1887

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Volume 85, Issue 6

Characterization of the genome and structural proteins of hepatitis C virus resolved from infected human liver Free

Abstract

In the absence of satisfactory cell culture systems for hepatitis C virus (HCV), virtually all that is known about the proteins of the virus has been learned by the study of recombinant proteins. Characterization of virus proteins from patients with HCV has been retarded by the low virus titre in blood and limited availability of infected tissue. Here, the authors have identified a primary infection in a liver transplanted into an immunodeficient patient with chronic HCV. The patient required re-transplant and the infected liver, removed 6 weeks after the initial transplant, had a very high titre of HCV, 5×10 International Units (IU) per gram of liver. The density distribution of HCV in iodixanol gradients showed a peak at 1·04 g ml with 73 % of virus below 1·08 g ml. Full-length HCV RNA was detected by Northern blotting and the ratio between positive- and negative-strand HCV RNA was determined as 60. HCV was partially purified by precipitation with heparin/Mn and a single species of each of the three structural proteins, core, E1 and E2, was detected by Western blotting. The molecular mass of core was 20 kDa, which corresponds to the mature form from recombinant sources. The molecular mass of glycoprotein E1 was 31 kDa before and 21 kDa after deglycosylation with PNGase F or endoglycosidase H. Glycoprotein E2 was 62 kDa before and 36 kDa after deglycosylation, but E2-P7 was not detected. This was in contrast to recombinant sources of E2 which contain E2-P7.

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2004-06-01
2024-04-19
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References

  1. André P., Pradel F. K., Deforges S. 7 other authors 2002; Characterization of low- and very-low-density hepatitis C virus RNA containing particles. J Virol 76:6919–6928 [CrossRef]
     [Google Scholar]
  2. Anonymous. 1997; Hepatitis C: global prevalence. Wkly Epidemiol Rec 72:341–344
     [Google Scholar]
  3. Aoyagi K., Ohue C., Iida K., Kimura T., Tanaka E., Kiyosawa K., Yagi S. 1999; Development of a simple and highly sensitive enzyme immunoassay for hepatitis C core antigen. J Clin Microbiol 37:1802–1808
     [Google Scholar]
  4. Bartenschlager R., Lohmann V. 2000; Replication of hepatitis C virus. J Gen Virol 81:1631–1648
     [Google Scholar]
  5. Bjøro K., Skaug K., Haaland T., Frøland S. S. 1999; Long-term outcome of chronic hepatitis C virus infection in primary hypogammaglobulinaemia. Q J Med 92:433–441 [CrossRef]
     [Google Scholar]
  6. Bradley D. W. 2000; Studies of non-A, non-B hepatitis and characterization of the hepatitis C virus in chimpanzees. Curr Top Microbiol Immunol 242:1–23
     [Google Scholar]
  7. Burstein M., Scholnick H. R., Morfin R. 1970; Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions. J Lipid Res 11:583–595
     [Google Scholar]
  8. Choo Q. L., Kuo G., Weiner A. J., Overby L. R., Bradley D. W., Houghton M. 1989; Isolation of a cDNA clone derived from a blood borne non-A, non-B viral hepatitis genome. Science 244:359–362 [CrossRef]
     [Google Scholar]
  9. Choo Q. L., Richman K. H., Han J. H. 11 other authors 1991; Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A 88:2451–2455 [CrossRef]
     [Google Scholar]
  10. Choukhi A., Pillez A., Drobecq H., Sergheraert C., Wychowski C., Dubuisson J. 1999; Characterization of aggregates of hepatitis C virus glycoproteins. J Gen Virol 80:3099–3107
     [Google Scholar]
  11. Christie J. M. L., Healey C. J., Watson J. 7 other authors 1997; Clinical outcome of hypogammaglobulinaemic patients following outbreak of acute hepatitis C: 2 year follow up. Clin Exp Immunol 110:4–8 [CrossRef]
     [Google Scholar]
  12. Dailey P. J., Collins M. L., Urdea M. S., Wilber J. C. 1998; Quantification of HCV RNA in liver tissue by bDNA assay. Methods Mol Med 19:119–129
     [Google Scholar]
  13. Di Bisceglie A. M., Chung Y. W., Dailey P. J., Shakil A. O., Krawczynski K., Hoofnagle J. H. 1994; Hepatitis C viral (HCV) markers in serum and liver of chronically infected patients. Hepatology 20:236A
     [Google Scholar]
  14. Dubuisson J. 2000; Folding, assembly and subcellular localization of hepatitis C virus glycoproteins. Curr Top Microbiol Immunol 242:135–148
     [Google Scholar]
  15. Dubuisson J., Hsu H. H., Cheung R. C., Greenberg H. B., Russell D. G., Rice C. M. 1994; Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and sindbis viruses. J Virol 68:6147–6160
     [Google Scholar]
  16. Dumonceaux J., Cormier E. G., Kajumo F., Donovan G. P., Roy-Chowdhury J., Fox I. J., Gardner J. P., Dragic T. 2003; Expression of unmodified hepatitis C virus envelope glycoprotein coding sequences leads to cryptic intron excision and cell surface expression of E1/E2 heterodimers comprising full-length and partially deleted E1. J Virol 77:13418–13424 [CrossRef]
     [Google Scholar]
  17. Fournillier-Jacob A., Cahour A., Escriou N., Girard M., Wychowski C. 1996; Processing of the E1 glycoprotein of hepatitis C virus expressed in mammalian cells. J Gen Virol 77:1055–1064 [CrossRef]
     [Google Scholar]
  18. Gaud U., Langer B., Petropoulou T., Thomas H., Karayiannis P. 2003; Changes in hypervariable region 1 of the envelope 2 glycoprotein of hepatitis C virus in children and adults with humoral immune defects. J Med Virol 69:350–356 [CrossRef]
     [Google Scholar]
  19. Gow P. J., Mutimer D. 2002; Successful outcome of liver transplantation in a patient with hepatitis C and common variable immune deficiency. Transpl Int 15:380–383 [CrossRef]
     [Google Scholar]
  20. Gowans E. 2000; Distribution of markers of hepatitis C virus infection throughout the body. Semin Liver Dis 20:85–102 [CrossRef]
     [Google Scholar]
  21. Graham J. M., Higgins J. A., Gillott T., Taylor T., Wilkinson J., Ford T., Billington D. 1996; A novel method for the rapid separation of plasma lipoproteins using self-generating gradients of iodixanol. Atherosclerosis 124:125–135 [CrossRef]
     [Google Scholar]
  22. Grakoui A., Wychowski C., Lin C., Feinstone S. M., Rice C. M. 1993; Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol 67:1385–1395
     [Google Scholar]
  23. Healey C. J., Sabharwal N. K., Daub J., Davidson F., Yap P. L., Fleming K. A., Chapman R. W. G., Simmonds P., Chapel H. 1996; Outbreak of acute hepatitis C following the use of anti hepatitis C virus screened intravenous immunoglobulin therapy. Gastroenterology 110:1120–1126 [CrossRef]
     [Google Scholar]
  24. Hijikata M., Shimizu Y. K., Kato H., Iwamoto A., Shih J. W., Alter H. J., Purcell R. H., Yoshikura H. 1993; Equilibrium centrifugation studies of hepatitis C virus: evidence for circulating immune complexes. J Virol 67:1953–1958
     [Google Scholar]
  25. Hüssy P., Langen H., Mous J., Jacobsen H. 1996; Hepatitis C virus core protein: carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase. Virology 224:93–104 [CrossRef]
     [Google Scholar]
  26. Kato N., Shimotohno K. 2000; Systems to culture hepatitis C virus. Curr Top Microbiol Immunol 242:261–278
     [Google Scholar]
  27. Koike K., Moriya K., Ishibashi K., Matsuura Y., Suzuki T., Saito I., Iino S., Kurokawa K., Miyamura T. 1995; Expression of hepatitis C virus envelope proteins in transgenic mice. J Gen Virol 76:3031–3038 [CrossRef]
     [Google Scholar]
  28. Kolykhalov A. A., Reed K. E., Rice C. M. 1998; Cloning and assembly of complex libraries of full-length HCV cDNA clones. In Hepatitis C Protocols . Methods in Molecular Medicine Vol 19 pp  289–301 Edited by Lau J. Y. N. New Jersey, USA: Humana Press;
     [Google Scholar]
  29. Lin C., Lindenbach B. D., Prágai B. M., McCourt D. W., Rice C. M. 1994; Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2 specific products with different C termini. J Virol 68:5063–5073
     [Google Scholar]
  30. Lin L., Fevery J., Yap S. H. 2002; A novel strand-specific RT-PCR for detection of hepatitis C virus negative-strand RNA (replicative intermediate): evidence of absence or very low level of HCV replication in peripheral blood mononuclear cells. J Virol Methods 100:97–105 [CrossRef]
     [Google Scholar]
  31. Liu Q., Tackney C., Bhat R. A., Prince A. M., Zhang P. 1997; Regulated processing of hepatitis C virus core protein is linked to subcellular localization. J Virol 71:657–662
     [Google Scholar]
  32. Mackett M. 1995; Construction and characterization of vaccinia virus recombinants. In DNA Cloning 4 pp  43–83 Edited by Glover C. M., Hames B. D. Oxford, UK: Oxford University Press;
     [Google Scholar]
  33. Mackness M. I., Durrington P. N. 1992; Lipoprotein separation and analysis for clinical studies. In Lipoprotein Analysis pp  1–42 Edited by Converse C. A., Skinner E. R. Oxford, UK: Oxford University Press;
     [Google Scholar]
  34. Major M. E., Rehermann B., Feinstone S. M. 2001; Hepatitis C viruses. In Fields Virology , 4th edn. pp  1127–1161 Edited by Knipe D. M., Howley P. M. Philadelphia: Lippincott Williams & Wilkins;
     [Google Scholar]
  35. McLauchlan J., Lemberg M. K., Hope G., Martoglio B. 2002; Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets. EMBO J 21:3980–3988 [CrossRef]
     [Google Scholar]
  36. Mercier B., Burlot L., Férec C. 1999; Simultaneous screening for HBV DNA and HCV RNA genomes in blood donations using a novel TaqMan PCR assay. J Virol Methods 77:1–9 [CrossRef]
     [Google Scholar]
  37. Meunier J. C., Fournillier A., Choukhi A., Cahour A., Cocquerel L., Dubuisson J., Wychowski C. 1999; Analysis of the glycosylation sites of hepatitis C virus (HCV) glycoprotein E1 and the influence of E1 glycans on the formation of the glycoprotein complex. J Gen Virol 80:887–896
     [Google Scholar]
  38. Miyai K. 1991; Structural organization of the liver. In Hepatotoxicology pp  1–65 Edited by Meeks R. G., Harrison S. D., Bull R. J. Boca Raton, FL: CRC Press;
     [Google Scholar]
  39. Nakamoto Y., Kaneko S., Honda M., Unoura M., Cheong J., Harada A., Matsushima K., Kobayashi K., Murakami S. 1994; Detection of the putative E2 protein of hepatitis C virus in human liver. J Med Virol 42:374–379 [CrossRef]
     [Google Scholar]
  40. Owsianka A., Clayton R. F., Price L. D. L., McKeating J. A., Patel A. H. 2001; Functional analysis of hepatitis C virus E2 glycoproteins and virus-like particles reveals structural dissimilarities between different forms of E2. J Gen Virol 82:1877–1883
     [Google Scholar]
  41. Papatheodoridis G. V., Patch D., Dusheiko G. M., Burroughs A. K. 1999; The outcome of hepatitis C virus infection after liver transplantation – is it influenced by the type of immunosuppression. J Hepatol 30:731–738 [CrossRef]
     [Google Scholar]
  42. Pietschmann T., Lohmann V., Kaul A., Krieger N., Rinck G., Rutter G., Strand D., Bartenschlager R. 2002; Persistent and transient replication of full-length hepatitis C virus genomes in cell culture. J Virol 76:4008–4021 [CrossRef]
     [Google Scholar]
  43. Prince A. M., Byron T. H., Parker T. S., Levine D. M. 1996; Visualization of hepatitis C virions and putative defective interfering particles isolated from low-density lipoproteins. J Viral Hepat 3:11–17 [CrossRef]
     [Google Scholar]
  44. Puig M., Mihalik K., Yu M. W., Feinstone S. M., Major M. E. 2002; Sensitivity and reproducibility of HCV quantitation in chimpanzee sera using TaqMan real-time PCR assay. J Virol Methods 105:253–263 [CrossRef]
     [Google Scholar]
  45. Ralston R., Thudium K., Berger K. 7 other authors 1993; Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses. J Virol 67:6753–6761
     [Google Scholar]
  46. Sakamoto N., Enomoto N., Kurosaki M., Marumo F., Sato C. 1994; Detection and quantification of hepatitis C virus RNA replication in the liver. J Hepatol 20:593–597 [CrossRef]
     [Google Scholar]
  47. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  48. Sarrazin C., Hendricks D. A., Sedarati F., Zeuzem S. 2001; Asseessment, by transcription-mediated amplification, of virologic response in patients with chronic hepatitis C virus treated with peginterferon alpha-2a. J Clin Microbiol 39:2850–2855 [CrossRef]
     [Google Scholar]
  49. Schlaak J. F., Trippler M., Ernst I., Meyer-Büschenfelde K. H., Gerken G. 1997; Chronic hepatitis C: the viral load per liver cell before treatment as a new marker to predict long-term response to IFN- α therapy. J Hepatol 27:917–921 [CrossRef]
     [Google Scholar]
  50. Shoji I., Suzuki T., Sato M., Aizaki H., Chiba T., Matsuura Y., Miyamura T. 1999; Internal processing of hepatitis C virus NS3 protein. Virology 254:315–323 [CrossRef]
     [Google Scholar]
  51. Simpson R. J., Reid G. E. 1998; Sequence analysis of gel-resolved proteins. In Gel Electrophoresis of Proteins , 3rd edn. pp  237–269 Edited by Hames B. D. Oxford, UK: Oxford University Press;
     [Google Scholar]
  52. Smith M. S. H., Webster A. D. B., Dhillon A. P., Dusheiko G., Boulton R., Savage K., Rolles K., Burroughs A. K. 1995; Orthotopic liver transplantation for chronic hepatitis in two patients with common variable immunodeficiency. Gastroenterology 108:879–884 [CrossRef]
     [Google Scholar]
  53. Terrault N. A., Dailey P. J., Ferrell L., Collins M. L., Wilber J. C., Urdea M. S., Bhandari B. N., Wright T. L. 1997; Hepatitis C virus: quantitation and distribution in liver. J Med Virol 51:217–224 [CrossRef]
     [Google Scholar]
  54. Thomssen R., Bonk S., Propfe C., Heermann K. H., Köchel H. G., Uy A. 1992; Association of hepatitis C virus in human sera with β -lipoprotein. Med Microbiol Immunol 181:293–300 [CrossRef]
     [Google Scholar]
  55. Thomssen R., Bonk S., Thiele A. 1993; Density heterogeneities of hepatitis C virus in human sera due to the binding of β -lipoproteins and immunoglobulins. Med Microbiol Immunol 182:329–334
     [Google Scholar]
  56. Watson J. P. 1996; Hepatitis C virus: studies of the molecular basis of virus/host interaction . PhD thesis University of Newcastle upon Tyne; UK:
  57. Widell A., Zhang Y. Y., Gäre B. A., Hammarström L. 1997; At least three hepatitis C virus strains implicated in Swedish and Danish patients with intravenous immunoglobulin associated hepatitis C. Transfusion 37:313–320 [CrossRef]
     [Google Scholar]
  58. Yasui K., Wakita T., Kohara K. T., Funahashi S. I., Ichikawa M., Kajita T., Moradpour D., Wands J. R., Kohara M. 1998; The native form and maturation process of hepatitis C virus core protein. J Virol 72:6048–6055
     [Google Scholar]
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Characterization of the genome and structural proteins of hepatitis C virus resolved from infected human liver
J. Gen. Virol. 85, 1497 (2004); https://doi.org/10.1099/vir.0.79967-0
/content/journal/jgv/10.1099/vir.0.79967-0
/content/journal/jgv/10.1099/vir.0.79967-0
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