Like most other positive-strand RNA viruses, hepatitis C virus (HCV) induces changes in the host cell's membranes, resulting in a membranous web. The non-structural proteins of the viral replication complex are thought to be associated with these newly synthesized membranes. We studied this phenomenon, using a Huh7.5 cell clone displaying high levels of replication of a subgenomic replicon of the JFH-1 strain. Electron microscopy of ultrathin sections of these cells revealed the presence of numerous double membrane vesicles (DMVs), resembling those observed for other RNA viruses such as poliovirus and coronavirus. Some sections had more discrete multivesicular units consisting of circular concentric membranes organized into clusters surrounded by a wrapping membrane. These structures were highly specific to HCV as they were not detected in naive Huh7.5 cells. Preparations enriched in these structures were separated from other endoplasmic reticulum-derived membranes by cell cytoplasm homogenization and ultracentrifugation on a sucrose gradient. They were found to contain the non-structural NS3 and NS5A HCV proteins, HCV RNA and LC3-II, a specific marker of autophagic membranes. By analogy to other viral models, HCV may induce DMVs by activating the autophagy pathway. This could represent a strategy to conceal the viral RNA and help the virus to evade double-stranded RNA-triggered host antiviral responses. More detailed characterization of these virus–cell interactions may facilitate the development of new treatments active against HCV and other RNA viruses that are dependent on newly synthesized cellular membranes for replication.


Article metrics loading...

Loading full text...

Full text loading...



  1. Ait-Goughoulte, M., Kanda, T., Meyer, K., Ryerse, J. S., Ray, R. B. & Ray, R.(2008). Hepatitis C virus genotype 1a growth and induction of autophagy. J Virol 82, 2241–2249.[CrossRef] [Google Scholar]
  2. Blight, K. J., Kolykhalov, A. A. & Rice, C. M.(2000). Efficient initiation of HCV RNA replication in cell culture. Science 290, 1972–1974.[CrossRef] [Google Scholar]
  3. Blight, K. J., McKeating, J. A. & Rice, C. M.(2002). Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 76, 13001–13014.[CrossRef] [Google Scholar]
  4. Chapel, C., Garcia, C., Bartosch, B., Roingeard, P., Zitzmann, N., Cosset, F. L., Dubuisson, J., Dwek, R. A., Trépo, C. & other authors(2007). Reduction of the infectivity of hepatitis C virus pseudoparticles by incorporation of misfolded glycoproteins induced by glucosidase inhibitors. J Gen Virol 88, 1133–1143.[CrossRef] [Google Scholar]
  5. Chevaliez, S., Bouvier-Alias, M. & Pawlotsky, J. M.(2009). Performance of the Abbott real-time PCR assay using m2000sp and m2000rt for hepatitis C virus RNA quantification. J Clin Microbiol 47, 1726–1732.[CrossRef] [Google Scholar]
  6. Chu, P. W. & Westaway, E. G.(1992). Molecular and ultrastructural analysis of heavy membrane fractions associated with the replication of Kunjin virus RNA. Arch Virol 125, 177–191.[CrossRef] [Google Scholar]
  7. Chu, P. W., Westaway, E. G. & Coia, G.(1992). Comparison of centrifugation methods for molecular and morphological analysis of membranes associated with RNA replication of the flavivirus Kunjin. J Virol Methods 37, 219–234.[CrossRef] [Google Scholar]
  8. David-Ferreira, J. F. & Manaker, R. A.(1965). An electron microscope study of the development of a mouse hepatitis virus in tissue culture cells. J Cell Biol 24, 57–78.[CrossRef] [Google Scholar]
  9. Dreux, M., Gastaminza, P., Wieland, S. F. & Chisari, F. V.(2009). The autophagy machinery is required to initiate hepatitis C virus replication. Proc Natl Acad Sci U S A 106, 14046–14051.[CrossRef] [Google Scholar]
  10. Egger, D., Pasamontes, L., Bolten, R., Boyko, V. & Bienz, K.(1996). Reversible dissociation of the poliovirus replication complex: functions and interactions of its components in viral RNA synthesis. J Virol 70, 8675–8683. [Google Scholar]
  11. Egger, D., Wölk, B., Gosert, R., Bianchi, L., Blum, H. E., Moradpour, D. & Bienz, K.(2002). Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J Virol 76, 5974–5984.[CrossRef] [Google Scholar]
  12. 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]
  13. Gosert, R., Egger, D., Lohmann, V., Bartenschlager, R., Blum, B. H., 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]
  14. Hourioux, C., Patient, R., Morin, A., Blanchard, E., Moreau, A., Trassard, S., Giraudeau, B. & Roingeard, P.(2007). The genotype 3-specific hepatitis C virus core protein residue phenylalanine 164 increases steatosis in an in vitro cellular model. Gut 56, 1302–1308.[CrossRef] [Google Scholar]
  15. Huang, H., Sun, F., Owen, D. M., Li, W., Chen, Y., Gale, M., Jr & 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]
  16. Jackson, W. T., Giddings, T. H., Jr, Taylor, M. P., Mulinyawe, S., Rabinovitch, M., Kopito, R. R. & Kirkegaard, K.(2005). Subversion of cellular autophagosomal machinery by RNA viruses. PLoS Biol 3, e156[CrossRef] [Google Scholar]
  17. Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y. & Yoshimori, T.(2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19, 5720–5728.[CrossRef] [Google Scholar]
  18. Kato, T., Date, T., Miyamoto, M., Furusaka, A., Tokushige, K., Mizokami, M. & Wakita, T.(2003). Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 125, 1808–1817.[CrossRef] [Google Scholar]
  19. Kirkegaard, K., Taylor, M. P. & Jackson, W. T.(2004). Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nat Rev Microbiol 2, 301–314.[CrossRef] [Google Scholar]
  20. Knoops, K., Kikkert, M., Worm, S. H., Zevenhoven-Dobbe, J. C., van der Meer, Y., Koster, A. J., Mommaas, A. M. & Snijder, E. J.(2008). SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol 6, e226[CrossRef] [Google Scholar]
  21. Krijnse-Locker, J., Ericsson, M., Rottier, P. J. & Griffiths, G.(1994). Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step. J Cell Biol 124, 55–70.[CrossRef] [Google Scholar]
  22. Lee, Y. R., Lei, H. Y., Liu, M. T., Wang, J. R., Chen, S. H., Jiang-Shieh, Y. F., Lin, Y. S., Yeh, T. M., Liu, C. C. & Liu, H. S.(2008). Autophagic machinery activated by dengue virus enhances virus replication. Virology 374, 240–248.[CrossRef] [Google Scholar]
  23. Lindenbach, B. D., Evans, M. J., Syder, A. J., Wölk, 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]
  24. Lohmann, V., Körner, F., Koch, J., Herian, U., Theilmann, L. & Bartenschlager, R.(1999). Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113.[CrossRef] [Google Scholar]
  25. Lyle, J. M., Clewell, A., Richmond, K., Richards, O. C., Hope, D. A., Schultz, S. C. & Kirkegaard, K.(2002). Similar structural basis for membrane localization and protein priming by an RNA-dependent RNA polymerase. J Biol Chem 277, 16324–16331.[CrossRef] [Google Scholar]
  26. Mackenzie, J.(2005). Wrapping things up about virus RNA replication. Traffic 6, 967–977.[CrossRef] [Google Scholar]
  27. Mackenzie, J. M., Jones, M. K. & Young, P. R.(1996). Immunolocalization of the dengue virus nonstructural glycoprotein NS1 suggests a role in viral RNA replication. Virology 220, 232–240.[CrossRef] [Google Scholar]
  28. Miller, S. & Krijnse-Locker, J.(2008). Modification of intracellular membrane structures for virus replication. Nat Rev Microbiol 6, 363–374.[CrossRef] [Google Scholar]
  29. Miyanari, Y., Hijikata, M., Yamaji, M., Hosaka, M., Takahashi, H. & Shimotohno, K.(2003). Hepatitis C virus non-structural proteins in the probable membranous compartment function in viral genome replication. J Biol Chem 278, 50301–50308.[CrossRef] [Google Scholar]
  30. Pawlotsky, J. M., Chevaliez, S. & McHutchison, J. G.(2007). The hepatitis C virus life cycle as a target for new antiviral therapies. Gastroenterology 132, 1979–1998.[CrossRef] [Google Scholar]
  31. Penin, F., Dubuisson, J., Rey, F. A., Moradpour, D. & Pawlotsky, J. M.(2004). Structural biology of hepatitis C virus. Hepatology 39, 5–19.[CrossRef] [Google Scholar]
  32. Prentice, E., Jerome, W. G., Yoshimori, T., Mizushima, N. & Denison, M. R.(2004). Coronavirus replication complex formation utilizes components of cellular autophagy. J Biol Chem 279, 10136–10141.[CrossRef] [Google Scholar]
  33. Quinkert, D., Bartenschlager, R. & Lohmann, V.(2005). Quantitative analysis of the hepatitis C virus replication complex. J Virol 79, 13594–13605.[CrossRef] [Google Scholar]
  34. Rehermann, B.(2009). Hepatitis C virus versus innate and adaptive immune responses: a tale of coevolution and coexistence. J Clin Invest 119, 1745–1754.[CrossRef] [Google Scholar]
  35. Schlegel, A., Giddings, T. H., Jr, Ladinsky, M. S. & Kirkegaard, K.(1996). Cellular origin and ultrastructure of membranes induced during poliovirus infection. J Virol 70, 6576–6588. [Google Scholar]
  36. Schwartz, M., Chen, J., Janda, M., Sullivan, M., den Boon, J. & Ahlquist, P.(2002). A positive-strand RNA virus replication complex parallels form and function of retrovirus capsids. Mol Cell 9, 505–514.[CrossRef] [Google Scholar]
  37. Shepard, C. W., Finelli, L. & Alter, M. J.(2005). Global epidemiology of hepatitis C virus infection. Lancet Infect Dis 5, 558–567.[CrossRef] [Google Scholar]
  38. Sir, D., Chen, W. L., Choi, J., Wakita, T., Yen, T. S. & Ou, J. H.(2008). Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response. Hepatology 48, 1054–1061.[CrossRef] [Google Scholar]
  39. Snijder, E. J., van der Meer, Y., Zevenhoven-Dobbe, J., Onderwater, J. J., van der Meulen, J., Koerten, H. K. & Mommaas, A. M.(2006). Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex. J Virol 80, 5927–5940.[CrossRef] [Google Scholar]
  40. Stone, M., Jia, S., Heo, W. D., Meyer, T. & Konan, K. V.(2007). Participation of rab5, an early endosome protein, in hepatitis C virus RNA replication machinery. J Virol 81, 4551–4563.[CrossRef] [Google Scholar]
  41. Tanida, I., Fukasawa, M., Ueno, T., Kominami, E., Wakita, T. & Hanada, K.(2009). Knockdown of autophagy-related gene decreases the production of infectious hepatitis C virus particles. Autophagy 5, 937–945.[CrossRef] [Google Scholar]
  42. Targett-Adams, P., Boulant, S. & McLauchlan, J.(2008). Visualization of double-stranded RNA in cells supporting hepatitis C virus RNA replication. J Virol 82, 2182–2195.[CrossRef] [Google Scholar]
  43. Taylor, M. P. & Kirkegaard, K.(2007). Modification of cellular autophagy protein LC3 by poliovirus. J Virol 81, 12543–12553.[CrossRef] [Google Scholar]
  44. Taylor, M. P. & Kirkegaard, K.(2008). Potential subversion of autophagosomal pathway by picornaviruses. Autophagy 4, 286–289.[CrossRef] [Google Scholar]
  45. Uchil, P. D. & Satchidanandam, V.(2003). Architecture of the flaviviral replication complex. Protease, nuclease, and detergents reveal encasement within double-layered membrane compartments. J Biol Chem 278, 24388–24398.[CrossRef] [Google Scholar]
  46. 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]
  47. Welsch, S., Miller, S., Romero-Brey, I., Merz, A., Bleck, C. K., Walther, P., Fuller, S. D., Antony, C., Krijnse-Locker, J. & Bartenschlager, R.(2009). Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe 5, 365–375.[CrossRef] [Google Scholar]
  48. Westaway, E. G., Mackenzie, J. M., Kenney, M. T., Jones, M. K. & Khromykh, A. A.(1997). Ultrastructure of Kunjin virus-infected cells: colocalization 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]
  49. Wong, J., Zhang, J., Si, X., Gao, G., Mao, I., McManus, B. M. & Luo, H.(2008). Autophagosome supports coxsackievirus B3 replication in host cells. J Virol 82, 9143–9153.[CrossRef] [Google Scholar]
  50. Zhong, J., Gastaminza, P., Cheng, G., Kapadia, S., Kato, T., Burton, D. R., Wieland, S. F., Uprichard, S. L., Wakita, T. & Chisari, F. V.(2005). Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A 102, 9294–9299.[CrossRef] [Google Scholar]

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