Expression Cloning of Host Factors Required for the HCV Replication Cycle

  • Sandra CiesekEmail author
  • Thomas von Hahn
Part of the Methods in Molecular Biology book series (MIMB, volume 1911)


Chronic infection with the hepatitis C virus (HCV) is a common cause of chronic liver disease, cirrhosis, and liver cancer. Like most viruses, HCV depends of a number of factors encoded by its host cell to execute its replication cycle and create. Expression cloning is one of several possible approaches that have been employed to identify novel host factors essential for the HCV replication cycle. It involves generation of a cDNA library from a cell type with a desired trait (such as ability to bind E2 or being susceptible to HCV infection), expression of that library in a different cell type missing the trait of interest, and selection for cell clones that have acquired that trait through expression of a specific cDNA. This chapter describes an expression cloning approach similar to the one that was used to identify the tight junction component claudin-1 as an essential HCV cell entry factor.

Key words

HCV Host factor Claudin-1 cDNA library Selection Cyclic packaging rescue V1 



The authors would like to acknowledge Theodora Hatziioannou and Paul Bieniasz of Aaron Diamond AIDS Research Center (NY, USA) who conceived, designed, generated, and provided the V1 vector enabling a CPR-based cloning strategy. The authors would also like to acknowledge the contribution of Charles Rice (The Rockefeller University, NY, USA) and Matthew Evans (Mount Sinai School of Medicine, NY, USA) who made very major contributions to devising the HCV-customized strategy described here.


  1. 1.
    Scarselli E, Ansuini H, Cerino R, Roccasecca RM, Acali S, Filocamo G et al (2002) The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J 21:5017–5025CrossRefGoogle Scholar
  2. 2.
    Watashi K, Hijikata M, Hosaka M, Yamaji M, Shimotohno K (2003) Cyclosporin a suppresses replication of hepatitis C virus genome in cultured hepatocytes. Hepatology 38:1282–1288CrossRefGoogle Scholar
  3. 3.
    Nakagawa M, Sakamoto N, Enomoto N, Tanabe Y, Kanazawa N, Koyama T et al (2004) Specific inhibition of hepatitis C virus replication by cyclosporin A. Biochem Biophys Res Commun 313:42–47CrossRefGoogle Scholar
  4. 4.
    Andre P, Komurian-Pradel F, Deforges S, Perret M, Berland JL, Sodoyer M et al (2002) Characterization of low- and very-low-density hepatitis C virus RNA-containing particles. J Virol 76:6919–6928CrossRefGoogle Scholar
  5. 5.
    Wozniak MA, Itzhaki RF, Faragher EB, James MW, Ryder SD, Irving WL et al (2002) Apolipoprotein E-epsilon 4 protects against severe liver disease caused by hepatitis C virus. Hepatology 36:456–463CrossRefGoogle Scholar
  6. 6.
    Toniutto P, Fabris C, Fumo E, Apollonio L, Caldato M, Mariuzzi L et al (2004) Carriage of the apolipoprotein E-epsilon4 allele and histologic outcome of recurrent hepatitis C after antiviral treatment. Am J Clin Pathol 122:428–433CrossRefGoogle Scholar
  7. 7.
    Price DA, Bassendine MF, Norris SM, Golding C, Toms GL, Schmid ML et al (2006) Apolipoprotein epsilon3 allele is associated with persistent hepatitis C virus infection. Gut 55:715–718CrossRefGoogle Scholar
  8. 8.
    Chang KS, 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 81:13783–13793CrossRefGoogle Scholar
  9. 9.
    Long G, Hiet MS, Windisch MP, Lee JY, Lohmann V, Bartenschlager R (2011) Mouse hepatic cells support assembly of infectious hepatitis C virus particles. Gastroenterology 141:1057–1066CrossRefGoogle Scholar
  10. 10.
    Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, Petracca R et al (1998) Binding of hepatitis C virus to CD81. Science 282:938–941CrossRefGoogle Scholar
  11. 11.
    Evans MJ, von Hahn T, Tscherne DM, Syder AJ, Panis M, Wolk B et al (2007) Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446:801–805CrossRefGoogle Scholar
  12. 12.
    Ploss A, Evans MJ, Gaysinskaya VA, Panis M, You H, de Jong YP et al (2009) Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 457:882–886CrossRefGoogle Scholar
  13. 13.
    Derdeyn CA, Decker JM, Sfakianos JN, Wu X, O'Brien WA, Ratner L et al (2000) Sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120. J Virol 74:8358–8367CrossRefGoogle Scholar
  14. 14.
    Demaison C, Parsley K, Brouns G, Scherr M, Battmer K, Kinnon C et al (2002) High-level transduction and gene expression in hematopoietic repopulating cells using a human immunodeficiency [correction of immunodeficiency] virus type 1-based lentiviral vector containing an internal spleen focus forming virus promoter. Hum Gene Ther 13:803–813CrossRefGoogle Scholar
  15. 15.
    Cowan S, Hatziioannou T, Cunningham T, Muesing MA, Gottlinger HG, Bieniasz PD (2002) Cellular inhibitors with Fv1-like activity restrict human and simian immunodeficiency virus tropism. Proc Natl Acad Sci U S A 99:11914–11919CrossRefGoogle Scholar
  16. 16.
    Zennou V, Bieniasz PD (2006) Comparative analysis of the antiretroviral activity of APOBEC3G and APOBEC3F from primates. Virology 349:31–40CrossRefGoogle Scholar
  17. 17.
    Reed LJ, Muench H (1938) A simple method of estimating fifty per cent endpoints. Am J Hyg 27:493–497Google Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Virology, University Hospital EssenUniversity of Duisburg-EssenEssenGermany
  2. 2.German Center for Infection Research (DZIF), External Partner Site EssenEssenGermany
  3. 3.Department of Gastroenterology, Hepatology und EndocrinologyHannover Medical SchoolHannoverGermany
  4. 4.Institute for Molecular BiologyHannover Medical SchoolHannoverGermany
  5. 5.German Center for Infection Research (DZIF)Hannover-Braunschweig SiteHannoverGermany

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