Evaluating Drug Resistant Mutations to HCV NS3 Protease Inhibitors in Iranian Naïve Patients

  • Tayebeh HashempourEmail author
  • Behzad Dehghani
  • Zahra Mousavi
  • Maryam Yahaghi
  • Zahra Hasanshahi
  • Javad Moayedi
  • Tahereh Akbari
  • Mohammad Ali DavarpanahEmail author


Hepatitis C virus (HCV) is an important causative agent of acute and chronic hepatitis. The non-structural protein 3 (NS3) of HCV retains two enzymatic domains which are essential for the virus life cycle. The serine protease inhibitors have developed to improve the responses of HCV-infected patients that have an effective impact on NS3. Nonetheless, drug-resistant variants are the prominent obstruction toward therapeutic success. Sixty-eight Iranian patients infected with HCV genotypes 1a and 3a and diagnosed with chronic active hepatitis were examined. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene; also, HCV viral load, molecular genotyping, and the ALT test were determined for all samples. The sequencing results were used to be analyzed by several reliable bioinformatics tools to determine the physicochemical properties, B cell epitopes, post-modification changes and secondary/tertiary structures; and evaluate the interactions with four drugs. Our results showed that 45% of patients were 1a genotype, the rest of them belonged to 3a genotype, and 70% of patients had abnormal ALT and AST levels. Several substitutions were observed in codons I52M, S102A, L132I, and S166A in 3a genotype and 40, 153 and 91 in 1a genotype. Interactions between references and sample sequences with available drugs showed that different genotypes or common mutations could not have any striking effect on the energy value of the interaction. This study displayed resistance mutations and genetic polymorphisms of NS3 region that are crucial in determining the efficiency of protease inhibitor class of drugs in Iranian HCV infected patients.


HCV NS3 Drug resistance Bioinformatics 



Hepatitis C virus


Non-structural protein 3


Hepatocellular carcinoma


Iran hepatitis network




Protease inhibitors


RNA-dependent RNA polymerase


Human immunodeficiency virus


Hepatitis B surface antigen


Amino acid



The authors would like to thank Shiraz University of Medical Sciences, Shiraz, Iran and Shariati Hospital, Dr. Shahin Merat, professor of gastrology and hepatology at Tehran University of Medical Sciences for providing us with absolute opportunity to be in touch with his patients, also Tehran University of Medical Sciences personnel for sampling, and Center for Development of Clinical Research of Nemazee Hospital and Dr. Nasrin Shokrpour for editorial assistance.


This study was funded by Shiraz University of Medical Sciences (Grant Number 94-01-01-9218).

Compliance with Ethical Standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with animals performed by any of the authors.


  1. Afrasiabi M, Hosseini SY, Yaghobi R, Fattahi MR, Ardebili M, Khodadad M (2015) Analysis of naturally occurring resistant mutations to hepatitis C virus NS3 protease inhibitors: a preliminary study in South of Iran. Jundishapur J Microbiol 8Google Scholar
  2. Alavian SM, Hajarizadeh B, Lankarani KB, Sharafi H, Daryani NE, Merat S, Mohraz M, Mardani M, Fattahi MR, Poustchi H, Nikbin M (2016) Recommendations for the clinical management of hepatitis C in Iran: a consensus-based national guideline. Hepat Mon 16Google Scholar
  3. Alborzi A, Hashempour T, Moayedi J, Musavi Z, Pouladfar G, Merat S (2017) Role of serum level and genetic variation of IL-28B in interferon responsiveness and advanced liver disease in chronic hepatitis C patients. Med Microbiol Immunol 206:165–174PubMedCrossRefGoogle Scholar
  4. Asselah T, Estrabaud E, Bieche I, Lapalus M, De Muynck S, Vidaud M, Saadoun D, Soumelis V, Marcellin P (2010) Hepatitis C: viral and host factors associated with non-response to pegylated interferon plus ribavirin. Liver Int 30:1259–1269PubMedPubMedCentralCrossRefGoogle Scholar
  5. Benkert P, Tosatto SCE, Schomburg D (2008) ‘QMEAN: a comprehensive scoring function for model quality assessment. Proteins 71:261–277PubMedCrossRefGoogle Scholar
  6. Blom N, Gammeltoft S, Brunak S (1999) Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 294:1351–1362PubMedCrossRefGoogle Scholar
  7. Blom N, Sicheritz-Pontén T, Gupta R, Gammeltoft S, Brunak S (2004) Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Proteomics 4:1633–1649PubMedCrossRefGoogle Scholar
  8. Borhani K, Bamdad T, Hashempour T (2017) Lenalidomide acts as an adjuvant for HCV DNA vaccine. Int Immunopharmacol 48:231–240PubMedCrossRefGoogle Scholar
  9. Butt AA, Kanwal F (2011) Boceprevir and telaprevir in the management of hepatitis C virus–infected patients. Clin Infect Dis 54:96–104PubMedCrossRefGoogle Scholar
  10. Cerino A, Mondelli M (1991) Identification of an immunodominant B cell epitope on the hepatitis C virus nonstructural region defined by human monoclonal antibodies. J Immunol 147:2692–2696PubMedPubMedCentralGoogle Scholar
  11. Chauhan JS, Rao A, Raghava GPS (2013) In silico platform for prediction of N-, O-and C-glycosites in eukaryotic protein sequences. PLoS ONE 8:e67008PubMedPubMedCentralCrossRefGoogle Scholar
  12. Chen C-C, Hwang J-K, Yang J-M (2006) 2: protein structure prediction server. Nucleic Acids Res 34:W152–W157PubMedPubMedCentralCrossRefGoogle Scholar
  13. Chung RT, Gale M, Polyak SJ, Lemon SM, Jake Liang T, Hoofnagle JH (2008) Mechanisms of action of interferon and ribavirin in chronic hepatitis C: summary of a workshop. Hepatology 47:306–320PubMedPubMedCentralCrossRefGoogle Scholar
  14. Coppola N, Pisaturo M, Sagnelli C, Sagnelli E, Angelillo IF (2014) Peg-interferon plus ribavirin with or without boceprevir or telaprevir for HCV genotype 1: a meta-analysis on the role of response predictors. PLoS ONE 9:e94542PubMedPubMedCentralCrossRefGoogle Scholar
  15. Costantino A, Spada E, Equestre M, Bruni R, Tritarelli E, Coppola N, Sagnelli C, Sagnelli E, Ciccaglione AR (2015) Naturally occurring mutations associated with resistance to HCV NS5B polymerase and NS3 protease inhibitors in treatment-naïve patients with chronic hepatitis C. Virol J 12:186PubMedPubMedCentralCrossRefGoogle Scholar
  16. Dehghani B, Ghasabi F, Hashempoor T, Joulaei H, Hasanshahi Z, Halaji M, Chatrabnous N, Mousavi Z, Moayedi J (2017) Functional and structural characterization of Ebola virus glycoprotein (1976–2015)—an in silico study. Int J Biomath 10:1750108CrossRefGoogle Scholar
  17. Dehghani B, Hashempour T, Hasanshahi Z (2019a) Interaction of human herpesvirus 8 viral interleukin-6 with human interleukin-6 receptor using in silico approach: the potential role in HHV-8 pathogenesis. Curr Proteomics 16:1–1CrossRefGoogle Scholar
  18. Dehghani B, Hashempour T, Hasanshahi Z (2019b) Using immunoinformatics and structural approaches to design a novel HHV8 vaccine. Int J Pept Res Ther 1–11Google Scholar
  19. Dehghani B, Hashempour T, Hasanshahi Z, Moayedi J (2019c) Bioinformatics analysis of domain 1 of HCV-core protein: Iran. Int J Pept Res Ther 1–18Google Scholar
  20. Gasteiger E, Christine H, Alexandre G, Marc RW, Ron DA, Amos B (2005) Protein identification and analysis tools on the ExPASy server. In: The proteomics protocols handbook. Springer, New YorkCrossRefGoogle Scholar
  21. Geourjon C, Deleage G (1995) SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Bioinformatics 11:681–684CrossRefGoogle Scholar
  22. Gupta R, Jung E, Brunak S (2017) Prediction of N-glycosylation sites in human proteins 2004.
  23. Hashempoor T, Alborzi AM, Moayedi J, Ajorloo M, Bamdad T, Sharifi AH, Lavergne JP, Haj-sheykholeslami A, Merat S (2018) A decline in anti-core + 1 antibody titer occurs in successful treatment of patients infected with hepatitis C virus. Jundishapur J Microbiol 11Google Scholar
  24. Hedegaard DL, Tully DC, Rowe IA, Reynolds GM, Bean DJ, Ke H, Davis C, Wilhelm A, Ogilvie CB, Power KA (2017) High resolution sequencing of hepatitis C virus reveals limited intra-hepatic compartmentalization in end-stage liver disease. J Hepatol 66:28–38PubMedPubMedCentralCrossRefGoogle Scholar
  25. Hoffmann L, Faffe DS, Lima JFC, Capitanio TA, Cabral BCA, Ürményi TP, Coelho HSM, Rondinelli E, Villela-Nogueira CA, Silva R (2015) No correspondence between resistance mutations in the HCV-NS3 protease at baseline and early telaprevir-based triple therapy. BBA Clin 3:146–151PubMedPubMedCentralCrossRefGoogle Scholar
  26. Iakoucheva LM, Radivojac P, Brown CJ, O’Connor TR, Sikes JG, Obradovic Z, Dunker A (2004) The importance of intrinsic disorder for protein phosphorylation. Nucleic Acids Res 32:1037–1049PubMedPubMedCentralCrossRefGoogle Scholar
  27. Kelley LA, Sternberg MJ (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4:363PubMedCrossRefGoogle Scholar
  28. Khudyakov Y, Khudyakova NS, Jue DL, Lambert SB, Fang S, Fields HA (1995) Linear B-cell epitopes of the NS3-NS4-NS5 proteins of the hepatitis C virusas modeled with synthetic peptides. Virology 206:666–672CrossRefGoogle Scholar
  29. Kirst ME, Li EC, Wang CX, Dong H-J, Liu C, Fried MW, Nelson DR, Wang GP (2013) Deep sequencing analysis of HCV NS3 resistance-associated variants and mutation linkage in liver transplant recipients. PLoS ONE 8:e69698PubMedPubMedCentralCrossRefGoogle Scholar
  30. Kumthip K, Maneekarn N (2015) The role of HCV proteins on treatment outcomes. Virology journal 12:217PubMedPubMedCentralCrossRefGoogle Scholar
  31. Lin MV, Charlton AN, Rouster SD, Zamor PJ, Sherman KE (2014) Hepatitis C virus NS3 mutations in haemophiliacs. Haemophilia 20:659–665PubMedPubMedCentralCrossRefGoogle Scholar
  32. López-Labrador FX, Moya A, Gonzàlez-Candelas F (2008) Mapping natural polymorphisms of hepatitis C virus NS3/4A protease and antiviral resistance to inhibitors in worldwide isolates. Antivir Ther 13:481PubMedPubMedCentralGoogle Scholar
  33. Moattari A, Dehghani B, Khodadad N, Tavakoli F (2015) In silico functional and structural characterization of H1N1 influenza a viruses hemagglutinin, 2010–2013, Shiraz, Iran. Acta Biotheor 63:183–202PubMedCrossRefPubMedCentralGoogle Scholar
  34. Natarajan S (2010) NS3 protease from flavivirus as a target for designing antiviral inhibitors against dengue virus. Genet Mol Biol 33:214–219PubMedPubMedCentralCrossRefGoogle Scholar
  35. Nishiya AS, de Almeida-Neto C, Ferreira SC, Alencar CS, Di-Lorenzo-Oliveira C, Levi JE, Salles NA, Jr AM, Sabino EC (2014) HCV genotypes, characterization of mutations conferring drug resistance to protease inhibitors, and risk factors among blood donors in Sao Paulo, Brazil. PLoS ONE 9:e86413PubMedPubMedCentralCrossRefGoogle Scholar
  36. Palumbo E (2011) Pegylated interferon and ribavirin treatment for hepatitis C virus infection. Ther Adv Chronic Dis 2:39–45PubMedPubMedCentralCrossRefGoogle Scholar
  37. Paolucci S, Fiorina L, Piralla A, Gulminetti R, Novati S, Barbarini G, Sacchi P, Gatti M, Dossena L, Baldanti F (2012) Naturally occurring mutations to HCV protease inhibitors in treatment-naive patients. Virol J 9:245PubMedPubMedCentralCrossRefGoogle Scholar
  38. Patel K, Pauli M, Abdelghany S (2011) Telaprevir: a novel, protease inhibitor for the treatment of hepatitis C virus. Formulary 46:205–213Google Scholar
  39. Perales C, Quer J, Gregori J, Esteban JI, Domingo E (2015) Resistance of hepatitis C virus to inhibitors: complexity and clinical implications. Viruses 7:5746–5766PubMedPubMedCentralCrossRefGoogle Scholar
  40. Raj VS, Hundie GB, Schürch AC, Smits SL, Pas SD, Le Pogam S, Janssen HL, de Knegt RJ, Osterhaus AD, Najera I, Boucher CA (2017) Identification of HCV resistant variants against direct acting antivirals in plasma and liver of treatment naïve patients. Sci Rep 7Google Scholar
  41. Rajagopalan R, Pan L, Schaefer C, Nicholas J, Lim S, Misialek S, Stevens S, Hooi L, Aleskovski N, Ruhrmund D (2017) Preclinical characterization and human microdose pharmacokinetics of ITMN-8187, a nonmacrocyclic inhibitor of the hepatitis C virus NS3 protease. Antimicrob Agents Chemother 61:e01569-16PubMedCrossRefGoogle Scholar
  42. Romano KP, Ali A, Royer WE, Schiffer CA (2010) Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding. Proc Natl Acad Sci 107:20986–20991PubMedCrossRefGoogle Scholar
  43. Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5:725PubMedPubMedCentralCrossRefGoogle Scholar
  44. Saadoun D, Rigon MR, Thibault V, Longuet M, Pol S, Blanc F, Pialoux G, Karras A, Bazin-Karra D, Cazorla C, Vittecoq D (2013) Peg-IFNα/ribavirin/protease inhibitor combination in hepatitis C virus associated mixed cryoglobulinemia vasculitis: results at week 24. Ann Rheum Dis 2012–202770Google Scholar
  45. Saha (2006) Prediction of continuous B-cell epitopes in an antigen using recurrent neural network. Proteins 65:40–48PubMedCrossRefGoogle Scholar
  46. Saha S, Raghava GPS (2004) BcePred: prediction of continuous B-cell epitopes in antigenic sequences using physico-chemical properties. In: International conference on artificial immune systems. Springer, New York, 197–204Google Scholar
  47. Sanford M (2015) Simeprevir: a review of its use in patients with chronic hepatitis C virus infection. Drugs 75:183–196PubMedCrossRefGoogle Scholar
  48. Susser S, Welsch C, Wang Y, Zettler M, Domingues FS, Karey U, Hughes E, Ralston R, Tong X, Herrmann E (2009) Characterization of resistance to the protease inhibitor boceprevir in hepatitis C virus–infected patients. Hepatology 50:1709–1718PubMedCrossRefGoogle Scholar
  49. Svarovskaia ES, Martin R, McHutchison JG, Miller MD, Mo H (2012) Abundant drug-resistant NS3 mutants detected by deep sequencing in hepatitis C virus-infected patients undergoing NS3 protease inhibitor monotherapy. J Clin Microbiol 50:3267–3274PubMedPubMedCentralCrossRefGoogle Scholar
  50. Tan S-L (2006) Hepatitis C viruses: genomes and molecular biology. Horizon Scientific Press, PooleGoogle Scholar
  51. Tong X, Bogen S, Robert Chase V, Girijavallabhan ZG, George Njoroge F, Prongay A, Saksena A, Skelton A, Xia E (2008) Characterization of resistance mutations against HCV ketoamide protease inhibitors. Antivir Res 77:177–185PubMedCrossRefGoogle Scholar
  52. Wilby KJ, Partovi N, Ford J-AE, Greanya ED, Yoshida EM (2012) Review of boceprevir and telaprevir for the treatment of chronic hepatitis C. Can J Gastroenterol Hepatol 26:205–210Google Scholar
  53. World Health Organization (2017) Global hepatitis report 2017. World Health Organization, GenevaGoogle Scholar
  54. Xue W, Ban Y, Liu H, Yao X (2013) Computational study on the drug resistance mechanism against HCV NS3/4A protease inhibitors vaniprevir and MK-5172 by the combination use of molecular dynamics simulation, residue interaction network, and substrate envelope analysis. J Chem Inf Model 54:621–633PubMedCrossRefPubMedCentralGoogle Scholar
  55. Zeminian LB, Padovani JL, Corvino SM, Silva GF, Pardini MIDMC, Grotto RMT (2013) Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors. Memórias do Inst Oswaldo Cruz 108:13–17CrossRefGoogle Scholar
  56. Zeuzem S, Sarrazin C, Rouzier R, Tarral A, Brion N, Gupta S, Deckman D, Fellows K, Hussain M, Cutler DL (2005) Anti-viral activity of SCH 503034, a HCV protease inhibitor, administered as monotherapy in hepatitis C genotype-1 (HCV-1) patients refractory to pegylated interferon (PEG-IFN-alpha). Hepatology 42:233A-34AGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Tayebeh Hashempour
    • 1
    Email author
  • Behzad Dehghani
    • 2
  • Zahra Mousavi
    • 1
  • Maryam Yahaghi
    • 3
  • Zahra Hasanshahi
    • 2
  • Javad Moayedi
    • 1
  • Tahereh Akbari
    • 3
  • Mohammad Ali Davarpanah
    • 2
    Email author
  1. 1.Clinical Microbiology Research Center, Nemazee HospitalShiraz University of Medical SciencesShirazIran
  2. 2.Shiraz HIV/AIDS Research Center, Institute of HealthShiraz University of Medical SciencesShirazIran
  3. 3.Department of Internal Medicine, Gastroenterohepatology Research CenterShiraz University of Medical SciencesShirazIran

Personalised recommendations