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• W3040697948 abstract "Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo. We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism. Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo. We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism. Viruses are well-known for their hijacking of cellular processes to enable their own replication while also evading or counteracting the host response to infection (1Thimme R. Lohmann V. Weber F. A target on the move: innate and adaptive immune escape strategies of hepatitis C virus.Antiviral Res. 2006; 69 (16413618): 129-14110.1016/j.antiviral.2005.12.001Crossref PubMed Scopus (102) Google Scholar). This is accomplished using diverse mechanisms that include viral regulation of host transcription and translation, as well as post-translational regulation of host factors (2Dawson C.W. Tramountanis G. Eliopoulos A.G. Young L.S. Epstein–Barr virus latent membrane protein 1 (LMP1) activates the phosphatidylinositol 3-kinase/Akt pathway to promote cell survival and induce actin filament remodeling.J. Biol. Chem. 2003; 278 (12446712): 3694-370410.1074/jbc.M209840200Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 3Cheeran M.C.-J. Hu S. Sheng W.S. Rashid A. Peterson P.K. Lokensgard J.R. Differential responses of human brain cells to West Nile virus infection.J. Neurovirol. 2005; 11 (16338745): 512-52410.1080/13550280500384982Crossref PubMed Scopus (79) Google Scholar). For example, hepatitis C virus (HCV) disrupts sensing by the host innate immune system by directly cleaving two critical adaptor proteins, mitochondrial antiviral signaling protein (MAVS) and Toll–interleukin-1 receptor domain containing adaptor-inducing interferon-β (TRIF) (4Li K. Foy E. Ferreon J.C. Nakamura M. Ferreon A.C.M. Ikeda M. Ray S.C. Gale M. Lemon S.M. Immune evasion by hepatitis C virus NS3/4A protease–mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF.Proc. Natl. Acad. Sci. U.S.A. 2005; 102 (15710891): 2992-299710.1073/pnas.0408824102Crossref PubMed Scopus (886) Google Scholar, 5Ferreon J.C. Ferreon A.C.M. Li K. Lemon S.M. Molecular determinants of TRIF proteolysis mediated by the hepatitis C virus NS3/4A protease.J. Biol. Chem. 2005; 280 (15767257): 20483-2049210.1074/jbc.M500422200Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 6Li X.-D. Sun L. Seth R.B. Pineda G. Chen Z.J. Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity.Proc. Natl. Acad. Sci. U.S.A. 2005; 102 (16301520): 17717-1772210.1073/pnas.0508531102Crossref PubMed Scopus (601) Google Scholar) HCV. Other viruses are also known to alter host metabolism to meet the energetic and metabolic needs of replication, for example, by altering the expression and/or localization of host proteins responsible for synthesis or trafficking of critical metabolites. One such example is the interaction of HCV nonstructural protein 5A (NS5A) with phosphatidylinositol 4-kinase α, which appears to regulate the phosphorylation status and function of NS5A while also stimulating the accumulation of phosphatidylinositol 4-phosphates that recruit viral and host factors required for RNA replication (7Berger K.L. Cooper J.D. Heaton N.S. Yoon R. Oakland T.E. Jordan T.X. Mateu G. Grakoui A. Randall G. Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication.Proc. Natl. Acad. Sci. U.S.A. 2009; 106 (19376974): 7577-758210.1073/pnas.0902693106Crossref PubMed Scopus (278) Google Scholar, 8Reiss S. Rebhan I. Backes P. Romero-Brey I. Erfle H. Matula P. Kaderali L. Poenisch M. Blankenburg H. Hiet M.-S. Longerich T. Diehl S. Ramirez F. Balla T. Rohr K. et al.Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment.Cell Host Microbe. 2011; 9 (21238945): 32-4510.1016/j.chom.2010.12.002Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). The clinical association of HCV infection with steatosis and hyperlipidemia positions it as a good experimental system for studying how viruses perturb and exploit the host lipid machinery (9Fabris C. Federico E. Soardo G. Falleti E. Pirisi M. Blood lipids of patients with chronic hepatitis: differences related to viral etiology.Clin. Chim. Acta. 1997; 261 (9201435): 159-16510.1016/S0009-8981(97)06532-7Crossref PubMed Scopus (71) Google Scholar, 10Jármay K. Karácsony G. Nagy A. Schaff Z. Changes in lipid metabolism in chronic hepatitis C.World J. Gastroenterol. 2005; 11 (16425410): 6422-642810.3748/wjg.v11.i41.6422Crossref PubMed Scopus (31) Google Scholar, 11Asselah T. Rubbia-Brandt L. Marcellin P. Negro F. Steatosis in chronic hepatitis C: why does it really matter?.Gut. 2006; 55 (16344578): 123-13010.1136/gut.2005.069757Crossref PubMed Scopus (311) Google Scholar, 12Corey K.E. Kane E. Munroe C. Barlow L.L. Zheng H. Chung R.T. Hepatitis C virus infection and its clearance alter circulating lipids: implications for long-term follow-up.Hepatology. 2009; 50 (19787818): 1030-103710.1002/hep.23219Crossref PubMed Scopus (127) Google Scholar, 13Harrison S.A. Rossaro L. Hu K.-Q. Patel K. Tillmann H. Dhaliwal S. Torres D.M. Koury K. Goteti V.S. Noviello S. Brass C.A. Albrecht J.K. McHutchison J.G. Sulkowski M.S. Serum cholesterol and statin use predict virological response to peginterferon and ribavirin therapy.Hepatology. 2010; 52 (20568303): 864-87410.1002/hep.23787Crossref PubMed Scopus (111) Google Scholar). Several studies of chronic HCV patients have confirmed that the presence of HCV affects host sterol metabolism (9Fabris C. Federico E. Soardo G. Falleti E. Pirisi M. Blood lipids of patients with chronic hepatitis: differences related to viral etiology.Clin. Chim. Acta. 1997; 261 (9201435): 159-16510.1016/S0009-8981(97)06532-7Crossref PubMed Scopus (71) Google Scholar, 10Jármay K. Karácsony G. Nagy A. Schaff Z. Changes in lipid metabolism in chronic hepatitis C.World J. Gastroenterol. 2005; 11 (16425410): 6422-642810.3748/wjg.v11.i41.6422Crossref PubMed Scopus (31) Google Scholar, 11Asselah T. Rubbia-Brandt L. Marcellin P. Negro F. Steatosis in chronic hepatitis C: why does it really matter?.Gut. 2006; 55 (16344578): 123-13010.1136/gut.2005.069757Crossref PubMed Scopus (311) Google Scholar, 12Corey K.E. Kane E. Munroe C. Barlow L.L. Zheng H. Chung R.T. Hepatitis C virus infection and its clearance alter circulating lipids: implications for long-term follow-up.Hepatology. 2009; 50 (19787818): 1030-103710.1002/hep.23219Crossref PubMed Scopus (127) Google Scholar, 13Harrison S.A. Rossaro L. Hu K.-Q. Patel K. Tillmann H. Dhaliwal S. Torres D.M. Koury K. Goteti V.S. Noviello S. Brass C.A. Albrecht J.K. McHutchison J.G. Sulkowski M.S. Serum cholesterol and statin use predict virological response to peginterferon and ribavirin therapy.Hepatology. 2010; 52 (20568303): 864-87410.1002/hep.23787Crossref PubMed Scopus (111) Google Scholar). Both proteomic and lipidomic profiling by targeted liquid chromatography - mass spectrometry (LC-MS) have shown that HCV perturbs multiple host lipid biosynthetic pathways (14Diamond D.L. Syder A.J. Jacobs J.M. Sorensen C.M. Walters K.-A. Proll S.C. McDermott J.E. Gritsenko M.A. Zhang Q. Zhao R. Metz T.O. Camp D.G. Waters K.M. Smith R.D. Rice C.M. et al.Temporal proteome and lipidome profiles reveal hepatitis C virus–associated reprogramming of hepatocellular metabolism and bioenergetics.PLoS Pathog. 2010; 6 (20062526)e100071910.1371/journal.ppat.1000719Crossref PubMed Scopus (283) Google Scholar). Additionally, unbiased lipidomic MS profiling by our laboratory previously discovered that HCV causes a 10-fold increase in intracellular desmosterol, an immediate precursor to cholesterol, without affecting the abundance of cholesterol (15Rodgers M.A. Villareal V.A. Schaefer E.A. Peng L.F. Corey K.E. Chung R.T. Yang P.L. Lipid metabolite profiling identifies desmosterol metabolism as a new antiviral target for hepatitis C virus.J. Am. Chem. Soc. 2012; 134 (22480142): 6896-689910.1021/ja207391qCrossref PubMed Scopus (35) Google Scholar, 16Villareal V.A. Fu D. Costello D.A. Xie X.S. Yang P.L. Hepatitis C virus selectively alters the intracellular localization of desmosterol.ACS Chem. Biol. 2016; 11 (27128812): 1827-183310.1021/acschembio.6b00324Crossref PubMed Scopus (13) Google Scholar, 17Costello D.A. Villareal V.A. Yang P.L. Desmosterol increases lipid bilayer fluidity during hepatitis C virus infection.ACS Infect. Dis. 2016; 2 (27933788): 852-86210.1021/acsinfecdis.6b00086Crossref PubMed Scopus (9) Google Scholar). We demonstrated that the HCV-induced accumulation of desmosterol is functionally important for HCV replication, as evidenced by strong reduction of accumulated HCV RNA when desmosterol is depleted from cells and restoration of HCV RNA levels upon addition of exogenous desmosterol but not cholesterol (15Rodgers M.A. Villareal V.A. Schaefer E.A. Peng L.F. Corey K.E. Chung R.T. Yang P.L. Lipid metabolite profiling identifies desmosterol metabolism as a new antiviral target for hepatitis C virus.J. Am. Chem. Soc. 2012; 134 (22480142): 6896-689910.1021/ja207391qCrossref PubMed Scopus (35) Google Scholar, 17Costello D.A. Villareal V.A. Yang P.L. Desmosterol increases lipid bilayer fluidity during hepatitis C virus infection.ACS Infect. Dis. 2016; 2 (27933788): 852-86210.1021/acsinfecdis.6b00086Crossref PubMed Scopus (9) Google Scholar). However, how HCV regulates host metabolism to cause an increase in the steady-state abundance of desmosterol remains unknown. Here, we establish a mechanism used by HCV to regulate desmosterol abundance. The mechanism involves post-translational regulation of the key enzyme in desmosterol metabolism, 24-dehydrocholesterol reductase (DHCR24), which converts desmosterol to cholesterol. Our study reveals that DHCR24 is proteolytically cleaved between Cys91 and Thr92 by HCV NS3-4A protease, resulting in inactivation of DHCR24 and accumulation of desmosterol in the replication membranes where NS3-4A and DHCR24 co-reside. This, in turn, is associated with robust HCV replication. Together, our studies suggest a model in which the HCV NS3-4A protease remodels the host lipid membrane by directly interacting with and cleaving a host biosynthetic enzyme to generate a chemical environment that promotes increased viral RNA replication. Desmosterol is produced in the Bloch branch of late-stage cholesterol biosynthesis by reduction of 7-dehydrodesmosterol by 7-dehydrodesmosterol reductase (DHCR7). Under normal physiological conditions, desmosterol does not accumulate and is rapidly converted to cholesterol by DHCR24 (Fig. 1A). Because HCV has no known gene products capable of catalyzing lipid synthesis or metabolism, we investigated whether HCV perturbs the expression of DHCR7 and DHCR24. For these experiments, we used autonomously replicating RNAs encoding either the full-length HCV polyprotein (full-length genomic replicon, or FGR) or encoding only the NS3-5B proteins (subgenomic replicon, or SGR) (Fig. 1B) (18Kato T. Date T. Miyamoto M. Furusaka A. Tokushige K. Mizokami M. Wakita T. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon.Gastroenterology. 2003; 125 (14724833): 1808-181710.1053/j.gastro.2003.09.023Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar). These and other replicon systems are well-established models for studying HCV gene expression and RNA replication in the absence of the complicating effects of viral entry, assembly, or egress. We first quantified DHCR7 and DHCR24 transcripts by reverse-transcription quantitative PCR (RT-qPCR) assay and found that the abundance of these transcripts is unaffected by the presence or absence of the HCV full-genomic and subgenomic replicon RNAs (Fig. 1C). This finding is consistent with transcript profiling studies showing that HCV infection induces no changes in DHCR7 or DHCR24 mRNA abundance (19Blackham S. Baillie A. Al-Hababi F. Remlinger K. You S. Hamatake R. McGarvey M.J. Gene expression profiling indicates the roles of host oxidative stress, apoptosis, lipid metabolism, and intracellular transport genes in the replication of hepatitis C virus.J. Virol. 2010; 84 (20200238): 5404-541410.1128/JVI.02529-09Crossref PubMed Scopus (100) Google Scholar, 20Bigger C.B. Guerra B. Brasky K.M. Hubbard G. Beard M.R. Luxon B.A. Lemon S.M. Lanford R.E. Intrahepatic gene expression during chronic hepatitis C virus infection in chimpanzees.J. Virol. 2004; 78 (15564486): 13779-1379210.1128/JVI.78.24.13779-13792.2004Crossref PubMed Scopus (229) Google Scholar) and indicates that the HCV-associated increase in intracellular desmosterol is not due to viral perturbation at the mRNA level. Next, we examined the steady-state abundance of DHCR7 or DHCR24 proteins by immunoblot analysis of whole cell lysates and detected no differences between cells harboring HCV replicon RNAs and negative control cells (Fig. 1D). Although we cannot exclude the possibility that noncoding RNAs might regulate translation of DHCR7 and/or DHCR24 mRNAs, this does not appear to happen to a significant extent based on the steady-state abundance of DHCR7 and DHCR24 proteins. These data show that HCV's effect on desmosterol homeostasis does not involve changes in the expression or abundance of DHCR7 and DHCR24. This led us to consider the possibility that HCV increases intracellular desmosterol by either increasing DHCR7 activity or decreasing DHCR24 activity. Because neither enzyme is known to be rate-determining for the pathway and because desmosterol is rapidly converted to cholesterol under normal physiological conditions, we deemed a virus-associated reduction in the DHCR24-catalyzed conversion of desmosterol to cholesterol to be the more likely scenario. To investigate this possibility, cells harboring HCV replicon RNAs and negative control cells were treated with medium containing deuterated desmosterol (desmosterol-d6), and conversion of the desmosterol-d6 to cholesterol-d6 was monitored over time by LC-MS analysis of extracted lipidomes. Conversion of desmosterol-d6 to cholesterol-d6 was reduced in cells harboring the HCV subgenomic replicon or treated with U18666A, a small molecule known to inhibit DHCR24's enzymatic activity and intracellular sterol transport (21Takano T. Tsukiyama-Kohara K. Hayashi M. Hirata Y. Satoh M. Tokunaga Y. Tateno C. Hayashi Y. Hishima T. Funata N. Sudoh M. Kohara M. Augmentation of DHCR24 expression by hepatitis C virus infection facilitates viral replication in hepatocytes.J. Hepatol. 2011; 55 (21184787): 512-52110.1016/j.jhep.2010.12.011Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 22Bierkamper G.G. Cenedella R.J. Induction of chronic epileptiform activity in the rat by an inhibitor of cholesterol synthesis, U18666A.Brain Res. 1978; 150 (678974): 343-35110.1016/0006-8993(78)90285-8Crossref PubMed Scopus (28) Google Scholar, 23Bae S.H. Paik Y.K. Cholesterol biosynthesis from lanosterol: development of a novel assay method and characterization of rat liver microsomal lanosterol Δ24-reductase.Biochem. J. 1997; 326 (9291139): 609-61610.1042/bj3260609Crossref PubMed Scopus (67) Google Scholar, 24Liscum L. Faust J.R. The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-β-[2-(diethylamino)ethoxy]androst-5-en-17-one.J. Biol. Chem. 1989; 264 (2745416): 11796-11806Abstract Full Text PDF PubMed Google Scholar), when compared with cells lacking the replicon (Mock) (Fig. 1E). This observation supports the proposal that HCV negatively regulates DHCR24 activity. Because HCV has no effect on DHCR7 or DHCR24 expression or abundance yet appears to affect the DHCR24-catalyzed reaction in cells, we next investigated whether DHCR24 is post-translationally modified in the presence of the virus. Immunoblot analysis of cells transfected with a C-terminal FLAG-tagged DHCR7 or DHCR24 (DHCR7–FLAG and DHCR24–FLAG respectively) revealed an additional, faster-migrating DHCR24-FLAG species in the presence of HCV SGR (Fig. 2A). We confirmed that formation of this faster-migrating DHCR24 species (DHCR24*) was not an artifact of ectopic expression of DHCR24–FLAG by demonstrating that DHCR24* is detected when endogenous DHCR24 is immunoprecipitated from cells harboring the SGR (Fig. 2B). To map which of the HCV proteins expressed by the subgenomic replicon (NS3, NS4A, NS4B, NS5A, and NS5B) is sufficient to induce formation of DHCR24*, we first co-expressed NS3-4A and NS5A individually with DHCR24–FLAG (Fig. 2C). NS3 contains a C-terminal helicase domain and an N-terminal serine protease domain that requires its cofactor NS4A for catalytic activity (25Raney K.D. Sharma S.D. Moustafa I.M. Cameron C.E. Hepatitis C virus non-structural protein 3 (HCV NS3): a multifunctional antiviral target.J. Biol. Chem. 2010; 285 (20457607): 22725-2273110.1074/jbc.R110.125294Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Although no inherent enzymatic function has been reported for NS5A, it is known to affect host lipids through interaction with phosphatidylinositol 4-kinase α (8Reiss S. Rebhan I. Backes P. Romero-Brey I. Erfle H. Matula P. Kaderali L. Poenisch M. Blankenburg H. Hiet M.-S. Longerich T. Diehl S. Ramirez F. Balla T. Rohr K. et al.Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment.Cell Host Microbe. 2011; 9 (21238945): 32-4510.1016/j.chom.2010.12.002Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar) and is hypothesized to have a significant role in coordinating genome replication and viral assembly (26Yin C. Goonawardane N. Stewart H. Harris M. A role for domain I of the hepatitis C virus NS5A protein in virus assembly.PLoS Pathog. 2018; 14 (29352312)e100683410.1371/journal.ppat.1006834Crossref PubMed Scopus (21) Google Scholar). We found that expression of the NS3-4A protease is sufficient to induce formation of DHCR24*–FLAG. Production of DHCR24*–FLAG requires active protease activity because no DHCR24*–FLAG is produced in the presence of the NS3-4A inhibitor danoprevir or when NS3 is expressed alone without NS4A. Further, production of DHCR24* is lost when a catalytic residue in the protease active site is mutated (NS3-4A–H57A) (28Grakoui A. McCourt D.W. Wychowski C. Feinstone S.M. Rice C.M. Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites.J. Virol. 1993; 67 (8386278): 2832-284310.1128/JVI.67.5.2832-2843.1993Crossref PubMed Google Scholar, 29Kolykhalov A.A. Mihalik K. Feinstone S.M. Rice C.M. Hepatitis C virus–encoded enzymatic activities and conserved RNA elements in the 3´ nontranslated region are essential for virus replication in vivo.J. Virol. 2000; 74 (10644379): 2046-205110.1128/jvi.74.4.2046-2051.2000Crossref PubMed Scopus (554) Google Scholar, 30Tsantrizos Y.S. Bolger G. Bonneau P. Cameron D.R. Goudreau N. Kukolj G. LaPlante S.R. Llinàs-Brunet M. Nar H. Lamarre D. Macrocyclic inhibitors of the NS3 protease as potential therapeutic agents of hepatitis C virus infection.Angew. Chem. Int. Ed. Engl. 2003; 42 (12671967): 1356-136010.1002/anie.200390347Crossref PubMed Scopus (155) Google Scholar) but is unaffected by a point mutation introduced to the NS3 helicase active site (NS3-4A–D290A) (29Kolykhalov A.A. Mihalik K. Feinstone S.M. Rice C.M. Hepatitis C virus–encoded enzymatic activities and conserved RNA elements in the 3´ nontranslated region are essential for virus replication in vivo.J. Virol. 2000; 74 (10644379): 2046-205110.1128/jvi.74.4.2046-2051.2000Crossref PubMed Scopus (554) Google Scholar) (Fig. 2D). Together, these results demonstrate that formation of DHCR24* requires the active NS3-4A serine protease domain. We further validated that the active NS3-4A protease is sufficient to generate DHCR24* by immunoprecipitating DHCR24–FLAG from uninfected cells and incubating this protein with recombinant NS3-4A. This successfully recapitulated formation of DHCR24*–FLAG in vitro (Fig. 2E). This in vitro reaction is blocked in the presence of telaprevir, a Food and Drug Administration–approved NS3-4A inhibitor, or a competitive peptide mimetic (31Lin C. Kwong A.D. Perni R.B. Discovery and development of VX-950, a novel, covalent, and reversible inhibitor of hepatitis C virus NS3.4A serine protease.Infect. Disord. Drug Targets. 2006; 6 (16787300): 3-1610.2174/187152606776056706Crossref PubMed Scopus (208) Google Scholar, 32Kwong A.D. Kauffman R.S. Hurter P. Mueller P. Discovery and development of telaprevir: an NS3-4A protease inhibitor for treating genotype 1 chronic hepatitis C virus.Nat. Biotechnol. 2011; 29 (22068541): 993-100310.1038/nbt.2020Crossref PubMed Scopus (201) Google Scholar) (Fig. 2E). Taken together, these experiments demonstrate that the NS3-4A protease causes production of DHCR24*. To examine the possibility that NS3-4A directly cleaves DHCR24, we isolated the two DHCR24 species observed by SDS-PAGE following incubation of immunoprecipitated DHCR24–FLAG with recombinant NS3-4A protease in vitro. “Bottom-up” LC-MS/MS analysis of the isolated species demonstrated that both bear the FLAG-derived DYKDDDDK sequence and thus are derived from DHCR24–FLAG (Fig. S1). Likewise, LC-MS/MS analysis of endogenous DHCR24 immunoprecipitated from cells harboring the HCV subgenomic replicon verified that both products are derived from DHCR24 (Fig. 2B). These results indicate that DHCR24 is a substrate for NS3-4A–mediated proteolysis. Due to several technical issues, including difficulty in driving complete conversion of immunoprecipitated DHCR24 to DHCR24*, poor ionization of the reaction products, and limited recovery of recombinant DHCR24 proteins due to high hydrophobicity, we were unable to identify the position of the cleavage site by LC-MS/MS. To facilitate expression and purification of DHCR24, we fused it to a maltose-binding protein (MBP) at its N terminus. We then expressed the MBP–DHCR24 fusion with a bacteria-derived cell-free, in vitro translation system (33Shimizu Y. Inoue A. Tomari Y. Suzuki T. Yokogawa T. Nishikawa K. Ueda T. Cell-free translation reconstituted with purified components.Nat. Biotechnol. 2001; 19 (11479568): 751-75510.1038/90802Crossref PubMed Scopus (1173) Google Scholar) and purified it by using magnetic amylose resin. Incubation of recombinant NS3-4A with the MBP–DHCR24 from this heterologous expression system produced a species akin to DHCR24* from HCV replicon cells (Fig. S2). Immunoblotting with an antibody that recognizes an epitope in the region between 68 and 85 of DHCR24 suggested that the cleavage site is located between the putative transmembrane region (residue 52) and the catalytic region (residue 110) of DHCR24 (Fig. 3A). To specifically investigate the 56–110 region of DHCR24 as a substrate for NS3-4A, a surrogate peptide spanning this region was expressed as a fusion between the soluble proteins MBP and GFP, with a FLAG tag at the C terminus (Fig. 3B). Incubation of this purified test substrate with recombinant NS3-4A produces cleavage fragments (Fig. 3C) that are not observed in the absence of NS3-4A or in the presence of the NS3-4A inhibitor telaprevir. Next, we used two 15-mer surrogate peptides corresponding to residues 52–66 (Fig. 3D) and 85–99 (Fig. 3E) of DHCR24 to map the NS3-4A proteolysis site. Reaction of recombinant NS3-4A with the substrate spanning residues 85–99 led to two distinct products and a proportionate decrease in the full-length substrate. We purified the two product species and subjected them to N-terminal sequencing by Edman degradation. This revealed an N-terminal sequence of TGRPG, corresponding to the sequence of DHCR24 beginning at Thr92, and suggested that the peptide bond between residues Cys91 and Thr92 is the target of NS3-4A cleavage (Fig. 3E). Consistent with this hypothesis, NS3-4A is well-known to have a strong preference for Cys in the P1 site, and we found that replacement of Pro with Cys, a substitution known to inhibit proteolysis of membrane-bound enzymes by proteases (34Frey S. Görlich D. A new set of highly efficient, tag-cleaving proteases for purifying recombinant proteins.J. Chromatogr. A. 2014; 1337 (24636565): 95-10510.1016/j.chroma.2014.02.029Crossref PubMed Scopus (48) Google Scholar, 35Wörmann M.E. Reichmann N.T. Malone C.L. Horswill A.R. Gründling A. Proteolytic cleavage inactivates the Staphylococcus aureus lipoteichoic acid synthase.J. Bacteriol. 2011; 193 (21784926): 5279-529110.1128/JB.00369-11Crossref PubMed Scopus (67) Google Scholar), prevented cleavage of the MBP–DHCR24(52–66)-GFP-FLAG substrate by NS3-4A in vitro (Fig. 3F). To examine the effect of proteolysis of DHCR24 by NS3-4A on the conversion of desmosterol to cholesterol in cells, we performed genome editing to “knock out” DHCR24 expression in Huh7.5 cells (Fig. S3) and then monitored the fate of desmosterol-d6 in cells ectopically expressing DHCR24–FLAG. As expected, WT DHCR24–FLAG supports conversion of desmosterol-d6 to cholesterol-d6 in Huh7.5-DHCR24KO cells. Co-expression of recombinant NS3-4A reduces the conversion of desmosterol-d6 to cholesterol-d6 to approximately half of the conversion observed in the control cells expressing only DHCR24–FLAG (Fig. 4A). This reduction in intracellular DHCR24 activity is furthermore associated with proteolytic cleavage of DHCR24 by the viral protease, as reflected by the appearance of a species corresponding to DHCR24*-FLAG (Fig. 4B). Importantly, the conversion of desmosterol-d6 to cholesterol-d6 is far less affected when telaprevir is used to inhibit NS3-4A protease activity or when the inactive NS3(H57A)-4A mutant is expressed (Fig. 4A). Conversion of desmosterol-d6 to cholesterol-d6 in these protease-inhibited samples does not differ in a statistically significant manner from the control samples in which DHCR24-FLAG is expressed alone without NS3-4A present. In contrast, the telaprevir-treated and NS3(H57A)-4A mutant samples do exhibit significantly reduced conversion of desmosterol-d6 to cholesterol-d6 compared to the samples in which NS3-4A and DHCR24 are co-expressed. These data thus provide a link between proteolytic activity of NS3-4A, cleavage of DHCR24, and changes in the DHCR24-catalyzed conversion of desmosterol to cholesterol. Since cleavage of DHCR24 between Cys91 and Thr92 is predicted to separate the N-terminal membrane-associated domain from the FAD-binding and catalytic domain, we hypothesized that this might affect DHCR24-catalyzed conversion of desmosterol to cholesterol by releasing the catalytic domain from the ER membrane. Supporting t" @default.
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• W3040697948 date "2020-08-01" @default.
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• W3040697948 title "Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase" @default.
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• W3040697948 doi "https://doi.org/10.1074/jbc.ra120.013455" @default.
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