{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T19:53:28Z","timestamp":1766087608176,"version":"build-2065373602"},"reference-count":63,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2020,9,18]],"date-time":"2020-09-18T00:00:00Z","timestamp":1600387200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon double reporter cell line suitable for testing different antiviral drugs and therapeutic interventions. This cells line allowed a rapid and accurate quantification of cell growth\/viability and HCV RNA replication, thus discriminating specific from unspecific antiviral effects caused by DAAs or cytotoxic compounds, respectively. By correlating cell number and virus replication, we could confirm the inhibitory effect on the latter of cell over confluency and characterize an array of lentiviral vectors expressing single, double, or triple cassettes containing different combinations of short hairpin (sh)RNAs, targeting both highly conserved viral genome sequences and cellular factors crucial for HCV replication. While all vectors were effective in reducing HCV replication, the ones targeting viral sequences displayed a stronger antiviral effect, without significant cytopathic effects. Such combinatorial platforms as well as the developed double reporter cell line might find application both in setting-up anti-HCV gene therapy approaches and in studies aimed at further dissecting the viral biology\/pathogenesis of infection.<\/jats:p>","DOI":"10.3390\/v12091044","type":"journal-article","created":{"date-parts":[[2020,9,18]],"date-time":"2020-09-18T10:22:23Z","timestamp":1600424543000},"page":"1044","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line"],"prefix":"10.3390","volume":"12","author":[{"given":"Hossein M.","family":"Elbadawy","sequence":"first","affiliation":[{"name":"Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mohi I.","family":"Mohammed Abdul","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Naif","family":"Aljuhani","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Adriana","family":"Vitiello","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, University of Padua, 35121 Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7237-6942","authenticated-orcid":false,"given":"Francesco","family":"Ciccarese","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, University of Padua, 35121 Padua, Italy"},{"name":"Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mohamed A.","family":"Shaker","sequence":"additional","affiliation":[{"name":"Pharmaceutics and Pharmaceutical Technology Department, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi Arabia"},{"name":"Pharmaceutics Department, Faculty of Pharmacy, Helwan University, Cairo 11795, Egypt"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Heba M.","family":"Eltahir","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Giorgio","family":"Pal\u00f9","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, University of Padua, 35121 Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Veronica","family":"Di Antonio","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, University of Padua, 35121 Padua, Italy"},{"name":"Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hanieh","family":"Ghassabian","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, University of Padua, 35121 Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5645-9126","authenticated-orcid":false,"given":"Claudia","family":"Del Vecchio","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, University of Padua, 35121 Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5136-7406","authenticated-orcid":false,"given":"Cristiano","family":"Salata","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, University of Padua, 35121 Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elisa","family":"Franchin","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, University of Padua, 35121 Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eleonora","family":"Ponterio","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, University of Padua, 35121 Padua, Italy"},{"name":"Fondazione Policlinico Universitario \"A. Gemelli\"\u2014I.R.C.C.S., 00168 Rome, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Saleh","family":"Bahashwan","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Khaled","family":"Thabet","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Faculty of Pharmacy, Minia University, Minia 61519, Egypt"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mekky M.","family":"Abouzied","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi Arabia"},{"name":"Department of Biochemistry, Faculty of Pharmacy, Minia University, Minia 61519, Egypt"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ahmed M.","family":"Shehata","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi Arabia"},{"name":"Department of Pharmacology and toxicology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62511, Egypt"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Cristina","family":"Parolin","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, University of Padua, 35121 Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Arianna","family":"Calistri","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, University of Padua, 35121 Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8177-3616","authenticated-orcid":false,"given":"Gualtiero","family":"Alvisi","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, University of Padua, 35121 Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,9,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1483","DOI":"10.1056\/NEJMoa1316366","article-title":"Ledipasvir and sofosbuvir for previously treated HCV genotype 1 infection","volume":"370","author":"Afdhal","year":"2014","journal-title":"N. Engl. J. Med."},{"key":"ref_2","first-page":"256","article-title":"Ledipasvir\/Sofosbuvir (harvoni): Improving options for hepatitis C virus infection","volume":"40","author":"Gritsenko","year":"2015","journal-title":"Pharm. Ther."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"244","DOI":"10.1038\/418244a","article-title":"RNA interference","volume":"418","author":"Hannon","year":"2002","journal-title":"Nature"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1038\/nbt936","article-title":"Rational siRNA design for RNA interference","volume":"22","author":"Reynolds","year":"2004","journal-title":"Nat. Biotechnol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1186\/1479-0556-9-15","article-title":"HCV entry receptors as potential targets for siRNA-based inhibition of HCV","volume":"9","author":"Jahan","year":"2011","journal-title":"Genet. Vaccines"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"669","DOI":"10.1128\/JVI.01496-06","article-title":"Small interfering RNA targeted to hepatitis C virus 5\u2019 nontranslated region exerts potent antiviral effect","volume":"81","author":"Kanda","year":"2007","journal-title":"J. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"433","DOI":"10.1007\/s00705-010-0887-6","article-title":"Inhibition of hepatitis C virus genotype 3a by siRNAs targeting envelope genes","volume":"156","author":"Khaliq","year":"2011","journal-title":"Arch. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Moon, J.S., Lee, S.H., Kim, E.J., Cho, H., Lee, W., Kim, G.W., Park, H.J., Cho, S.W., Lee, C., and Oh, J.W. (2016). Inhibition of hepatitis C virus in mice by a small interfering RNA targeting a highly conserved sequence in viral IRES pseudoknot. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0146710"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"12884","DOI":"10.1073\/pnas.0704894104","article-title":"Cellular cofactors affecting hepatitis C virus infection and replication","volume":"104","author":"Randall","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/978-1-4939-2432-5_1","article-title":"Gene therapies for hepatitis C virus","volume":"848","author":"Verstegen","year":"2015","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2783","DOI":"10.1073\/pnas.252758799","article-title":"RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells","volume":"100","author":"Wilson","year":"2003","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1965","DOI":"10.3889\/oamjms.2018.372","article-title":"Silencing HCV replication in its reservoir","volume":"6","author":"Youssef","year":"2018","journal-title":"Open Access Maced. J. Med. Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1007\/s12192-016-0747-8","article-title":"Inhibition of hepatitis C virus using siRNA targeted to the virus and Hsp90","volume":"22","author":"Braga","year":"2017","journal-title":"Cell Stress Chaperones"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1724","DOI":"10.1038\/mt.2012.107","article-title":"Inhibition of hepatitis C virus replication by intracellular delivery of multiple siRNAs by nanosomes","volume":"20","author":"Chandra","year":"2012","journal-title":"Mol. Ther."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1016\/j.jhep.2005.02.046","article-title":"Inhibition of hepatitis C virus translation and subgenomic replication by siRNAs directed against highly conserved HCV sequence and cellular HCV cofactors","volume":"43","author":"Korf","year":"2005","journal-title":"J. Hepatol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1737","DOI":"10.1038\/mt.2012.119","article-title":"Safe, long-term hepatic expression of anti-HCV shRNA in a nonhuman primate model","volume":"20","author":"Suhy","year":"2012","journal-title":"Mol. Ther."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Reiss, S., Harak, C., Romero-Brey, I., Radujkovic, D., Klein, R., Ruggieri, A., Rebhan, I., Bartenschlager, R., and Lohmann, V. (2013). The lipid kinase phosphatidylinositol-4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A. PLoS Pathog., 9.","DOI":"10.1371\/journal.ppat.1003359"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1016\/j.chom.2010.12.002","article-title":"Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment","volume":"9","author":"Reiss","year":"2011","journal-title":"Cell Host Microbe"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1638","DOI":"10.1002\/hep.23281","article-title":"Cyclosporine A inhibits hepatitis C virus nonstructural protein 2 through cyclophilin A","volume":"50","author":"Ciesek","year":"2009","journal-title":"Hepatology"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Kaul, A., Stauffer, S., Berger, C., Pertel, T., Schmitt, J., Kallis, S., Zayas, M., Lopez, M.Z., Lohmann, V., and Luban, J. (2009). Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics. PLoS Pathog., 5.","DOI":"10.1371\/annotation\/29dffb2a-5d37-4196-b166-b0b15bc2f3fd"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"6554","DOI":"10.1128\/JVI.02550-08","article-title":"Critical role of cyclophilin A and its prolyl-peptidyl isomerase activity in the structure and function of the hepatitis C virus replication complex","volume":"83","author":"Liu","year":"2009","journal-title":"J. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"5269","DOI":"10.1128\/JVI.02614-07","article-title":"Cyclophilin A is an essential cofactor for hepatitis C virus infection and the principal mediator of cyclosporine resistance in vitro","volume":"82","author":"Yang","year":"2008","journal-title":"J. Virol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1577","DOI":"10.1126\/science.1113329","article-title":"Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA","volume":"309","author":"Jopling","year":"2005","journal-title":"Science"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"3300","DOI":"10.1038\/emboj.2008.244","article-title":"microRNA-122 stimulates translation of hepatitis C virus RNA","volume":"27","author":"Henke","year":"2008","journal-title":"EMBO J."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1220","DOI":"10.1042\/BST0361220","article-title":"Regulation of hepatitis C virus by microRNA-122","volume":"36","author":"Jopling","year":"2008","journal-title":"Biochem. Soc. Trans."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"7372","DOI":"10.1128\/JVI.00513-12","article-title":"Base pairing between hepatitis C virus RNA and microRNA 122 3\u2019 of its seed sequence is essential for genome stabilization and production of infectious virus","volume":"86","author":"Shimakami","year":"2012","journal-title":"J. Virol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.antiviral.2014.08.015","article-title":"Long-term safety and efficacy of microRNA-targeted therapy in chronic hepatitis C patients","volume":"111","author":"Maan","year":"2014","journal-title":"Antivir. Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"829","DOI":"10.1111\/apt.13342","article-title":"Randomised clinical trial: Alisporivir combined with peginterferon and ribavirin in treatment-naive patients with chronic HCV genotype 1 infection (ESSENTIAL II)","volume":"42","author":"Zeuzem","year":"2015","journal-title":"Aliment. Pharm."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Bianco, A., Reghellin, V., Donnici, L., Fenu, S., Alvarez, R., Baruffa, C., Peri, F., Pagani, M., Abrignani, S., and Neddermann, P. (2012). Metabolism of phosphatidylinositol 4-kinase IIIalpha-dependent PI4P Is subverted by HCV and is targeted by a 4-anilino quinazoline with antiviral activity. PLoS Pathog., 8.","DOI":"10.1371\/journal.ppat.1002576"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"962","DOI":"10.1002\/hep.20819","article-title":"Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes","volume":"42","author":"Simmonds","year":"2005","journal-title":"Hepatology"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"12047","DOI":"10.1128\/JVI.75.24.12047-12057.2001","article-title":"Sequences in the 5\u2019 nontranslated region of hepatitis C virus required for RNA replication","volume":"75","author":"Friebe","year":"2001","journal-title":"J. Virol."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"ElHefnawi, M., Kim, T., Kamar, M.A., Min, S., Hassan, N.M., El-Ahwany, E., Kim, H., Zada, S., Amer, M., and Windisch, M.P. (2016). In silico design and experimental validation of siRNAs targeting conserved regions of multiple hepatitis c virus genotypes. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0159211"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e312","DOI":"10.1038\/mtna.2016.24","article-title":"Development of lentiviral vectors simultaneously expressing multiple siRNAs against CCR5, vif and tat\/rev genes for an HIV-1 gene therapy approach","volume":"5","author":"Spanevello","year":"2016","journal-title":"Mol. Nucleic Acids"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"6866","DOI":"10.1021\/bi060086u","article-title":"Human cytomegalovirus (HCMV) DNA polymerase processivity factor ppUL44 dimerizes in the cytosol before translocation to the nucleus","volume":"45","author":"Alvisi","year":"2006","journal-title":"Biochemistry"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1455","DOI":"10.1042\/BCJ20170897","article-title":"Intersectin goes nuclear: Secret life of an endocytic protein","volume":"475","author":"Alvisi","year":"2018","journal-title":"Biochem. J."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1114","DOI":"10.1016\/j.bbamcr.2018.05.006","article-title":"Contribution of the residue at position 4 within classical nuclear localization signals to modulating interaction with importins and nuclear targeting","volume":"1865","author":"Smith","year":"2018","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1391","DOI":"10.1053\/j.gastro.2008.12.034","article-title":"Analysis of CD8+ T-cell-mediated inhibition of hepatitis C virus replication using a novel immunological model","volume":"136","author":"Jo","year":"2009","journal-title":"Gastroenterology"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"3406","DOI":"10.1093\/nar\/gkg595","article-title":"Mfold web server for nucleic acid folding and hybridization prediction","volume":"31","author":"Zuker","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Trevisan, M., Di Antonio, V., Radeghieri, A., Palu, G., Ghildyal, R., and Alvisi, G. (2018). Molecular requirements for self-interaction of the respiratory syncytial virus matrix protein in living mammalian cells. Viruses, 10.","DOI":"10.3390\/v10030109"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"380","DOI":"10.1128\/JVI.79.1.380-392.2005","article-title":"Kissing-loop interaction in the 3\u2019 end of the hepatitis C virus genome essential for RNA replication","volume":"79","author":"Friebe","year":"2005","journal-title":"J. Virol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/j.virol.2014.05.016","article-title":"The lysine methyltransferase SMYD3 interacts with hepatitis C virus NS5A and is a negative regulator of viral particle production","volume":"462\u2013463","author":"Eberle","year":"2014","journal-title":"Virology"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1016\/j.chom.2012.05.013","article-title":"Dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection","volume":"12","author":"Ruggieri","year":"2012","journal-title":"Cell Host Microbe"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1038\/nature11289","article-title":"Viral immune modulators perturb the human molecular network by common and unique strategies","volume":"487","author":"Pichlmair","year":"2012","journal-title":"Nature"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1126\/science.285.5424.110","article-title":"Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line","volume":"285","author":"Lohmann","year":"1999","journal-title":"Science"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"2902","DOI":"10.1093\/nar\/20.11.2902","article-title":"Electroporation in \u2018intracellular\u2019 buffer increases cell survival","volume":"20","author":"Moorman","year":"1992","journal-title":"Nucleic Acids Res."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"e179","DOI":"10.1093\/nar\/gni178","article-title":"Real-time quantification of microRNAs by stem-loop RT-PCR","volume":"33","author":"Chen","year":"2005","journal-title":"Nucleic Acids Res."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"9567","DOI":"10.1128\/JVI.00669-09","article-title":"The flexible loop of the human cytomegalovirus DNA polymerase processivity factor ppUL44 is required for efficient DNA binding and replication in cells","volume":"83","author":"Alvisi","year":"2009","journal-title":"J. Virol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1038\/ng1117","article-title":"A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference","volume":"33","author":"Rubinson","year":"2003","journal-title":"Nat. Genet."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1181","DOI":"10.1128\/JVI.80.3.1181-1190.2006","article-title":"Effect of cell growth on hepatitis C virus (HCV) replication and a mechanism of cell confluence-based inhibition of HCV RNA and protein expression","volume":"80","author":"Nelson","year":"2006","journal-title":"J. Virol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"34337","DOI":"10.1074\/jbc.M110.161802","article-title":"Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977","volume":"285","author":"Murakami","year":"2010","journal-title":"J. Biol. Chem."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1111\/jvh.13005","article-title":"The comprehensive outcomes of hepatitis C virus infection: A multi-faceted chronic disease","volume":"25","author":"Younossi","year":"2018","journal-title":"J. Viral Hepat."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"114","DOI":"10.7326\/0003-4819-158-2-201301150-00576","article-title":"Comparative effectiveness of antiviral treatment for hepatitis C virus infection in adults: A systematic review","volume":"158","author":"Chou","year":"2013","journal-title":"Ann. Intern. Med."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1016\/j.jhep.2019.04.017","article-title":"Sustained virologic response to direct-acting antiviral therapy in patients with chronic hepatitis C and hepatocellular carcinoma: A systematic review and meta-analysis","volume":"71","author":"Ji","year":"2019","journal-title":"J. Hepatol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"831","DOI":"10.1007\/s00705-013-1803-7","article-title":"Current and future therapies for hepatitis C virus infection: From viral proteins to host targets","volume":"159","author":"Imran","year":"2014","journal-title":"Arch. Virol."},{"key":"ref_55","first-page":"99","article-title":"High-efficiency generation of induced pluripotent mesenchymal stem cells from human dermal fibroblasts using recombinant proteins","volume":"7","author":"Chen","year":"2016","journal-title":"Stem Cell Res."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"2207","DOI":"10.2147\/IDR.S172226","article-title":"Baseline hepatitis C virus resistance-associated substitutions present at frequencies lower than 15% may be clinically significant","volume":"11","author":"Perales","year":"2018","journal-title":"Infect. Drug Resist."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"756","DOI":"10.1111\/j.1365-2893.2006.00752.x","article-title":"siRNA-resistance in treated HCV replicon cells is correlated with the development of specific HCV mutations","volume":"13","author":"Konishi","year":"2006","journal-title":"J. Viral Hepat."},{"key":"ref_58","first-page":"9671031","article-title":"Combinations of siRNAs against La Autoantigen with NS5B or hVAP-A have additive effect on inhibition of HCV replication","volume":"2016","author":"Mandal","year":"2016","journal-title":"Hepat. Res. Treat."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"9739","DOI":"10.1073\/pnas.0504296102","article-title":"Efficient hepatitis C virus cell culture system: What a difference the host cell makes","volume":"102","author":"Bartenschlager","year":"2005","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"3075","DOI":"10.1099\/vir.0.81334-0","article-title":"Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon","volume":"86","author":"McLauchlan","year":"2005","journal-title":"J. Gen. Virol."},{"key":"ref_61","unstructured":"Vitiello, A. Alvisi G. Material not intended for publication, 2019."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"557","DOI":"10.1038\/sj.mt.6300382","article-title":"Lentiviral vector design for multiple shRNA expression and durable HIV-1 inhibition","volume":"16","author":"Liu","year":"2008","journal-title":"Mol. Ther."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"18","DOI":"10.5501\/wjv.v2.i2.18","article-title":"High-throughput RNA interference screens integrative analysis: Towards a comprehensive understanding of the virus-host interplay","volume":"2","author":"Amberkar","year":"2013","journal-title":"World J. Virol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/12\/9\/1044\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T10:11:27Z","timestamp":1760177487000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/12\/9\/1044"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,9,18]]},"references-count":63,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2020,9]]}},"alternative-id":["v12091044"],"URL":"https:\/\/doi.org\/10.3390\/v12091044","relation":{},"ISSN":["1999-4915"],"issn-type":[{"type":"electronic","value":"1999-4915"}],"subject":[],"published":{"date-parts":[[2020,9,18]]}}}