1887

Journal of General Virology header logo

Volume 102, Issue 3

Cell viability assay as a tool to study activity and inhibition of hepatitis C p7 channels Free

Abstract

The p7 viroporin of the hepatitis C virus (HCV) forms an intracellular proton-conducting transmembrane channel in virus-infected cells, shunting the pH of intracellular compartments and thus helping virus assembly and release. This activity is essential for virus infectivity, making viroporins an attractive target for drug development. The protein sequence and drug sensitivity of p7 vary between the seven major genotypes of the hepatitis C virus, but the essential channel activity is preserved. Here, we investigated the effect of several inhibitors on recombinant HCV p7 channels corresponding to genotypes 1a–b, 2a–b, 3a and 4a using patch-clamp electrophysiology and cell-based assays. We established a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cell viability assay for recombinant p7 expressed in HEK293 cells to assess channel activity and its sensitivity to inhibitors. The results from the cell viability assay were consistent with control measurements using established assays of haemadsorption and intracellular pH, and agreed with data from patch-clamp electrophysiology. Hexamethylene amiloride (HMA) was the most potent inhibitor of p7 activity, but possessed cytotoxic activity at higher concentrations. Rimantadine was active against p7 of all genotypes, while amantadine activity was genotype-dependent. The alkyl-chain iminosugars B-DNJ, N-DNJ and N-DGJ were tested and their activity was found to be genotype-specific. In the current study, we introduce cell viability assays as a rapid and cost-efficient technique to assess viroporin activity and identify channel inhibitors as potential novel antiviral drugs.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): cell viability assay , haemadsorption , hepatitis C genotypes , intracellular pH , p7 inhibitors , patch-clamp electrophysiology and viroporins
© 2021 The Authors
Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001571
2021-03-12
2024-05-06
Loading full text...

Full text loading...

/deliver/fulltext/jgv/102/3/vir001571.html?itemId=/content/journal/jgv/10.1099/jgv.0.001571&mimeType=html&fmt=ahah

References

  1. Messina JP, Humphreys I, Flaxman A, Brown A, Cooke GS et al. Global distribution and prevalence of hepatitis C virus genotypes. Hepatology 2015; 61:77–87 [View Article][PubMed]
     [Google Scholar]
  2. Mohamoud YA, Mumtaz GR, Riome S, Miller D, Abu-Raddad LJ. The epidemiology of hepatitis C virus in Egypt: a systematic review and data synthesis. BMC Infect Dis 2013; 13:288 [View Article][PubMed]
     [Google Scholar]
  3. De Francesco R. Molecular virology of the hepatitis C virus. J Hepatol 1999; 31 Suppl 1:47–53 [View Article][PubMed]
     [Google Scholar]
  4. Dubuisson J. Hepatitis C virus proteins. World J Gastroenterol 2007; 13:2406–2415 [View Article][PubMed]
     [Google Scholar]
  5. Suzuki T, Aizaki H, Murakami K, Shoji I, Wakita T. Molecular biology of hepatitis C virus. J Gastroenterol 2007; 42:411–423 [View Article][PubMed]
     [Google Scholar]
  6. Murphy DG, Sablon E, Chamberland J, Fournier E, Dandavino R et al. Hepatitis C virus genotype 7, a new genotype originating from central Africa. J Clin Microbiol 2015; 53:967–972 [View Article][PubMed]
     [Google Scholar]
  7. Popescu C-I, Rouillé Y, Dubuisson J. Hepatitis C virus assembly imaging. Viruses 2011; 3:2238–2254 [View Article][PubMed]
     [Google Scholar]
  8. Gonzalez ME, Carrasco L. Viroporins. FEBS Lett 2003; 552:28–34 [View Article][PubMed]
     [Google Scholar]
  9. Carrère-Kremer S, Montpellier-Pala C, Cocquerel L, Wychowski C, Penin F et al. Subcellular localization and topology of the p7 polypeptide of hepatitis C virus. J Virol 2002; 76:3720–3730 [View Article][PubMed]
     [Google Scholar]
  10. Griffin S, Stgelais C, Owsianka AM, Patel AH, Rowlands D et al. Genotype-dependent sensitivity of hepatitis C virus to inhibitors of the p7 ion channel. Hepatology 2008; 48:1779–1790 [View Article][PubMed]
     [Google Scholar]
  11. Steinmann E, Penin F, Kallis S, Patel AH, Bartenschlager R et al. Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions. PLoS Pathog 2007; 3:e103 [View Article][PubMed]
     [Google Scholar]
  12. Wozniak AL, Griffin S, Rowlands D, Harris M, Yi M et al. Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production. PLoS Pathog 2010; 6:e1001087 [View Article][PubMed]
     [Google Scholar]
  13. Griffin SDC, Beales LP, Clarke DS, Worsfold O, Evans SD et al. The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, amantadine. FEBS Lett 2003; 535:34–38 [View Article]
     [Google Scholar]
  14. Pavlovic D, Fischer W, Hussey M, Durantel D, Durantel S et al. Long alkylchain iminosugars block the HCV p7 ion channel. Adv Exp Med Biol 2005; 564:3–4 [View Article][PubMed]
     [Google Scholar]
  15. Pavlović D, Neville DCA, Argaud O, Blumberg B, Dwek RA et al. The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives. Proc Natl Acad Sci U S A 2003; 100:6104–6108 [View Article][PubMed]
     [Google Scholar]
  16. Premkumar A, Wilson L, Ewart GD, Gage PW. Cation-selective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride. FEBS Lett 2004; 557:99–103 [View Article][PubMed]
     [Google Scholar]
  17. Soranzo T, Cortès S, Gilde F, Kreir M, Picart C et al. Functional characterization of p7 viroporin from hepatitis C virus produced in a cell-free expression system. Protein Expr Purif 2016; 118:83–91 [View Article][PubMed]
     [Google Scholar]
  18. StGelais C, Tuthill TJ, Clarke DS, Rowlands DJ, Harris M et al. Inhibition of hepatitis C virus p7 membrane channels in a liposome-based assay system. Antiviral Res 2007; 76:48–58 [View Article][PubMed]
     [Google Scholar]
  19. OuYang B, Xie S, Berardi MJ, Zhao X, Dev J et al. Unusual architecture of the p7 channel from hepatitis C virus. Nature 2013; 498:521–525 [View Article][PubMed]
     [Google Scholar]
  20. Montserret R, Saint N, Vanbelle C, Salvay AG, Simorre J-P et al. Nmr structure and ion channel activity of the p7 protein from hepatitis C virus. J Biol Chem 2010; 285:31446–31461 [View Article][PubMed]
     [Google Scholar]
  21. Breitinger U, Farag NS, Ali NKM, Breitinger H-GA. Patch-Clamp study of hepatitis C p7 channels reveals genotype-specific sensitivity to inhibitors. Biophys J 2016; 110:2419–2429 [View Article][PubMed]
     [Google Scholar]
  22. Cook GA, Opella SJ. NMR studies of p7 protein from hepatitis C virus. Eur Biophys J 2010; 39:1097–1104 [View Article][PubMed]
     [Google Scholar]
  23. Patargias G, Zitzmann N, Dwek R, Fischer WB. Protein-protein interactions: modeling the hepatitis C virus ion channel p7. J Med Chem 2006; 49:648–655 [View Article][PubMed]
     [Google Scholar]
  24. Kalita MM, Griffin S, Chou JJ, Fischer WB. Genotype-specific differences in structural features of hepatitis C virus (HCV) p7 membrane protein. Biochim Biophys Acta 2015; 1848:1383–1392 [View Article][PubMed]
     [Google Scholar]
  25. Dev J, Brüschweiler S, Ouyang B, Chou JJ. Transverse relaxation dispersion of the p7 membrane channel from hepatitis C virus reveals conformational breathing. J Biomol NMR 2015; 61:369–378 [View Article][PubMed]
     [Google Scholar]
  26. Oestringer BP, Bolivar JH, Claridge JK, Almanea L, Chipot C et al. Hepatitis C virus sequence divergence preserves p7 viroporin structural and dynamic features. Sci Rep 2019; 9:8383 [View Article][PubMed]
     [Google Scholar]
  27. El-Zayadi A-R, Attia M, Barakat EMF, Badran HM, Hamdy H et al. Response of hepatitis C genotype-4 naïve patients to 24 weeks of Peg-interferon-alpha2b/ribavirin or induction-dose interferon-alpha2b/ribavirin/amantadine: a non-randomized controlled study. Am J Gastroenterol 2005; 100:2447–2452 [View Article][PubMed]
     [Google Scholar]
  28. Maynard M, Pradat P, Bailly F, Rozier F, Nemoz C et al. Amantadine triple therapy for non-responder hepatitis C patients. clues for controversies (ANRS HC 03 BITRI). J Hepatol 2006; 44:484–490 [View Article][PubMed]
     [Google Scholar]
  29. Carlsen THR, Pedersen J, Prentoe JC, Giang E, Keck Z-Y et al. Breadth of neutralization and synergy of clinically relevant human monoclonal antibodies against HCV genotypes 1A, 1B, 2A, 2B, 2C, and 3A. Hepatology 2014; 60:1551–1562 [View Article][PubMed]
     [Google Scholar]
  30. Meuleman P, Bukh J, Verhoye L, Farhoudi A, Vanwolleghem T et al. In vivo evaluation of the cross-genotype neutralizing activity of polyclonal antibodies against hepatitis C virus. Hepatology 2011; 53:755–762 [View Article][PubMed]
     [Google Scholar]
  31. Konerman MA, Lok ASF. Hepatitis C treatment and barriers to eradication. Clin Transl Gastroenterol 2016; 7:e193 [View Article][PubMed]
     [Google Scholar]
  32. Griffin S. "Too little, too late?" Will inhibitors of the hepatitis C virus p7 ion channel ever be used in the clinic?. Future Med Chem 2014; 6:1893–1907 [View Article][PubMed]
     [Google Scholar]
  33. Chizhmakov IV, Ogden DC, Geraghty FM, Hayhurst A, Skinner A, Betakova T et al. Differences in conductance of M2 proton channels of two influenza viruses at low and high pH. J Physiol 2003; 546:427–438 [View Article][PubMed]
     [Google Scholar]
  34. Farag NS, Breitinger U, El-Azizi M, Breitinger HG. The p7 viroporin of the hepatitis C virus contributes to liver inflammation by stimulating production of interleukin-1β. Biochim Biophys Acta Mol Basis Dis 1863; 2017:712–720
     [Google Scholar]
  35. Griffin SDC, Harvey R, Clarke DS, Barclay WS, Harris M et al. A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria. J Gen Virol 2004; 85:451–461 [View Article][PubMed]
     [Google Scholar]
  36. Kumar P, Nagarajan A, Uchil PD. Analysis of cell viability by the MTT assay. Cold Spring Harb Protoc 2018; 2018:pdb.prot095505 01 06 2018 [View Article][PubMed]
     [Google Scholar]
  37. Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999; 4:67–73 [View Article][PubMed]
     [Google Scholar]
  38. Griffin SDC, Beales LP, Clarke DS, Worsfold O, Evans SD et al. The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, amantadine. FEBS Lett 2003; 535:34–38 [View Article][PubMed]
     [Google Scholar]
  39. Chizhmakov IV, Geraghty FM, Ogden DC, Hayhurst A, Antoniou M, Hay AJ et al. Selective proton permeability and pH regulation of the influenza virus M2 channel expressed in mouse erythroleukaemia cells. J Physiol 1996; 494:329–336 [View Article][PubMed]
     [Google Scholar]
  40. Wang C, Lamb RA, Pinto LH. Direct measurement of the influenza A virus M2 protein ion channel activity in mammalian cells. Virology 1994; 205:133–140 [View Article][PubMed]
     [Google Scholar]
  41. Wang C, Lamb RA, Pinto LH. Activation of the M2 ion channel of influenza virus: a role for the transmembrane domain histidine residue. Biophys J 1995; 69:1363–1371 [View Article][PubMed]
     [Google Scholar]
  42. Wang C, Takeuchi K, Pinto LH, Lamb RA. Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block. J Virol 1993; 67:5585–5594 [View Article][PubMed]
     [Google Scholar]
  43. Johnson DE, Ostrowski P, Jaumouillé V, Grinstein S. The position of lysosomes within the cell determines their luminal pH. J Cell Biol 2016; 212:677–692 [View Article][PubMed]
     [Google Scholar]
  44. Yapici NB, Bi Y, Li P, Chen X, Yan X et al. Highly stable and sensitive fluorescent probes (LysoProbes) for lysosomal labeling and tracking. Sci Rep 2015; 5:8576 [View Article][PubMed]
     [Google Scholar]
  45. Ohuchi M, Cramer A, Vey M, Ohuchi R, Garten W et al. Rescue of vector-expressed fowl plague virus hemagglutinin in biologically active form by acidotropic agents and coexpressed M2 protein. J Virol 1994; 68:920–926 [View Article][PubMed]
     [Google Scholar]
  46. Takeuchi K, Lamb RA. Influenza virus M2 protein ion channel activity stabilizes the native form of fowl plague virus hemagglutinin during intracellular transport. J Virol 1994; 68:911–919 [View Article][PubMed]
     [Google Scholar]
  47. Steinmann E, Whitfield T, Kallis S, Dwek RA, Zitzmann N et al. Antiviral effects of amantadine and iminosugar derivatives against hepatitis C virus. Hepatology 2007; 46:330–338 [View Article][PubMed]
     [Google Scholar]
  48. Medeiros R, Escriou N, Naffakh N, Manuguerra JC, van der Werf S. Hemagglutinin residues of recent human A(H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes. Virology 2001; 289:74–85 [View Article][PubMed]
     [Google Scholar]
  49. Krüger N, Hoffmann M, Drexler JF, Müller MA, Corman VM et al. Functional properties and genetic relatedness of the fusion and hemagglutinin-neuraminidase proteins of a mumps virus-like bat virus. J Virol 2015; 89:4539–4548 [View Article][PubMed]
     [Google Scholar]
  50. Sun C, Wen H, Chen Y, Chu F, Lin B et al. Roles of the highly conserved amino acids in the globular head and stalk region of the Newcastle disease virus HN protein in the membrane fusion process. Biosci Trends 2015; 9:56–64 [View Article][PubMed]
     [Google Scholar]
  51. Fukushima K, Takahashi T, Ueyama H, Takaguchi M, Ito S et al. Amino acid substitutions contributing to α2,6-sialic acid linkage binding specificity of human parainfluenza virus type 3 hemagglutinin-neuraminidase. FEBS Lett 2015; 589:1278–1282 [View Article][PubMed]
     [Google Scholar]
  52. Porotto M, Greengard O, Poltoratskaia N, Horga MA, Moscona A. Human parainfluenza virus type 3 HN-receptor interaction: effect of 4-guanidino-Neu5Ac2en on a neuraminidase-deficient variant. J Virol 2001; 75:7481–7488 [View Article][PubMed]
     [Google Scholar]
  53. Foster TL, Verow M, Wozniak AL, Bentham MJ, Thompson J et al. Resistance mutations define specific antiviral effects for inhibitors of the hepatitis C virus p7 ion channel. Hepatology 2011; 54:79–90 [View Article][PubMed]
     [Google Scholar]
  54. Foster TL, Thompson GS, Kalverda AP, Kankanala J, Bentham M et al. Structure-guided design affirms inhibitors of hepatitis C virus p7 as a viable class of antivirals targeting virion release. Hepatology 2014; 59:408–422 [View Article][PubMed]
     [Google Scholar]
  55. Mihm U, Grigorian N, Welsch C, Herrmann E, Kronenberger B et al. Amino acid variations in hepatitis C virus p7 and sensitivity to antiviral combination therapy with amantadine in chronic hepatitis C. Antivir Ther 2006; 11:507–519[PubMed]
     [Google Scholar]
  56. Mihm U, Herrmann E, Sarrazin C, Zeuzem S. Review article: predicting response in hepatitis C virus therapy. Aliment Pharmacol Ther 2006; 23:1043–1054 [View Article][PubMed]
     [Google Scholar]
  57. Zhao L, Wang S, Du L, Dev J, Zhou L et al. Structural basis of interaction between the hepatitis C virus p7 channel and its blocker hexamethylene amiloride. Protein Cell 2016; 7:300–304 [View Article][PubMed]
     [Google Scholar]
  58. Hong W, Lang Y, Li T, Zeng Z, Song Y et al. A p7 ion Channel-derived peptide inhibits hepatitis C virus infection in vitro. J Biol Chem 2015; 290:23254–23263 [View Article][PubMed]
     [Google Scholar]
  59. Horne G, Wilson FX. Therapeutic applications of iminosugars: current perspectives and future opportunities. Prog Med Chem 2011; 50:135–176 [View Article][PubMed]
     [Google Scholar]
  60. Miller JL, Tyrrell BE, Zitzmann N. Mechanisms of antiviral activity of iminosugars against dengue virus. Adv Exp Med Biol 2018; 1062:277–301 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.001571
Loading
Cell viability assay as a tool to study activity and inhibition of hepatitis C p7 channels
J. Gen. Virol. 102, 001571 (2021); https://doi.org/10.1099/jgv.0.001571
/content/journal/jgv/10.1099/jgv.0.001571
/content/journal/jgv/10.1099/jgv.0.001571
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error