1887

Abstract

Hepatitis C virus (HCV) particles found are heterogeneous in density and size, but their detailed characterization has been restricted by the low titre of HCV in human serum. Previously, our group has found that HCV circulates in blood in association with very-low-density lipoprotein (VLDL). Our aim in this study was to characterize HCV RNA-containing membranes and particles in human liver by both density and size and to identify the subcellular compartment(s) where the association with VLDL occurs. HCV was purified by density using iodixanol gradients and by size using gel filtration. Both positive-strand HCV RNA (present in virus particles) and negative-strand HCV RNA (an intermediate in virus replication) were found with densities below 1.08 g ml. Viral structural and non-structural proteins, host proteins ApoB, ApoE and caveolin-2, as well as cholesterol, triglyceride and phospholipids were also detected in these low density fractions. After fractionation by size with Superose gel filtration, HCV RNA and viral proteins co-fractionated with endoplasmic reticulum proteins and VLDL. Fractionation on Toyopearl, which separates particles with diameters up to 200 nm, showed that 78 % of HCV RNA from liver was >100 nm in size, with a positive-/negative-strand ratio of 6 : 1. Also, 8 % of HCV RNA was found in particles with diameters between 40 nm and 70 nm and a positive-/negative-strand ratio of 45 : 1. This HCV was associated with ApoB, ApoE and viral glycoprotein E2, similar to viral particles circulating in serum. Our results indicate that the association between HCV and VLDL occurs in the liver.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.2008/000083-0
2008-10-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/89/10/2507.html?itemId=/content/journal/jgv/10.1099/vir.0.2008/000083-0&mimeType=html&fmt=ahah

References

  1. Aizaki H., Lee K. J., Sung V. M. H., Ishiko H., Lai M. M. C. 2004; Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts. Virology 324:450–461 [CrossRef]
    [Google Scholar]
  2. André P., Komurian-Pradel F., Deforges S., Perret M., Berland J. L., Sodoyer M., Pol S., Brechot C., Paranhos-Baccala G., Lotteau V. 2002; Characterization of low- and very-low-density hepatitis C virus RNA-containing particles. J Virol 76:6919–6928 [CrossRef]
    [Google Scholar]
  3. Anonymous 2002 Gel filtration, Principles and Methods Uppsala, Sweden: Amersham Biosciences;
    [Google Scholar]
  4. Chang M., Williams O., Mittler J., Quintanilla A., Carithers R. L., Perkins J., Corey L., Gretch D. R. 2003; Dynamics of hepatitis C virus replication in human liver. Am J Pathol 163:433–444 [CrossRef]
    [Google Scholar]
  5. Chang K.-S., Jiang J., Cai Z., Luo G. 2007; Human apolipoprotein E is required for infectivity and production of hepatitis C virus in cell culture. J Virol 81:13783–13793 [CrossRef]
    [Google Scholar]
  6. Christie J. M. L., Healey C. J., Watson J., Wong V. S., Duddridge M., Snowden N., Rosenberg W. M. C., Fleming K. A., Chapel H., Chapman R. W. G. 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]
  7. Diaz O., Delers F., Maynard M., Demignot S., Zoulim F., Chambaz J., Trépo C., Lotteau V., André P. 2006; Preferential association of hepatitis C virus with apolipoprotein B48-containing lipoproteins. J Gen Virol 87:2983–2991 [CrossRef]
    [Google Scholar]
  8. Dubuisson J., Penin F., Moradpour D. 2002; Interaction of hepatitis C virus proteins with host cell membranes and lipids. Trends Cell Biol 12:517–523 [CrossRef]
    [Google Scholar]
  9. El-Hage N., Luo G. 2003; Replication of hepatitis C virus RNA occurs in a membrane-bound replication complex containing nonstructural viral proteins and RNA. J Gen Virol 84:2761–2769 [CrossRef]
    [Google Scholar]
  10. Fenwick F., Bassendine M. F., Agarwal K., Bevitt D. J., Pumeechockchai W., Burt A. D., Toms G. L. 2006; Immunohistochemical assessment of hepatitis C virus antigen in cholestatic hepatitis after liver transplantation. J Clin Pathol 59:174–178 [CrossRef]
    [Google Scholar]
  11. Folch J., Lees M., Stanley G. H. S. 1957; A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497–509
    [Google Scholar]
  12. Gastaminza P., Kapadia S., Chisari F. V. 2006; Differential biophysical properties of infectious intracellular and secreted hepatitis C virus particles. J Virol 80:11074–11081 [CrossRef]
    [Google Scholar]
  13. Gastaminza P., Cheng G., Wieland S., Zhong J., Liao W., Chisari F. V. 2008; Cellular determinants of hepatitis C virus assembly, maturation, degradation and secretion. J Virol 82:2120–2129 [CrossRef]
    [Google Scholar]
  14. Gosert R., Egger D., Lohmann V., Bartenschlager R., Blum H. E., Bienz K., Moradpour D. 2003; Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J Virol 77:5487–5492 [CrossRef]
    [Google Scholar]
  15. Graham J., Ford T., Rickwood D. 1994; The preparation of subcellular organelles from mouse liver in self-generated gradients of iodixanol. Anal Biochem 220:367–373 [CrossRef]
    [Google Scholar]
  16. Huang H., Sun F., Owen D. M., Li W., Chen Y., Gale M., Ye J. 2007; Hepatitis C virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins. Proc Natl Acad Sci U S A 104:5848–5853 [CrossRef]
    [Google Scholar]
  17. Ishida S., Kaito M., Kohara M., Tsukiyama-Kohora K., Fujita N., Ikoma J., Adachi Y., Watanabe S. 2001; Hepatitis C virus core particle detected by immunoelectron microscopy and optical rotation technique. Hepatol Res 20:335–347 [CrossRef]
    [Google Scholar]
  18. Kalamvoki M., Georgopoulou U., Mavromara P. 2006; The NS5A protein of the hepatitis C virus genotype 1a is cleaved by caspases to produce C-terminal-truncated forms of the protein that reside mainly in the cytosol. J Biol Chem 281:13449–13462 [CrossRef]
    [Google Scholar]
  19. Kanto T., Hayashi N., Takehara T., Hagiwara H., Mita E., Naito M., Kasahara A., Fusamoto H., Kamada T. 1995; Density analysis of hepatitis C virus particle population in the circulation of infected hosts: implications for virus neutralization or persistence. J Hepatol 22:440–448 [CrossRef]
    [Google Scholar]
  20. Kapadia S. B., Chisari F. V. 2005; Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids. Proc Natl Acad Sci U S A 102:2561–2566 [CrossRef]
    [Google Scholar]
  21. Kato T., Furusaka A., Miyamoto M., Date T., Yasui K., Hiramoto J., Nagayama K., Tanaka T., Wakita T. 2001; Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient. J Med Virol 64:334–339 [CrossRef]
    [Google Scholar]
  22. Komurian-Pradel F., Perret M., Deiman B., Sodoyer M., Lotteau V., Paranhos-Baccala G., André P. 2004; Strand-specific quantitative real-time PCR to study replication of hepatitis C virus genome. J Virol Methods 116:103–106 [CrossRef]
    [Google Scholar]
  23. Lindenbach B. D., Rice C. M. 2001; Flaviviridae : the viruses and their replication. In Fields Virology, 4th edn. vol 2 pp 991–1041Edited by Knipe D. M. Philadelphia: Lippincott-Raven;
    [Google Scholar]
  24. Lindenbach B. D., Evans M. J., Syder A. J., Wolk B., Tellinghuisen T. L., Liu C. C., Maruyama T., Hynes R. O., Burton D. R. other authors 2005; Complete replication of hepatitis C virus in cell culture. Science 309:623–626 [CrossRef]
    [Google Scholar]
  25. Lohmann V., Korner F., Koch J. O., Herian U., Theilmann L., Bartenschlager R. 1999; Replication of subgenomic hepatits C virus RNAs in a hepatoma cell line. Science 285:110–113 [CrossRef]
    [Google Scholar]
  26. Mackness M. I., Durrington P. N. 1992; Lipoprotein separation and analysis for clinical studies. In Lipoprotein Analysis: A Practical Approach pp 1–42Edited by Converse C. A., Skinner E. R. Oxford: Oxford University Press;
    [Google Scholar]
  27. März W., Siekmeier R., Scharnagl H., Seiffert U. B., Gross W. 1993; Fast lipoprotein chromatography: new method of analysis for plasma lipoproteins. Clin Chem 39:2276–2281
    [Google Scholar]
  28. Mercier B., Burlot L., Ferec C. 1999; Simultaneous screening for HBV DNA and HCV RNA genomes in blood donations using a novel TaqMan assay. J Virol Methods 77:1–9 [CrossRef]
    [Google Scholar]
  29. Miyanari Y., Atsuzawa K., Usuda N., Watashi K., Hishiki T., Zayas M., Bartenschlager R., Wakita T., Hijikata M., Shimotohno K. 2007; The lipid droplet is an important organelle for hepatitis C virus production. Nat Cell Biol 9:1089–1097 [CrossRef]
    [Google Scholar]
  30. Moradpour D., Penin F., Rice C. M. 2007; Replication of hepatitis C virus. Nat Rev Microbiol 5:453–463 [CrossRef]
    [Google Scholar]
  31. Neddermann P., Clementi A., De Francesco R. 1999; Hyperphosphorylation of the hepatitis C virus NS5A protein requires an active NS3 protease, NS4A, NS4B, and NS5A encoded on the same polyprotein. J Virol 73:9984–9991
    [Google Scholar]
  32. Negro F., Giostra G., Krawczynski K., Quadri R., Brandt L. R., Mentha G., Colucci G., Perrin L., Hadengue A. 1998; Detection of intrahepatic hepatitis C virus replication by strand-specific semi-quantitative RT-PCR: preliminary application to the liver transplantation model. J Hepatol 29:1–11 [CrossRef]
    [Google Scholar]
  33. Nielsen S. U., Bassendine M. F., Burt A. D., Bevitt D. J., Toms G. L. 2004; Characterization of the genome and structural proteins of hepatitis C virus resolved from infected human liver. J Gen Virol 85:1497–1507 [CrossRef]
    [Google Scholar]
  34. Nielsen S. U., Bassendine M. F., Burt A. D., Martin C., Pumeechockchai W., Toms G. L. 2006; Association between hepatitis C virus and very low-density lipoprotein (VLDL)/LDL analyzed in iodixanol density gradients. J Virol 80:2418–2428 [CrossRef]
    [Google Scholar]
  35. Nomura-Takigawa Y., Nagano-Fujii M., Deng L., Kitazawa S., Ishido S., Sada K., Hotta H. 2006; Non-structural protein 4A of hepatitis C virus accumulates on mitochondria and renders the cells prone to undergoing mitochondria-mediated apoptosis. J Gen Virol 87:1935–1945 [CrossRef]
    [Google Scholar]
  36. Okazaki M., Usui S., Ishigami M., Sakai N., Nakamura T., Matsuzawa Y., Yamashita S. 2005; Identification of unique lipoprotein subclasses for visceral obesity by component analysis of cholesterol profile in high performance liquid chromatography. Arterioscler Thromb Vasc Biol 25:578–584 [CrossRef]
    [Google Scholar]
  37. Petit M. A., Lièvre M., Peyrol S., De Sequeira S., Berthillon P., Ruigrok R. W. H., Trépo C. 2005; Enveloped particles in the serum of chronic hepatitis C patients. Virology 336:144–153 [CrossRef]
    [Google Scholar]
  38. 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]
  39. Plonne D., Cartwright I., Lins W., Dargel R., Graham J. M., Higgins J. A. 1999; Separation of the intracellular secretory compartment of rat liver and isolated rat hepatocytes in a single step using self-generating gradients of iodixanol. Anal Biochem 276:88–96 [CrossRef]
    [Google Scholar]
  40. Pownall H. J., Gotto A. M., Packard C. J., Shepherd J., Redgrave T. G., Ginsberg H. N., Dixon J. L., Goldberg I. J., Eisenberg S. other authors 1999; Plasma lipoproteins, chemistry and biology. In Lipoproteins in Health and Disease pp 1–110Edited by Betteridge D. J., Illingworth D. R., Shepherd. London: Arnold;
    [Google Scholar]
  41. 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]
  42. Pumeechockchai W., Bevitt D. J., Agarwal K., Petropoulou T., Langer B. C. A., Belohradsky B., Bassendine M. F., Toms G. L. 2002; Hepatitis C virus particles of different density in the blood of chronically infected immuncompetent and immunodeficient patients: implications for virus clearance by antibody. J Med Virol 68:335–342 [CrossRef]
    [Google Scholar]
  43. Quinkert D., Bartenschlager R., Lohmann V. 2005; Quantitative analysis of the hepatitis C virus replication complex. J Virol 79:13594–13605 [CrossRef]
    [Google Scholar]
  44. Roingeard P., Hourioux C., Blanchard E., Brand D., Goughoulte M. A. 2004; Hepatitis C virus ultrastructure and morphogenesis. Biol Cell 96:103–108 [CrossRef]
    [Google Scholar]
  45. Rustaeus S., Lindberg K., Stillemark P., Claesson C., Asp L., Larsson T., Boren J., Olofsson S.-O. 1999; Assembly of very low density lipoprotein: a two-step process of apolipoprotein B core lipidation. J Nutr 129:463S–466S
    [Google Scholar]
  46. Shavinskaya A., Boulant S., Penin F., McLauchlan J., Bartenschlager R. 2007; The lipid droplet binding domain of hepatitis C virus core protein is a major determinant for efficient virus assembly. J Biol Chem 282:37158–37169 [CrossRef]
    [Google Scholar]
  47. Shi S. T., Lee K. J., Aizaki H., Hwang S. B., Lai M. M. C. 2003; Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2. J Virol 77:4160–4168 [CrossRef]
    [Google Scholar]
  48. Targett-Adams P., Chambers D., Gledhill S., Hope R. G., Coy J. F., Girod A., McLauchlan J. 2003; Live cell analysis and targeting of the lipid droplet-binding adipocyte differentiation-related protein. J Biol Chem 278:15998–16007 [CrossRef]
    [Google Scholar]
  49. 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]
  50. Tran K., Thorne-Tjomsland G., DeLong C. J., Cui Z., Shan J., Burton L., Jamieson J. C., Yao Z. 2002; Intracellular assembly of very low density lipoproteins containing apolipoprotein B100 in rat hepatoma McA-RH7777 cells. J Biol Chem 277:31187–31200 [CrossRef]
    [Google Scholar]
  51. Uchil P. D., Satchidanandam V. 2003; Architecture of the flaviviral replication complex. J Biol Chem 278:24388–24398 [CrossRef]
    [Google Scholar]
  52. Wakita T., Pietschmann T., Kato T., Date T., Miyamoto M., Zhao Z., Murthy K., Habermann A., Kräusslich H. G. other authors 2005; Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11:791–796 [CrossRef]
    [Google Scholar]
  53. Waris G., Sarker S., Siddiqui A. 2004; Two-step affinity purification of the hepatitis C virus ribonucleoprotein complex. RNA 10:321–329 [CrossRef]
    [Google Scholar]
  54. Westaway E. G., Mackenzie J. M., Kenney M. T., Jones M. K., Khromykh A. A. 1997; Ultrastructure of Kunjin virus infected cells: co-localisation of NS1 and NS3 with double stranded RNA, and of NS2B with NS3, in virus induced membrane structures. J Virol 71:6650–6661
    [Google Scholar]
  55. Yao H., Ye J. 2008; Long chain acyl-coA synthetase 3-mediated phosphatidylcholine synthesis is required for assembly of very low density lipoproteins in human hepatoma Huh7 cells. J Biol Chem 283:849–854 [CrossRef]
    [Google Scholar]
  56. Zahn A., Allain J.-P. 2005; Hepatitis C virus and hepatitis B virus bind to heparin: purification of largely IgG-free virions from infected plasma by heparin chromatography. J Gen Virol 86:677–685 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.2008/000083-0
Loading
/content/journal/jgv/10.1099/vir.0.2008/000083-0
Loading

Data & Media loading...

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