010; 5 (20442781): e1037010.1371/journal.pone.0010370Crossref PubMed Scopus (47) Google Scholar, 2He R. Mott B.T. Rosenthal A.S. Genna D.T. Posner G.H. Arav-Boger R. An artemisinin-derived dimer has highly potent anti-cytomegalovirus (CMV) and anti-cancer activities.PLoS One. 2011; 6 (21904628): e2433410.1371/journal.pone.0024334Crossref PubMed Scopus (56) Google Scholar, 7Oiknine-Djian E. Weisblum Y. Panet A. Wong H.N. Haynes R.K. Wolf D.G. The artemisinin derivative artemisone is a potent inhibitor of human cytomegalovirus replication.Antimicrob. Agents Chemother. 2018; 62 (29712656): e00218-e0028810.1128/AAC.00288-18Crossref PubMed Scopus (27) Google Scholar, 8Reiter C. Fröhlich T. Gruber L. Hutterer C. Marschall M. Voigtländer C. Friedrich O. Kappes B. Efferth T. Tsogoeva S.B. Highly potent artemisinin-derived dimers and trimers: synthesis and evaluation of their antimalarial, antileukemia and antiviral activities.Bioorg. Med. Chem. 2015; 23 (26260339): 5452-545810.1016/j.bmc.2015.07.048Crossref PubMed Scopus (73) Google Scholar). The endoperoxide bridge within the artemisinin trioxane pharmacophore is critical for compound activity (9O'Neill P.M. Barton V.E. Ward S.A. The molecular mechanism of action of artemisinin—the debate continues.Molecules. 2010; 15 (20336009): 1705-172110.3390/molecules15031705Crossref PubMed Scopus (395) Google Scholar, 10Hartwig C.L. Rosenthal A.S. D'Angelo J. Griffin C.E. Posner G.H. Cooper R.A. Accumulation of artemisinin trioxane derivatives within neutral lipids of Plasmodium falciparum malaria parasites is endoperoxide-dependent.Biochem. Pharmacol. 2009; 77 (19022224): 322-33610.1016/j.bcp.2008.10.015Crossref PubMed Scopus (105) Google Scholar), and its chemical disruption ("deoxyartemisinin") abolishes the anti-HCMV activity (2He R. Mott B.T. Rosenthal A.S. Genna D.T. Posner G.H. Arav-Boger R. An artemisinin-derived dimer has highly potent anti-cytomegalovirus (CMV) and anti-cancer activities.PLoS One. 2011; 6 (21904628): e2433410.1371/journal.pone.0024334Crossref PubMed Scopus (56) Google Scholar, 11He R. Forman M. Mott B.T. Venkatadri R. Posner G.H. Arav-Boger R. The unique and highly-selective anti-cytomegalovirus activities of artemisinin-derived dimer diphenyl phosphate stem from combination of dimer unit and a diphenyl phosphate moiety.Antimicrob. Agents Chemother. 2013; 57 (23774439): 4208-421710.1128/AAC.00893-13Crossref PubMed Scopus (25) Google Scholar). Until now, an artemisinin-resistant HCMV has not been selected, suggesting that virus inhibition primarily involves host-directed functions critical for virus replication (12Roy S. He R. Kapoor A. Forman M. Mazzone J.R. Posner G.H. Arav-Boger R. Inhibition of human cytomegalovirus replication by artemisinins: effects mediated through cell cycle modulation.Antimicrob. Agents Chemother. 2015; 59 (25870074): 3870-387910.1128/AAC.00262-15Crossref PubMed Scopus (28) Google Scholar, 13Efferth T. Romero M.R. Wolf D.G. Stamminger T. Marin J.J. Marschall M. The antiviral activities of artemisinin and artesunate.Clin. Infect. Dis. 2008; 47 (18699744): 804-81110.1086/591195Crossref PubMed Scopus (348) Google Scholar). The mechanisms of HCMV inhibition by artemisinins are different from the DNA polymerase inhibitor, ganciclovir (GCV), as they inhibit GCV-resistant HCMV. The combination of artemisinins and GCV is synergistic against HCMV (7Oiknine-Djian E. Weisblum Y. Panet A. Wong H.N. Haynes R.K. Wolf D.G. The artemisinin derivative artemisone is a potent inhibitor of human cytomegalovirus replication.Antimicrob. Agents Chemother. 2018; 62 (29712656): e00218-e0028810.1128/AAC.00288-18Crossref PubMed Scopus (27) Google Scholar, 14Cai H. Kapoor A. He R. Venkatadri R. Forman M. Posner G.H. Arav-Boger R. In vitro combination of anti-cytomegalovirus compounds acting through different targets: role of the slope parameter and insights into mechanisms of action.Antimicrob. Agents Chemother. 2014; 58 (24277030): 986-99410.1128/AAC.01972-13Crossref PubMed Scopus (42) Google Scholar). The in vitro anti-HCMV activity of artesunate correlated with cell cycle stage (12Roy S. He R. Kapoor A. Forman M. Mazzone J.R. Posner G.H. Arav-Boger R. Inhibition of human cytomegalovirus replication by artemisinins: effects mediated through cell cycle modulation.Antimicrob. Agents Chemother. 2015; 59 (25870074): 3870-387910.1128/AAC.00262-15Crossref PubMed Scopus (28) Google Scholar), efficacious in contact-inhibited human foreskin fibroblasts (HFFs) but reduced in subconfluent HFFs. In contact-inhibited cells, HCMV induced cell cycle progression to G1/S at 24 h postinfection (hpi), but artesunate reverted it to early G0/G1 and decreased virus-induced expression of cyclin-dependent kinases (CDK1, -2, and -4). The cellular/microbial targets of artemisinins have been of major interest to several disciplines, including infectious diseases and cancer. Studies have highlighted the complexity and promiscuity of these drugs toward multiple proteins in Plasmodium falciparum, but specific targets underlying the anti-infective or anti-cancer properties remain inconclusive (15Wang J. Zhang C.J. Chia W.N. Loh C.C. Li Z. Lee Y.M. He Y. Yuan L.X. Lim T.K. Liu M. Liew C.X. Lee Y.Q. Zhang J. Lu N. Lim C.T. et al.Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum.Nat. Commun. 2015; 6 (26694030): 1011110.1038/ncomms10111Crossref PubMed Scopus (332) Google Scholar, 16Efferth T. Molecular pharmacology and pharmacogenomics of artemisinin and its derivatives in cancer cells.Curr. Drug Targets. 2006; 7 (16611029): 407-42110.2174/138945006776359412Crossref PubMed Scopus (229) Google Scholar). To better define the anti-HCMV activities of artemisinins and identify cellular functions targeted by these drugs, we synthesized a biotin-labeled trioxane semisynthetically derived from artemisinin at the C10 position. Immunoprecipitation (IP)-MS followed by molecular, biological, and biochemical studies confirmed the type III-intermediate filament protein vimentin as an artemisinin target, resulting in post-translational modification and stabilization of vimentin. Because artemisinins inhibit CMV in vitro and in vivo, we determined the requirement of vimentin for virus replication. Reportedly, vimentin enables HCMV trafficking into the nucleus early after infection (17Miller M.S. Hertel L. Onset of human cytomegalovirus replication in fibroblasts requires the presence of an intact vimentin cytoskeleton.J. Virol. 2009; 83 (19403668): 7015-702810.1128/JVI.00398-09Crossref PubMed Scopus (40) Google Scholar). However, its functions during later stages of infection have not been studied. Our data show distinct roles of vimentin at different stages of HCMV replication. In the early stage of infection, vimentin level is stable, likely providing support for virus transport into the nucleus, but subsequently, HCMV strategy is to destabilize and degrade vimentin. This latter process requires several mechanisms, including vimentin phosphorylation and induction of calpain activity. Binding of artesunate to vimentin counteracts and reverses virus-induced vimentin degradation, primarily through decreasing its phosphorylation and inducing calpain activity, effects culminating in virus inhibition. Trioxane C10 primary alcohol (1Arav-Boger R. He R. Chiou C.J. Liu J. Woodard L. Rosenthal A. Jones-Brando L. Forman M. Posner G.H. Artemisinin-derived dimers have greatly improved anti-cytomegalovirus activity compared to artemisinin monomers.PLoS One. 2010; 5 (20442781): e1037010.1371/journal.pone.0010370Crossref PubMed Scopus (47) Google Scholar), derived from dihydroartemisinin (DHA), the active metabolite of all monomeric artemisinins, was coupled with carboxylic acid (2He R. Mott B.T. Rosenthal A.S. Genna D.T. Posner G.H. Arav-Boger R. An artemisinin-derived dimer has highly potent anti-cytomegalovirus (CMV) and anti-cancer activities.PLoS One. 2011; 6 (21904628): e2433410.1371/journal.pone.0024334Crossref PubMed Scopus (56) Google Scholar) to produce biotin-labeled trioxane (552 kDa) (Fig. 1A). The biotin-labeled product will be referred to as 552. Lysates from noninfected HFFs were treated overnight at 4 °C with DMSO, 552 (20 μm), or 552 with DHA as a competitor (200 μm), which was added 1 h prior to 552. 552-protein complexes were captured with streptavidin agarose beads followed by protein separation on SDS-PAGE and silver staining (Fig. 1B). Specific bands enriched in the 552 lane were cut and analyzed by MS. Vimentin was identified as one of the main 552-binding proteins (Table 1). To confirm the MS findings, IP with streptavidin agarose beads was performed in noninfected HFF cell lysates treated with DMSO, 552 (20 μm), or 552 with DHA as a competitor (200 μm). In the drug competition condition, DHA was added 1 h before 552. Lysates were incubated overnight, followed by Western blotting with anti-vimentin antibody. Vimentin was detected in the 552-treated lysates, but not in the DMSO or DHA (competitor)-treated samples (Fig. 1C). Next, equal quantities of purified His-vimentin protein (100 ng) were incubated for 1 h with increasing concentrations of DHA, followed by incubation with 552 for 1 h. Proteins were blotted and probed with streptavidin-HRP. Competition with increasing concentrations of DHA resulted in reduced biotinylated adducts (Fig. 1D), whereas vimentin level was similar in all conditions. A surface plasmon resonance (SPR) assay was performed after immobilizing purified His-vimentin on the CM5 sensor surface (Biacore T200, GE Healthcare). Artesunate (monomeric artemisinin) and an inactive metabolite, deoxyartemisinin, that lacks anti-HCMV activity were tested at concentrations ranging from 0.93 to 33.3 μm along with at least two zero concentrations. Artesunate showed fast association (ka) and dissociation (kd) rates at all tested concentrations based on each sensorgram (Fig. 1E). The binding affinity (KD) of artesunate binding to vimentin was calculated to be 12.3 ± 1.7 μm by steady-state affinity fitting with data at equilibrium (Fig. 1F). 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.Table 1MS analysis of proteins bound to 552 in HFFsProteinMolecular massNo. of peptidesAccession no.CoveragekDa%Cytoskeleton-associated protein 46611gi 1992031721.0Vimentin5431gi 6241428962.0Annexin A2 isoform 2392gi 2098628315.9Annexin A1396gi 450210123.0Actin, cytoplasmic 1426gi 450188519.0Cathepsin D preproprotein452gi 45031434.1Serpin B3454gi 590207211Elongation factor 1-α1503gi 45034716.5Pyruvate kinase isozymes M1/M2662gi 3321647754.8Heat shock 70-kDa protein 1–like703gi 1242564966.2Junction plakoglobin822gi 120564682.8 Open table in a new tab To begin understanding whether binding of artemisinins to vimentin results in HCMV inhibition, we tested the effects of infection with two genotypically distinct strains of HCMV (TB40 and Towne) on vimentin expression and modulation. Vimentin level was gradually and reproducibly reduced in infected HFFs. At 4 hpi, vimentin level was stable, but starting at 24 hpi with HCMV-TB40 (Fig. 2A), its level decreased. Vimentin is a highly phosphorylated protein, and its phosphorylation strongly correlates with its disassembly (18Eriksson J.E. He T. Trejo-Skalli A.V. Härmälä-Braskén A.S. Hellman J. Chou Y.H. Goldman R.D. Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments.J. Cell Sci. 2004; 117 (14762106): 919-93210.1242/jcs.00906Crossref PubMed Scopus (231) Google Scholar). We selected two sites to measure vimentin phosphorylation during infection, Ser-55 and Ser-83 (19Inagaki N. Goto H. Ogawara M. Nishi Y. Ando S. Inagaki M. Spatial patterns of Ca2+ signals define intracellular distribution of a signaling by Ca2+/calmodulin-dependent protein kinase II.J. Biol. Chem. 1997; 272 (9312133): 25195-2519910.1074/jbc.272.40.25195Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Ser-55 is phosphorylated by CDK1, which also recruits Polo-like kinase (PLK1) to phosphorylated vimentin Ser-55, resulting in PLK1 activation and further vimentin phosphorylation at Ser-83 (20Yamaguchi T. Goto H. Yokoyama T. Silljé H. Hanisch A. Uldschmid A. Takai Y. Oguri T. Nigg E.A. Inagaki M. Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis.J. Cell Biol. 2005; 171 (16260496): 431-43610.1083/jcb.200504091Crossref PubMed Scopus (105) Google Scholar). The decrease in vimentin level during HCMV infection correlated with increased phosphorylation at the respective time points. Ser-55 phosphorylation was increased from 24 hpi onward, and Ser-83 was phosphorylated at 48 and 72 hpi (Fig. 2A). Artesunate treatment resulted in reduced expression of HCMV Towne-encoded IE1/2 and pp65 as well as vimentin Ser-83 phosphorylation and recovered vimentin level (Fig. 2B). The changes in vimentin level and phosphorylation were similar to those observed with HCMV-TB40 (Fig. 2A), indicating that the two viral strains did not differ in their effects on vimentin. Deoxyartemisinin did not change vimentin level or Ser-83 phosphorylation and did not inhibit pp65 expression (Fig. 2C). The disappearance of the Ser-83 band at 50 kDa and the Ser-55 band at 57 kDa by phosphatase assay confirmed that these were phosphorylated proteins (Fig. 2D). In addition, MS analysis of the 50 kDa band identified vimentin. 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). We investigated the effects of artesunate on vimentin level at different times during infection. When added from the time of infection up until 72 hpi, artesunate treatment resulted in remarkable vimentin stabilization (Fig. 2F). When added from 24 to 42 hpi or from 42 to 72 hpi, artesunate had a minor effect on vimentin level. Vimentin stabilization required artesunate to be present in the cell before the onset of viral DNA replication up until 72 hpi. Vimentin is a cytoskeleton protein, and its filaments are seen in attached and flattened cells (21Mendez M.G. Kojima S. Goldman R.D. Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition.FASEB J. 2010; 24 (20097873): 1838-185110.1096/fj.09-151639Crossref PubMed Scopus (574) Google Scholar, 22Goldman R.D. Khuon S. Chou Y.H. Opal P. Steinert P.M. The function of intermediate filaments in cell shape and cytoskeletal integrity.J. Cell Biol. 1996; 134 (8769421): 971-98310.1083/jcb.134.4.971Crossref PubMed Scopus (300) Google Scholar). We tested the localization of vimentin during HCMV infection and artesunate treatment. An indirect immunofluorescence (IFA) was performed at 24 and 72 hpi with HCMV TB40 (Fig. 3 and Fig. S1), revealing an altered staining pattern and reduced vimentin signal during infection and recovery of vimentin signal with artesunate. The inactive artemisinin metabolite could not rescue vimentin signal at 24 or 72 hpi. Altogether, the data indicate that vimentin is disassembled during infection and artesunate maintains its assembly condition. To confirm the role of artesunate in vimentin stabilization, we performed a cellular thermal shift assay (23Jafari R. Almqvist H. Axelsson H. Ignatushchenko M. Lundbäck T. Nordlund P. Martinez M.D. The cellular thermal shift assay for evaluating drug target interactions in cells.Nat. Protoc. 2014; 9 (25101824): 2100-212210.1038/nprot.2014.138Crossref PubMed Scopus (507) Google Scholar, 24Martinez M.D. Jafari R. Ignatushchenko M. Seki T. Larsson E.A. Dan C. Sreekumar L. Cao Y. Nordlund P. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay.Science. 2013; 341 (23828940): 84-8710.1126/science.1233606Crossref PubMed Scopus (842) Google Scholar). HFFs were treated with artesunate (30 μm) or DMSO, washed with PBS, trypsinized and resuspended in PBS containing protease inhibitors, and incubated in a thermal cycler at the indicated temperatures. Vimentin level was measured by Western blotting. In the DMSO-treated samples, vimentin level decreased with increasing temperatures, whereas artesunate stabilized vimentin at all tested temperatures (Fig. 4A). Comparison of the cellular thermal shift by artesunate and deoxyartemisinin (30 μm) at 56 and 60 °C showed that only artesunate stabilized vimentin (Fig. 4B). Neither artesunate nor deoxyartemisinin had an effect on p53 stabilization (Fig. 4B). Vimentin undergoes homodimerization, followed by antiparallel association between homodimers to form soluble tetramers. Further assembly of vimentin filaments involves lateral association between eight tetramers to form a unit length filament, which is followed by longitudinal annealing of these into short filaments and radial compaction of the annealed filaments to produce mature filaments. These structures are dynamic and reorganize during mitosis or cell stress (25Snider N.T. Omary M.B. Post-translational modifications of intermediate filament proteins: mechanisms and functions.Nat. Rev. Mol. Cell Biol. 2014; 15 (24556839): 163-17710.1038/nrm3753Crossref PubMed Scopus (288) Google Scholar). Using glutaraldehyde cross-linking, we found that high-molecular weight forms of vimentin, representing soluble tetramers and possibly unit length filaments, were increased with artesunate treatment in noninfected and HCMV-infected HFFs (Fig. 4C). In sum, artesunate treatment results in vimentin stabilization. We investigated whether vimentin was degraded during HCMV infection through the proteasome. Using the proteasome inhibitor MG132, vimentin level did not recover at 72 hpi (Fig. 4D, top). Another experiment performed at 24 and 48 hpi revealed again that the vimentin level did not recover with MG132 treatment, whereas p53 level was recovered as expected (Fig. 4D, bottom), indicating that MG132 activity was intact. The data suggest proteasome-independent degradation of vimentin in HCMV-infected cells. Vimentin is subject to proteolytic cleavage by calpains (Ca2+-activated neutral proteases) (26Yoshida H. Murachi T. Tsukahara I. Degradation of actin and vimentin by calpain II, a Ca2+-dependent cysteine proteinase, in bovine lens.FEBS Lett. 1984; 170 (6327382): 259-26210.1016/0014-5793(84)81324-1Crossref PubMed Scopus (51) Google Scholar, 27Davis M.A. Fairgrieve M.R. Den Hartigh A. Yakovenko O. Duvvuri B. Lood C. Thomas W.E. Fink S.L. Gale Jr., M. Calpain drives pyroptotic vimentin cleavage, intermediate filament loss, and cell rupture that mediates immunostimulation.Proc. Natl. Acad. Sci. U. S. A. 2019; 116 (30796192): 5061-507010.1073/pnas.1818598116Crossref PubMed Scopus (34) Google Scholar), and HCMV reportedly activates calpains 1 and 2, which regulate p21cip (28Chen Z. Knutson E. Kurosky A. Albrecht T. Degradation of p21cip1 in cells productively infected with human cytomegalovirus.J. Virol. 2001; 75 (11264351): 3613-362510.1128/JVI.75.8.3613-3625.2001Crossref PubMed Scopus (63) Google Scholar). We measured calpain activity using a calpain-induced substrate cleavage assay. At 72 hpi, HCMV induced calpain activity (Fig. 4E). In the presence of the calpain inhibitor E64D (100 μm, 6 h), protease activity was reduced. HCMV-infected artesunate-treated cells showed similar relative fluorescence signal as infected-only cells, indicating that artesunate does not inhibit calpain. Next, we performed an in vitro calpain digestion assay using purified vimentin protein to determine whether artesunate-bound vimentin was rendered calpain-resistant. Recombinant human vimentin (1 μm) was digested with 2.5 units of calpain 1 for 10 min, and resultant vimentin fragments were visualized after Coomassie Blue staining. Without calpain, vimentin was uncleaved (Fig. 4F, lane 1). Calpain 1 was in a high-glycerol buffer, which altered the migration pattern of vimentin (lane 2). When incubated with calpain 1 under appropriate reaction conditions, vimentin cleavage generated a series of bands ranging from 15 to 37 kDa (lane 3). The calpain inhibitor E64D prevented vimentin cleavage (lane 6). Importantly, preincubation of vimentin with artesunate protected it from cleavage by calpain 1 (Lane 4 vs Lane 3). GCV did not confer protection against vimentin degradation (lane 5). Altogether, we confirm that vimentin cleavage results, at least in part, from HCMV-induced calpain activity. Artesunate binding to vimentin may prevent calpains from accessing several sites and digesting vimentin during infection, providing protection of vimentin structure/activity. We reported that artemisinins inhibit HCMV after virus entry, and their inhibitory effects persist during most stages of HCMV replication (6He R. Park K. Cai H. Kapoor A. Forman M. Mott B. Posner G.H. Arav-Boger R. Artemisinin-derived dimer diphenyl phosphate is an irreversible inhibitor of human cytomegalovirus replication.Antimicrob. Agents Chemother. 2012; 56 (22547612): 3508-351510.1128/AAC.00519-12Crossref PubMed Scopus (30) Google Scholar). We therefore investigated the role of vimentin during HCMV replication. The onset of infection and HCMV migration to the nucleus were reportedly delayed in the absence of an intact vimentin network (17Miller M.S. Hertel L. Onset of human cytomegalovirus replication in fibroblasts requires the presence of an intact vimentin cytoskeleton.J. Virol. 2009; 83 (19403668): 7015-702810.1128/JVI.00398-09Crossref PubMed Scopus (40) Google Scholar), but later effects of vimentin on virus replication were not studied. Using lentivirus shRNAs, we knocked down vimentin in HFFs to a degree that would still enable virus entry and replication for inhibition studies (Fig. 5A). Virus entry into vimentin-deficient cells was reduced compared with entry into control transduced cells, indicated by pp65 level (Fig. 5B). For in vivo relevance of these findings, we infected vimentin knockout and control mice (129S) with mouse CMV (MCMV). All tested tissues from vimentin knockout mice showed significantly reduced virus titer, indicating the requirement of vimentin for initiation of MCMV replication (Fig. 5C). We next measured the effects of artesunate on HCMV replication in vimentin knockdown and control cells. Cells were infected with HCMV Towne (MOI = 3), and a pp28-luciferase assay was performed at 96 hpi. Artesunate activity against HCMV was reduced in vimentin knockdown cells, whereas GCV activity was comparable between the two cell lines (Fig. 5D). A virus yield assay revealed reduced virus progeny from vimentin knockdown cells starting from 48 hpi (Fig. 5E). Compared with control cells, inhibition of HCMV with artesunate was less effective in vimentin-deficient cells (5.2-fold versus 1.7-fold, respectively). Thus, vimentin level directly correlates with the anti-HCMV activity of artesunate. Given the reported timing of HCMV inhibition by artemisinins (post-entry), reduced levels of vimentin during the progression of infection, and the correlation between artemisinins' activity and vimentin stabilization, we attempted to dissociate the early role of vimentin from its later effects during HCMV replication. Vimentin was overexpressed in U373 glioma cells, followed by infection with the pp28-luciferase Towne. Luciferase activity measured at 96 hpi was significantly reduced in vimentin-overexpressing cells compared with controls (Fig. 5F). Vimentin overexpression was confirmed by Western blotting (Fig. 5G, top). Viral IE1/2 and pp65 expression was reduced at 72 hpi in vimentin-overexpressing cells (Fig. 5G, bottom), despite similar virus entry among all conditions (Fig. 5H). The data corroborate an earlier report that intermediate filament (IF) bundling in cells from a patient with giant axonal neuropathy results