Comparison of U2OS and Huh-7 cells for identifying host factors that affect hepatitis C virus RNA replication Free

Abstract

Host cell factors are critical to all stages of the hepatitis C virus (HCV) life cycle. While many cellular proteins that regulate HCV genome synthesis have been identified, the mechanisms engaged in this process are incompletely understood. To identify novel cellular proteins involved in HCV RNA replication, we screened a library of small interfering RNAs (siRNAs) targeting 299 cellular factors, which principally function in RNA interactions. For the screen, a robust system was established using two cell lines (derived from Huh-7 and U2OS cells) that replicated tricistronic subgenomic replicons (SGRs). We found that the U2OS cell line gave lower levels of intracellular HCV RNA replication compared with Huh-7 cells and was more readily transfected by siRNAs. Consequently, increased gene silencing and greater effects on HCV replication were observed in the U2OS cell line. Thus, U2OS cells provided a suitable and more sensitive alternative to Huh-7 cells for siRNA studies on HCV RNA replication. From the screen, several cellular proteins that enhanced and suppressed HCV RNA replication were identified. One of the genes found to downregulate viral RNA synthesis, ISG15, is expressed in response to alpha interferon and may therefore partly contribute to the clearance of virus from infected individuals. A second gene that inhibited HCV RNA levels was the 5′–3′ exoRNase XRN1, which suggested a role for cellular RNA degradation pathways in modulating the abundance of viral genomes. Therefore, this study provides an important framework for future detailed analyses of these and other cellular proteins.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.022210-0
2010-09-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/91/9/2238.html?itemId=/content/journal/jgv/10.1099/vir.0.022210-0&mimeType=html&fmt=ahah

References

  1. Anderson, P. & Kedersha, N.(2006). RNA granules. J Cell Biol 172, 803–808.[CrossRef] [Google Scholar]
  2. Berger, K. L., Cooper, J. D., Heaton, N. S., Yoon, R., Oakland, T. E., Jordan, T. X., Mateu, G., Grakoui, A. & Randall, G.(2009). Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication. Proc Natl Acad Sci U S A 106, 7577–7582.[CrossRef] [Google Scholar]
  3. Chable-Bessia, C., Meziane, O., Latreille, D., Robinson, R. T., Zamborlini, A., Wagschal, A., Jacquet, J. M., Reynes, J., Levy, Y. & other authors(2009). Suppression of HIV-1 replication by microRNA effectors. Retrovirology 6, 26[CrossRef] [Google Scholar]
  4. Chevalier, C., Saulnier, A., Benureau, Y., Flechet, D., Delgrange, D., Colbere-Garapin, F., Wychowski, C. & Martin, A.(2007). Inhibition of hepatitis C virus infection in cell culture by small interfering RNAs. Mol Ther 15, 1452–1462.[CrossRef] [Google Scholar]
  5. Coute, Y., Kindbeiter, K., Belin, S., Dieckmann, R., Duret, L., Bezin, L., Sanchez, J. C. & Diaz, J. J.(2008). ISG20L2, a novel vertebrate nucleolar exoribonuclease involved in ribosome biogenesis. Mol Cell Proteomics 7, 546–559. [Google Scholar]
  6. Egger, D., Wolk, 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]
  7. Fraser, C. S. & Doudna, J. A.(2007). Structural and mechanistic insights into hepatitis C viral translation initiation. Nat Rev Microbiol 5, 29–38.[CrossRef] [Google Scholar]
  8. 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]
  9. Honda, M., Brown, E. A. & Lemon, S. M.(1996a). Stability of a stem-loop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA. RNA 2, 955–968. [Google Scholar]
  10. Honda, M., Ping, L. H., Rijnbrand, R. C., Amphlett, E., Clarke, B., Rowlands, D. & Lemon, S. M.(1996b). Structural requirements for initiation of translation by internal ribosome entry within genome-length hepatitis C virus RNA. Virology 222, 31–42.[CrossRef] [Google Scholar]
  11. Ji, H., Fraser, C. S., Yu, Y., Leary, J. & Doudna, J. A.(2004). Coordinated assembly of human translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA. Proc Natl Acad Sci U S A 101, 16990–16995.[CrossRef] [Google Scholar]
  12. Jones, D. M., Patel, A. H., Targett-Adams, P. & McLauchlan, J.(2009). The hepatitis C virus NS4B protein can trans-complement viral RNA replication and modulates production of infectious virus. J Virol 83, 2163–2177.[CrossRef] [Google Scholar]
  13. Kanaoka, Y. & Nojima, H.(1994). SCR: novel human suppressors of cdc2/cdc13 mutants of Schizosaccharomyces pombe harbour motifs for RNA binding proteins. Nucleic Acids Res 22, 2687–2693.[CrossRef] [Google Scholar]
  14. Kanda, T., Steele, R., Ray, R. & Ray, R. B.(2007). Small interfering RNA targeted to hepatitis C virus 5′ nontranslated region exerts potent antiviral effect. J Virol 81, 669–676.[CrossRef] [Google Scholar]
  15. Kapadia, S. B., Brideau-Andersen, A. & Chisari, F. V.(2003). Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc Natl Acad Sci U S A 100, 2014–2018.[CrossRef] [Google Scholar]
  16. Kronke, J., Kittler, R., Buchholz, F., Windisch, M. P., Pietschmann, T., Bartenschlager, R. & Frese, M.(2004). Alternative approaches for efficient inhibition of hepatitis C virus RNA replication by small interfering RNAs. J Virol 78, 3436–3446.[CrossRef] [Google Scholar]
  17. 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]
  18. Lohmann, V., Hoffmann, S., Herian, U., Penin, F. & Bartenschlager, R.(2003). Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. J Virol 77, 3007–3019.[CrossRef] [Google Scholar]
  19. Malakhova, O. A. & Zhang, D. E.(2008). ISG15 inhibits Nedd4 ubiquitin E3 activity and enhances the innate antiviral response. J Biol Chem 283, 8783–8787.[CrossRef] [Google Scholar]
  20. 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]
  21. Mottola, G., Cardinali, G., Ceccacci, A., Trozzi, C., Bartholomew, L., Torrisi, M. R., Pedrazzini, E., Bonatti, S. & Migliaccio, G.(2002). Hepatitis C virus nonstructural proteins are localized in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons. Virology 293, 31–43.[CrossRef] [Google Scholar]
  22. Muhlrad, D., Decker, C. J. & Parker, R.(1994). Deadenylation of the unstable mRNA encoded by the yeast MFA2 gene leads to decapping followed by 5′→3′ digestion of the transcript. Genes Dev 8, 855–866.[CrossRef] [Google Scholar]
  23. Ng, T. I., Mo, H., Pilot-Matias, T., He, Y., Koev, G., Krishnan, P., Mondal, R., Pithawalla, R., He, W. & other authors(2007). Identification of host genes involved in hepatitis C virus replication by small interfering RNA technology. Hepatology 45, 1413–1421.[CrossRef] [Google Scholar]
  24. Okumura, A., Lu, G., Pitha-Rowe, I. & Pitha, P. M.(2006). Innate antiviral response targets HIV-1 release by the induction of ubiquitin-like protein ISG15. Proc Natl Acad Sci U S A 103, 1440–1445.[CrossRef] [Google Scholar]
  25. Okumura, A., Pitha, P. M. & Harty, R. N.(2008). ISG15 inhibits Ebola VP40 VLP budding in an L-domain-dependent manner by blocking Nedd4 ligase activity. Proc Natl Acad Sci U S A 105, 3974–3979.[CrossRef] [Google Scholar]
  26. Paek, K. Y., Kim, C. S., Park, S. M., Kim, J. H. & Jang, S. K.(2008). RNA-binding protein hnRNP D modulates internal ribosome entry site-dependent translation of hepatitis C virus RNA. J Virol 82, 12082–12093.[CrossRef] [Google Scholar]
  27. Parker, R. & Sheth, U.(2007). P bodies and the control of mRNA translation and degradation. Mol Cell 25, 635–646.[CrossRef] [Google Scholar]
  28. Pestova, T. V., Shatsky, I. N., Fletcher, S. P., Jackson, R. J. & Hellen, C. U.(1998). A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. Genes Dev 12, 67–83.[CrossRef] [Google Scholar]
  29. Prabhu, R., Vittal, P., Yin, Q., Flemington, E., Garry, R., Robichaux, W. H. & Dash, S.(2005). Small interfering RNA effectively inhibits protein expression and negative strand RNA synthesis from a full-length hepatitis C virus clone. J Med Virol 76, 511–519.[CrossRef] [Google Scholar]
  30. Quintavalle, M., Sambucini, S., Di Pietro, C., De Francesco, R. & Neddermann, P.(2006). The α isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation. J Virol 80, 11305–11312.[CrossRef] [Google Scholar]
  31. Quintavalle, M., Sambucini, S., Summa, V., Orsatti, L., Talamo, F., De Francesco, R. & Neddermann, P.(2007). Hepatitis C virus NS5A is a direct substrate of casein kinase I-alpha, a cellular kinase identified by inhibitor affinity chromatography using specific NS5A hyperphosphorylation inhibitors. J Biol Chem 282, 5536–5544.[CrossRef] [Google Scholar]
  32. Randall, G., Panis, M., Cooper, J. D., Tellinghuisen, T. L., Sukhodolets, K. E., Pfeffer, S., Landthaler, M., Landgraf, P., Kan, S. & other authors(2007). Cellular cofactors affecting hepatitis C virus infection and replication. Proc Natl Acad Sci U S A 104, 12884–12889.[CrossRef] [Google Scholar]
  33. Sadler, A. J. & Williams, B. R.(2008). Interferon-inducible antiviral effectors. Nat Rev Immunol 8, 559–568.[CrossRef] [Google Scholar]
  34. Seo, M. Y., Abrignani, S., Houghton, M. & Han, J. H.(2003). Small interfering RNA-mediated inhibition of hepatitis C virus replication in the human hepatoma cell line Huh-7. J Virol 77, 810–812.[CrossRef] [Google Scholar]
  35. Soriano, V., Peters, M. G. & Zeuzem, S.(2009). New therapies for hepatitis C virus infection. Clin Infect Dis 48, 313–320.[CrossRef] [Google Scholar]
  36. Steitz, T. A.(2008). A structural understanding of the dynamic ribosome machine. Nat Rev Mol Cell Biol 9, 242–253.[CrossRef] [Google Scholar]
  37. Supekova, L., Supek, F., Lee, J., Chen, S., Gray, N., Pezacki, J. P., Schlapbach, A. & Schultz, P. G.(2008). Identification of human kinases involved in hepatitis C virus replication by small interference RNA library screening. J Biol Chem 283, 29–36.[CrossRef] [Google Scholar]
  38. Targett-Adams, P. & McLauchlan, J.(2005). Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon. J Gen Virol 86, 3075–3080.[CrossRef] [Google Scholar]
  39. 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]
  40. Tellinghuisen, T. L., Evans, M. J., von Hahn, T., You, S. & Rice, C. M.(2007). Studying hepatitis C virus: making the best of a bad virus. J Virol 81, 8853–8867.[CrossRef] [Google Scholar]
  41. Tsukiyama-Kohara, K., Iizuka, N., Kohara, M. & Nomoto, A.(1992). Internal ribosome entry site within hepatitis C virus RNA. J Virol 66, 1476–1483. [Google Scholar]
  42. Wang, C., Sarnow, P. & Siddiqui, A.(1993). Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism. J Virol 67, 3338–3344. [Google Scholar]
  43. Yokota, T., Sakamoto, N., Enomoto, N., Tanabe, Y., Miyagishi, M., Maekawa, S., Yi, L., Kurosaki, M., Taira, K. & other authors(2003). Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs. EMBO Rep 4, 602–608.[CrossRef] [Google Scholar]
  44. Zhao, C., Denison, C., Huibregtse, J. M., Gygi, S. & Krug, R. M.(2005). Human ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways. Proc Natl Acad Sci U S A 102, 10200–10205.[CrossRef] [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.022210-0
Loading
/content/journal/jgv/10.1099/vir.0.022210-0
Loading

Data & Media loading...

Supplements

vol. , part 9, pp. 2238 - 2248

Genes from plate 1

Genes from plate 2

Genes from plate 3

Genes from plate 4

U2OS cells are more effectively transfected than Huh-7 cells

Results from the siRNA library screen [Single PDF file](3284 KB)



PDF

Most cited Most Cited RSS feed