Expression Cloning of Host Factors Required for the HCV Replication Cycle
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 wordsHCV 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.
- 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
- 17.Reed LJ, Muench H (1938) A simple method of estimating fifty per cent endpoints. Am J Hyg 27:493–497Google Scholar