Abstract
The establishment of a cell culture system for hepatitis C virus based on the JFH-1 strain and human hepatoma cell lines has been instrumental for the study of the viral replication cycle. The robustness of the JFH1-based cell culture models enabled many laboratories around the world to perform HCV infections in cell culture, accelerating the identification of cellular and viral targets to develop novel antiviral compounds. Although other robust infection systems based on different molecular clones and different cell lines have been developed since then, here we describe the protocols corresponding to infections with JFH-1 and JFH1-derived viruses carried out in our laboratory to produce virus stocks and persistently infected cell cultures. We also describe the experimental setups used to determine virus spreading capacity (multiple cycle infections) as well as to dissect early and late aspects of HCV infection (single cycle infections).
Materials and Reagents
Equipment
Procedure
Part I. Multiple cycle infections Multiple cycle infections are also defined as low multiplicity of infection (MOI, i.e. number of infectious particles/cell) experiments. They are generally used to produce virus stocks and to generate persistently infected cell cultures. They also constitute a first approach to characterize the capacity of a virus to spread under different experimental conditions or to compare the spreading capacity of different viruses.
Part II. Virus titration by endpoint dilution and immunofluorescence This procedure is based on the detection of infection foci (infected cell clusters) by immunofluorescence microscopy. Each infection focus is attributed to one infectious unit, defined as focus forming unit (FFU). The number of infection foci obtained by inoculation of naive cells with serial sample dilutions is used to determine the infectivity titer.
Part III. Single cycle infection experiments Single cycle infection experiments, also termed high multiplicity infections (MOI of 10 FFU/cell), attempt modeling synchronic infections of one virion infecting one target cell and are generally used to dissect early and late aspects of the hepatitis C virus life cycle. In contrast to multiple cycle infections, we recommend measuring different parameters such as intracellular and extracellular infectivity and RNA. Simultaneous analysis of these parameters in this experimental setup will enable determining the efficiency of different aspects of the infection. Since high infectivity titer virus stocks are required to perform these experiments and sufficient titers are difficult to reach with the parental JFH-1 strain, we typically use D183 virus stocks for these experiments (see Part I, section A).
Representative data
Figure 1. Immunofluorescence microscopy of Huh-7 cells infected with serial dilutions of infected cell supernatant and visualization of individual infection foci in cells inoculated with 1:10 diluted samples. Red channel anti-E2 antibody, blue channel cell nuclei stained with DAPI. The typical yield of virus stocks should be around 104 FFU/ml for stocks generated with JFH-1 virus and >106 FFU/ml with D183 virus. Persistently infected cell cultures should produce consistently titers >103 FFU/ml for the first month after reaching 100% infection. Single cycle infection experiments should display peak extracellular infectivity titers >104 FFU/ml, typically 48 h post-infection.
Notes
Recipes
Acknowledgments
This protocol is based on work initially developed at Dr. Francis V. Chisari´s laboratory at The Scripps Research Institute (La Jolla, CA) in collaboration with Dr. Takaji Wakita´s group at the Tokyo Metropolitan Institute of Medical Science (Tokyo, Japan) and was originally generated and improved with the essential contribution of Jin Zhong (currently at Pasteur Institute in Shangai), Sharookh Kapadia (currently at Genentech), Guofeng Cheng (currently at Gilead), Susan Uprichard (currently at U. of Illinois, Chicago) and specially Stefan Wieland (currently at Basel University Hospital Basel). L.M. was funded by a JAE-Pre fellowship from Consejo Superior de Investigaciones Científicas and CIBERehd (Instituto de Salud Carlos III). C.V. is funded by a Fundación “La Caixa”/CNB fellowship. This work was supported by the grants Plan Nacional De Investigación Científica, Desarrollo e Innovación Tecnológica from the Spanish Ministry of Science and Innovation (SAF2010-19270) and a Marie Curie Career Integration Grant (PCIG-9-GA-2011-293664) from the European Union 7th Framework Programme for Research.
References
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